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https://openalex.org/W2756060471
https://www.nature.com/articles/s41598-017-11969-5.pdf
English
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Bacteriological diagnosis of childhood TB: a prospective observational study
Scientific reports
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Bacteriological diagnosis of childhood TB: a prospective observational study y Andrew J. Brent   1,2,3,4, Daisy Mugo1, Robert Musyimi1, Agnes Mutiso1, Susan C. Morpeth1 Michael Levin3 & J. Anthony G. Scott   1,2,5 Received: 16 June 2017 Accepted: 1 September 2017 Published: xx xx xxxx Received: 16 June 2017 Accepted: 1 September 2017 Published: xx xx xxxx Childhood TB diagnosis is challenging. Studies in adults suggest Microscopic Observation Drug Susceptibility (MODS) culture or the Xpert MTB/RIF assay might be used to expand bacteriological diagnosis. However data from children are more limited. We prospectively compared MODS and Xpert MTB/RIF with standard microscopy and culture using the BD MGIT 960 system among 1442 Kenyan children with suspected TB. 97 specimens from 54 children were TB culture-positive: 91 (94%) by MGIT and 74 (76%) by MODS (p = 0.002). 72 (74%) culture-positive and 7 culture-negative specimens were Xpert MTB/RIF positive. Xpert MTB/RIF specificity was 100% (99.7–100%) among 1164 specimens from 892 children in whom TB was excluded, strongly suggesting all Xpert MTB/RIF positives are true positives. The sensitivity of MGIT, MODS and Xpert MTB/RIF was 88%, 71% and 76%, respectively, among all 104 true positive (culture and/or Xpert MTB/RIF positive) specimens. MGIT, MODS and Xpert MTB/RIF on the initial specimen identified 40/51 (78%), 33/51 (65%) and 33/51 (65%) culture-confirmed pulmonary TB cases, respectively; Xpert MTB/RIF detected 5 additional culture-negative cases. The high sensitivity and very high specificity of the Xpert MTB/RIF assay supports its inclusion in the reference standard for bacteriological diagnosis of childhood TB in research and clinical practice. Definitive diagnosis of tuberculosis (TB) rests on detection of M. tuberculosis from clinical specimens, but is difficult in children. While sputum smear microscopy underpins the WHO DOTS and Stop TB strategies for TB diagnosis in adults1, obtaining sputum is more difficult in children, and the yield of microscopy alone is very poor due to small numbers of bacilli in clinical specimens. Bacteriological diagnosis therefore depends on mycobac- terial culture2. Strengthening laboratory capacity is a key component of the Stop TB Strategy and Global Plan to Stop TB1,3. Commercial liquid culture methods like the BACTEC Mycobacteria Growth Indicator Tube (MGIT) system (BD Diagnostics, Sparks, MD, USA) offer higher sensitivity and more rapid results than traditional solid media. However, barriers to their uptake include cost, a lack of technically trained personnel, and the need for biosafety level 3 facilities3. Correction: Author Correction www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 Bacteriological diagnosis of childhood TB: a prospective observational study Provider initiated testing and counseling for HIV was performed according to Kenyan national guidelines, which recommend testing for all inpatients, and for all patients investigated for TB, on an opt-out basis. g Children who were able to expectorate provided up to three spontaneous sputum samples. Sputum induction was performed on the remainder. If sputum induction was contraindicated (e.g. due to severe respiratory dis- tress), gastric aspiration was performed. Sputum induction and gastric aspiration were performed according to international recommendations2. Further investigations including extra-pulmonary or repeat sputum sampling were performed at the discretion of the clinical team caring for the patient. p g p We classified children as Confirmed TB, Highly Probable TB, Possible TB or Not TB according to their clin- ical, radiological and microbiological findings. Categories were defined a priori and based closely on published definitions (Fig. 1)13,14. Treatment protocols followed Kenyan national guidelines. Children treated for TB were followed up until completion of treatment at 6 months. Other children were also followed up for 6 months, or until TB could be confidently excluded. Final diagnostic assignments were revised in the light of follow up data. Laboratory procedures. Specimens were transported to the laboratory at 2–8 °C and processed the same day. Sputum specimens were decontaminated using the modified Petroff’s method and 4% sodium hydroxide15; centrifuged; and the sediment re-suspended in 2 ml culture broth containing Middlebrook 7H9 medium, oxalic acid, albumin, dextrose, and catalase (OADC, Becton Dickinson), and polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA, Becton Dickinson)16. After vigorous vortexing to ensure sample homog- enization, two drops were used to make a smear for Ziehl Neelsen staining, and the remainder divided equally for MGIT, MODS, and Xpert MTB/RIF. Samples for Xpert MTB/RIF analysis were promptly frozen at −80 °C for analysis at the end of the study. y y Liquid mycobacterial culture was performed using the MGIT 960 system and MODS assay. A laboratory technician (RM) first received one month MODS training at the Universidad Peruana Cayetano Heredia in Lima, Peru. Standard protocols were followed15,16, except that MODS plates were examined 3 times a week rather than daily for logistic reasons. y g Positive cultures from both methods were identified as M. tuberculosis complex (MTBC) or non-MTBC by the BD MGIT TBc Identification test (BD Diagnostics, Sparks, MD, USA). Bacteriological diagnosis of childhood TB: a prospective observational study Microcolony culture techniques such as the Microscopic Observation Drug Susceptibility (MODS) assay have several potential advantages over conventional culture methods in low resource settings, including high sensi- tivity; more rapid results that include drug susceptibility testing (DST); lower cost; and less stringent biosafety requirements making it applicable in biosafety level 2 facilities4. However only limited data exist on their use in children5–7, and no studies have compared MODS with MGIT for diagnosis of TB in young children; nor eval- uated MODS yield from induced sputum samples, despite good evidence and international recommendations supporting sputum induction for childhood TB diagnosis2,8.hf g g The Xpert MTB/RIF real-time PCR assay (Cepheid, Sunnyvale, CA, USA) offers a rapid and operationally simpler alternative to culture for detection of M. tuberculosis and rifampicin resistance9,10. A recent meta-analysis suggests modest sensitivity and high specificity for paediatric TB diagnosis11, and the latest WHO childhood TB guidelines (2014) recommend Xpert MTB/RIF as an alternative to conventional microscopy and culture - but acknowledge the “very low quality of evidence”2. 1KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya. 2University of Oxford, Oxford, UK. 3Imperial College London, London, UK. 4Oxford University Hospitals NHS Foundation Trust, Oxford, UK. 5London School of Hygiene & Tropical Medicine, London, UK. Correspondence and requests for materials should be addressed to A.J.B. (email: dr.a.brent@gmail.com) SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 1 www.nature.com/scientificreports/ We compared microscopy, MGIT and MODS culture, and the Xpert MTB/RIF assay for diagnosis of child- hood TB in Kenya. Participants. We prospectively assessed each diagnostic method among children presenting to Coast Provincial General Hospital and Kilifi County Hospital in Coast Province, Kenya. Between July 2010 and December 2011 children aged <15 years were investigated for TB if one or more of the following features of sus- pected TB were present and they were not already on TB treatment: unexplained persistent cough for >2 weeks; pneumonia not responding to first line antibiotics; unexplained fever for >2 weeks; unexplained progressive weight loss or failure to thrive for >4 weeks; a history of close contact with a suspected or confirmed case of pul- monary TB; or clinical suspicion of TB for any other reason. Clinical procedures. Each child underwent a structured history and examination, chest radiography and tuberculin skin testing (TST) according to WHO guidelines2. Severe malnutrition was defined as weight for age z-score of <−3 or the presence of nutritional oedema12. Bacteriological diagnosis of childhood TB: a prospective observational study They were then further speciated and probed for isoniazid and rifampicin resistance mutations by PCR, using the Hain Genotype® line probe assay platform (Hain Lifescience GmbH, Nehren, Germany). The Xpert MTB/RIF assay (version G4) was performed at the end of the study on specimens from all children treated for confirmed, highly probable or possible TB, and from children in whom TB had been excluded. Laboratory procedures were externally monitored through the UK NEQAS quality assurance scheme and annual Good Clinical Laboratory Practice audits (Qualogy, UK). q y y gy In order to compare the performance of MGIT and MODS, and to prevent observer bias in the interpretation of either result, laboratory technologists were blinded to the identity of the MODS portion of each specimen. Specimens were instead identified by an electronically generated random numeric code, the key to which was held by the Principal Investigator. Statistical analysis. Data were analyzed at both the patient and the specimen level. We first performed a per patient analysis to compare the sensitivity of smear microscopy, MGIT, MODS and Xpert MTB/RIF for identification of culture confirmed pulmonary TB cases. We then included all specimens (pulmonary and extra-pulmonary) in a per specimen analysis to calculate the sensitivity of each method against the existing refer- ence standard of a positive M. tuberculosis culture (by MGIT and/or MODS).i p y To explore the specificity of Xpert MTB/RIF we calculated the proportion of specimens from Not TB cases that were Xpert MTB/RIF positive. Having established the very high specificity of the Xpert MTB/RIF assay, we then repeated the per specimen analysis using a composite reference standard incorporating Xpert MTB/RIF, such that a specimen was considered positive if either culture or Xpert MTB/RIF identified M. tuberculosis. i We used McNemar’s χ2 test to compare proportions between tests on paired aliquots of the same sample. To investigate any learning effect as laboratory staff became more experienced with MODS we performed the χ2 test for trend in the proportion of M. tuberculosis culture positive specimens identified by MODS in each quarter, and the Wilcoxon rank sum test to compare MODS time to detection (TTD) in the first and second years of the study. We used the Wilcoxon signed rank test to compare TTD of MTBC by MODS and MGIT. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Bacteriological diagnosis of childhood TB: a prospective observational study Associations with a positive Xpert MTB/RIF assay were explored using univariable and multivariable logistic regression models adjusted for clustering at the patient level. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 2 Figure 1.  Childhood TB Case Definitions. Ethical approval and informed consent. The study was approved by the Kenya National Ethics Committee. All study procedures were performed in accordance with relevant guidelines and regulations. A parent or guardian provided written informed consent. Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 3 www.nature.com/scientificreports/ Results We identified 1500 children with features of suspected TB, 1442 of whom were investigated for TB (Fig. 2). Active Figure 2. Patient enrolment and disease assignments. Confirmed TB Highly Probable TB Treated for Possible TB Not TB (TB excluded) Not classifiable (n = 54) (n = 63) (n = 95) (n = 892) (n = 338) Patient demographics Age Median (IQR1), months 53 (21–112) 32 (13–61) 17 (10–68) 16 (9–40) 17 (9–48) 0 to 4 years 27 (50%) 47 (75%) 67 (70%) 750 (84%) 271 (80%) 5 to 9 years 14 (26%) 10 (16%) 15 (16%) 95 (11%) 45 (13%) 10 to 14 years 13 (24%) 6 (9%) 13 (14%) 47 (5%) 22 (7%) Male sex 33 (61%) 32 (51%) 48 (51%) 469 (53%) 188 (56%) Clinical features Cough > 2 weeks 40 (74%) 37 (59%) 71 (75%) 425 (48%) 194 (57%) Fever > 2 weeks 37 (69%) 24 (38%) 66 (69%) 375 (42%) 167 (49%) Weight loss/FTT2 > 4 weeks 32 (59%) 29 (46%) 51 (54%) 385 (43%) 168 (50%) Pneumonia not responding to first line antibiotics 22 (41%) 21 (33%) 35 (37%) 260 (29%) 144 (43%) Close TB contact 24 (44%) 25 (40%) 23 (24%) 145 (16%) 62 (18%) HIV infected 15 (28%) 15 (24%) 35 (37%) 121 (14%) 88 (26%) Severely malnourished 19 (35%) 31 (49%) 40 (42%) 303 (34%) 119 (35%) Pulmonary TB3 51 (94%) 57 (90%) 77 (81%) — — Extra-pulmonary TB3 7 (13%) 6 (10%) 20 (21%) — — Table 1. Baseline characteristics of children with and without TB. 1IQR, interquartile range. 2FTT, failure to thrive. 3Six patients with pulmonary TB also had extrapulmonary TB (4 with culture confirmed TB, 2 that were treated for possible TB). Figure 2. Patient enrolment and disease assignments. Figure 2. Patient enrolment and disease assignments. Confirmed TB Highly Probable TB Treated for Possible TB Not TB (TB excluded) Not classifiable (n = 54) (n = 63) (n = 95) (n = 892) (n = 338) Patient demographics Age Median (IQR1), months 53 (21–112) 32 (13–61) 17 (10–68) 16 (9–40) 17 (9–48) 0 to 4 years 27 (50%) 47 (75%) 67 (70%) 750 (84%) 271 (80%) 5 to 9 years 14 (26%) 10 (16%) 15 (16%) 95 (11%) 45 (13%) 10 to 14 years 13 (24%) 6 (9%) 13 (14%) 47 (5%) 22 (7%) Male sex 33 (61%) 32 (51%) 48 (51%) 469 (53%) 188 (56%) Clinical features Cough > 2 weeks 40 (74%) 37 (59%) 71 (75%) 425 (48%) 194 (57%) Fever > 2 weeks 37 (69%) 24 (38%) 66 (69%) 375 (42%) 167 (49%) Weight loss/FTT2 > 4 weeks 32 (59%) 29 (46%) 51 (54%) 385 (43%) 168 (50%) Pneumonia not responding to first line antibiotics 22 (41%) 21 (33%) 35 (37%) 260 (29%) 144 (43%) Close TB contact 24 (44%) 25 (40%) 23 (24%) 145 (16%) 62 (18%) HIV infected 15 (28%) 15 (24%) 35 (37%) 121 (14%) 88 (26%) Severely malnourished 19 (35%) 31 (49%) 40 (42%) 303 (34%) 119 (35%) Pulmonary TB3 51 (94%) 57 (90%) 77 (81%) — — Extra-pulmonary TB3 7 (13%) 6 (10%) 20 (21%) — — Table 1. Baseline characteristics of children with and without TB. 1IQR, interquartile range. 2FTT, failure to thrive. 3Six patients with pulmonary TB also had extrapulmonary TB (4 with culture confirmed TB, 2 that were treated for possible TB). Table 1. Baseline characteristics of children with and without TB. 1IQR, interquartile range. 2FTT, failure to thrive. 3Six patients with pulmonary TB also had extrapulmonary TB (4 with culture confirmed TB, 2 that were treated for possible TB). Resultsi Comparison of MGIT and MODS culture sensitivity against culture reference standard incorporating both MGIT and MODS. subsequent sputum sample. MGIT, MODS and Xpert MTB/RIF each demonstrated a modest incremental yield from additional specimens (Table 3). None of the 8 initial culture-negative specimens from confirmed PTB cases were Xpert MTB/RIF positive. However Xpert MTB/RIF did detect M. tuberculosis in sputum from 5 additional children with culture negative pulmonary TB (3 with clinically Highly Probable TB and 2 who were treated for Possible TB). subsequent sputum sample. MGIT, MODS and Xpert MTB/RIF each demonstrated a modest incremental yield from additional specimens (Table 3). None of the 8 initial culture-negative specimens from confirmed PTB cases were Xpert MTB/RIF positive. However Xpert MTB/RIF did detect M. tuberculosis in sputum from 5 additional children with culture negative pulmonary TB (3 with clinically Highly Probable TB and 2 who were treated for Possible TB). Per specimen analysis of pulmonary and extra-pulmonary specimens combined. A total of 2085 specimens were obtained for mycobacterial culture. We were unable to do MODS on 182 samples due to holiday staffing shortages (169) or insufficient sample (13). A total of 1903 specimens were therefore included in the anal- ysis, of which 1802 (95%) were sputum specimens, 33 (2%) were smear positive, and 97 (5%) were M. tuberculosis culture positive (Supplementary Appendix, Table S1). MGIT was more sensitive than MODS overall (93.8% vs 76.3%, p = 0.002), among all sputum specimens, among induced sputum specimens, and among specimens from both younger and older children (Table 4).h y g ( ) There was weak evidence of an increase in MODS sensitivity (χ2 test for trend p = 0.038) and a decrease in TTD over time, with a median TTD of 16 (IQR 7 to 21) days in 2010 compared with 10 (7 to 14) days in 2011 (p = 0.052; Supplementary Appendix, Fig. S1). Overall, TTD was slightly shorter for MODS than MGIT (11 [IQR 6 to 15] days versus 12 [7 to 17] days, p = 0.001). ] y [ ] y , p ) Discordant culture results were obtained for 29 specimens. Independent bacteriological confirmation of M. tuberculosis was available for 21/29 of these specimens, either by the Xpert MTB/RIF assay on the original specimen (15/29) and/or by isolation of M. tuberculosis from an independent specimen from the same patient (Supplementary Appendix, Table S2). Resultsi yield* 1st specimen 51 13 (25%) 25% 40 (78%) 78% 33 (65%) 65% 43 (84%) 84% 33 (65%) 65% 2nd specimen 32 10 (31%) 33% 22 (69%) 82% 15 (68%) 73% 24 (75%) 90% 18 (56%) 69% 3rd specimen 23 7 (30%) 33% 14 (61%) 84% 13 (57%) 75% 15 (65%) 92% 13 (57%) 71% Specimen N Smear microscopy MGIT culture MODS culture MODS + MGIT culture Xpert MTB/RIF Test + (%) Cum. yield* Test + (%) Cum. yield* Test + (%) Cum. yield* Test + (%) Cum. yield* Test + (%) Cum. yield* 1st specimen 51 13 (25%) 25% 40 (78%) 78% 33 (65%) 65% 43 (84%) 84% 33 (65%) 65% 2nd specimen 32 10 (31%) 33% 22 (69%) 82% 15 (68%) 73% 24 (75%) 90% 18 (56%) 69% 3rd specimen 23 7 (30%) 33% 14 (61%) 84% 13 (57%) 75% 15 (65%) 92% 13 (57%) 71% Table 3. Cumulative sensitivity (incremental yield) of serial sputum specimens from patients with culture confirmed pulmonary TB. *Cum. yield = cumulative sensitivity of serial sputum specimens. In 4 (8%) cases of culture-confirmed pulmonary TB, culture confirmation was obtained from a subsequent specimen (n = 1) or from a specimen excluded from the analysis because MODS culture was not performed (n = 3). Table 3. Cumulative sensitivity (incremental yield) of serial sputum specimens from patients with culture confirmed pulmonary TB. *Cum. yield = cumulative sensitivity of serial sputum specimens. In 4 (8%) cases of culture-confirmed pulmonary TB, culture confirmation was obtained from a subsequent specimen (n = 1) or from a specimen excluded from the analysis because MODS culture was not performed (n = 3). MGIT positive MGIT negative MGIT Sensitivity % (95% CI) MODS Sensitivity % (95% CI) p value MODS positive MODS negative MODS positive MODS negative Specimen type All specimens 68 23 6 1807 93.8 (87.0–97.7) 76.3 (66.6–84.3) 0.002 All sputum specimens 61 22 6 1713 93.3 (85.9–97.5) 75.3 (65.0–83.8) 0.003 Induced sputum 40 18 6 1603 90.6 (80.7–96.5) 71.9 (59.2–82.4) 0.014 Age of patient <5 years 28 14 5 1398 89.4 (76.9–96.5) 70.2 (55.1–82.7) 0.039 5–14 years 36 8 1 380 97.8 (88.2–99.9) 82.2 (67.9–92.0) 0.039 Table 4. Comparison of MGIT and MODS culture sensitivity against culture reference standard incorporating both MGIT and MODS. Table 4. Comparison of MGIT and MODS culture sensitivity against culture reference standard incorporating both MGIT and MODS. Table 4. Resultsi We identified 1500 children with features of suspected TB, 1442 of whom were investigated for TB (Fig. 2). Active TB was diagnosed in 212 (14.7%) children, including 54 (25.5%) with Culture-confirmed TB, 63 (29.7%) with Highly Probable TB, and 95 (44.8%) with Possible TB. Most cases (185, 87%) had pulmonary TB (PTB). Baseline demographic and clinical characteristics are summarized in Table 1. In keeping with previous studies, the propor- tion of cases that were bacteriologically confirmed was higher among older children. Per patient analysis of pulmonary TB cases. There were 51 cases of Culture-confirmed pulmonary TB. Smear microscopy, MGIT and MODS on the initial sputum specimen identified M. tuberculosis in 13 (25%), 40 (78%) and 33 (65%) cases, respectively (Table 2). Sensitivity was highest for each method among smear positive samples; Xpert MTB/RIF identified 100% of smear-positive cases and 53% of smear-negative cases on the first sputum specimen (Table 2). p p Second and third sputum specimens were obtained from 237 (16%) and 102 (7%) children, respectively. Eight (16%) culture confirmed PTB cases were culture negative from the initial sample but grew M. tuberculosis from a SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 4 www.nature.com/scientificreports/ AFB smear positive (N = 13) AFB smear negative (N = 38) Total (N = 51) N Sensitivity (95% CI) N Sensitivity (95% CI) N Sensitivity (95% CI) AFB smear microscopy 13 N/A N/A N/A 13 25.5 (14.3–39.6) MGIT culture 13 100.0 (75.3–100.0) 27 71.1 (54.1–84.6) 40 78.4 (64.7–88.7) MODS culture 13 100.0 (75.3–100.0) 20 52.6 (35.8–69.0) 33 64.7 (50.1–77.6) MGIT + MODS culture 13 100.0 (75.3–100.0) 30 78.9 (62.7–90.4) 43 84.3 (71.4–93.0) Xpert MTB/RIF 13 100.0 (75.3–100.0) 20 52.6 (35.8–69.0) 33 64.7 (50.1–77.6) Table 2. Sensitivity of each method for detection of M. tuberculosis in the initial sputum specimen from patients with culture confirmed pulmonary TB. Table 2. Sensitivity of each method for detection of M. tuberculosis in the initial sputum specimen from patients with culture confirmed pulmonary TB. Specimen N Smear microscopy MGIT culture MODS culture MODS + MGIT culture Xpert MTB/RIF Test + (%) Cum. yield* Test + (%) Cum. yield* Test + (%) Cum. yield* Test + (%) Cum. yield* Test + (%) Cum. Discussion Th l k f l The lack of reliable diagnostic tools is the single greatest obstacle to improved childhood TB case management, particularly in low resource settings where the disease burden is greatest. Expansion of TB laboratory diagnostic capacity provides an opportunity to improve both diagnosis and surveillance in many settings3. However good quality data on the performance of each method are required to optimize their use and interpretation. q y p q p p Few studies have compared laboratory methods for childhood TB diagnosis. Limitations of existing studies include small numbers of confirmed TB cases5–7,14,17–22, particularly among young children in whom diagnosis is h ll i 5 7 14 19 22 d l i f ‘ ld d d’ h d f i ll i d/ b i l y Few studies have compared laboratory methods for childhood TB diagnosis. Limitations of existing studies include small numbers of confirmed TB cases5–7,14,17–22, particularly among young children in whom diagnosis is most challenging5–7,14,19,22, and exclusion of ‘gold standard’ methods for specimen collection and/or mycobacterial lt 5 7 17 20 23 26 St th f thi t d i l d th f b th t i d ti d li id b t i l culture5 7,17,20,23 26. Strengths of this study include the use of both sputum induction and liquid mycobacterial culture in keeping with current international recommendations2, and rigorous diagnostic assignments based on detailed clinical assessment and follow up. Prospective recruitment of all children who met broad, pre-defined inclusion criteria also ensures generalizability to a wide range of settings and clinical syndromes, including among children under 5 years who accounted for 81% of those investigated and half of all confirmed cases.i y gi While comparison of culture yield between studies is hampered by widely varying clinical definitions, speci- men types and culture methods, the proportion of culture-confirmed cases in our study is consistent with other studies using liquid mycobacterial culture for paediatric diagnosis5,8,14,18,20,26–30.h In our study MGIT was more sensitive than MODS culture. This is in keeping with the only other published study comparing MGIT with MODS for paediatric TB diagnosis5. Importantly we were also able to demonstrate the higher yield of MGIT for culture of paediatric induced sputum samples. These results contrast with other studies in adults in which MODS compared more favourably with MGIT4. Resultsi The clinical picture strongly supported the presence of M. tuberculosis complex in the remaining 8 culture-discordant samples: 5 MGIT+/MODS− specimens came from children with clinically Highly Probable TB prior to culture results; 2 MGIT−/MODS+ specimens came from chil- dren treated empirically for TB prior to the culture result although they did not meet the stringent definition SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 5 www.nature.com/scientificreports/ Culture positive Culture negative Culture Sensitivity % (95% CI) Xpert MTB/RIF Sensitivity % (95% CI) p value Xpert positive Xpert negative Xpert positive Xpert negative Specimen type All specimens 72 25 7 1452 93.3 (86.6–97.3) 76.0 (66.6–83.8) 0.002 All sputum specimens 68 21 7 1419 92.7 (85.6–97.0) 78.1 (68.5–85.9) 0.008 Induced sputum 47 17 5 1356 92.8 (83.9–97.6) 75.4 (63.5–84.9) 0.011 Age of patient <5 years 32 15 7 1152 87.0 (75.1–94.6) 72.2 (58.4–83.5) 0.088 5–14 years 40 10 0 300 100 (92.9–100) 80.0 (66.3–90.0) 0.002 Table 5. Comparison of culture and Xpert MTB/RIF sensitivity against composite reference standard of both culture and Xpert MTB/RIF. Table 5. Comparison of culture and Xpert MTB/RIF sensitivity against composite reference standard of bot culture and Xpert MTB/RIF. of Highly Probable TB; and one MGIT+/MODS− specimen that grew M. bovis BCG came from a 4 month old HIV-infected child who had a clinical syndrome consistent with disseminated BCG disease (Supplementary Appendix, Table S2). All were the only positive cultures in their batch, arguing against cross-contamination caus- ing the discordance. Even if cross-contamination were the cause and these specimens considered culture negative, MGIT would remain more sensitive than MODS (95.5% vs 80.9%, p = 0.005).i of Highly Probable TB; and one MGIT+/MODS− specimen that grew M. bovis BCG came from a 4 month old HIV-infected child who had a clinical syndrome consistent with disseminated BCG disease (Supplementary Appendix, Table S2). All were the only positive cultures in their batch, arguing against cross-contamination caus- ing the discordance. Even if cross-contamination were the cause and these specimens considered culture negative, MGIT would remain more sensitive than MODS (95.5% vs 80.9%, p = 0.005).i Xpert MTB/RIF identified 72/97M. tuberculosis culture positive specimens, giving a sensitivity compared to culture of 74.2% (95% CI 64.3 to 82.6). Xpert MTB/RIF was also positive on 5/86 (6%) specimens from patients with culture-negative but clinically highly probable TB, and 2/179 (1%) specimens from children treated for possible TB. Resultsi No other specimens were Xpert MTB/RIF positive, including none of 1164 specimens obtained from children in whom TB was subsequently excluded - giving a specificity of 100% (1 sided 97.5% CI 99.7 to 100%) and strongly suggesting positive Xpert MTB/RIF results should be interpreted as true positives. Against the composite reference standard of a positive TB culture and/or Xpert MTB/RIF result, the sensi- tivity of TB culture was 93.3% (86.6 to 97.3), compared with 76.0% (66.6 to 83.8) for Xpert MTB/RIF (p = 0.002; Table 5). The sensitivity of MGIT and MODS culture alone were 87.5% (79.6 to 93.2) and 71.2% (61.4 to 79.6), respectively. Table 6 shows Xpert MTB/RIF sensitivity against the composite reference standard, broken down by smear status, specimen type, age and HIV status. Sensitivity was higher for smear positive specimens (100% compared with 65% for smear negative specimens, p < 0.001), with a trend towards higher sensitivity among sputum sam- ples. Neither age nor HIV status were associated with Xpert MTB/RIF yield in univariable or multivariable analyses.i y Xpert MTB/RIF identified rifampicin resistance in 4 specimens from the same child, all of which were con- firmed resistant to rifampicin by MODS and the GenoType MTBDRplus assay. MODS did not identify any other rifampicin resistant isolates. ignificantly, a combination of culture and Xpert MTB/RIF increased bacteriological yield without compromising pecificity.ii i Importantly, we were able to confirm the very high specificity of Xpert MTB/RIF in a large sample of speci- mens from children in whom TB had been excluded by careful clinical, radiological and laboratory assessment and follow up. Positive Xpert MTB/RIF results have been reported in a small number of child TB suspects who did well at follow up without TB treatment11,24. However the very high precision with which we were able to demonstrate 100% specificity of Xpert MTB/RIF in a carefully characterized group of children without TB sug- gests that these apparent false positive results from TB suspects in very high burden settings may have represented self-limiting pulmonary tuberculosis. This is well recognized in the pre-chemotherapy literature31. h Our primary analysis compared Xpert MTB/RIF sensitivity with the current culture reference standard. Having demonstrated the very high specificity of Xpert MTB/RIF we then repeated the analysis using a compos- ite reference standard of positive culture or Xpert MTB/RIF. Composite reference standards should be used with care32. However, given the equivalent high specificities of Xpert MTB/RIF and mycobacterial culture, incorpora- tion bias in favour of Xpert MTB/RIF is unlikely. Furthermore, misclassification bias arising from the imperfect sensitivity of culture may bias estimates of the accuracy of Xpert MTB/RIF culture alone is used as the refer- ence33,34. We believe the composite reference standard therefore provides a fairer comparison of the two methods, and a better estimate of the true sensitivity of the Xpert MTB/RIF assay. One potential limitation of our study is that molecular typing was not performed on MGIT-MODS discordant culture isolates to exclude cross-contamination. Nevertheless there was strong microbiological and/or clinical evidence to corroborate the positive culture in every case. In addition, negative controls in every MGIT batch and MODS plate provided no evidence of cross-contamination throughout the study, consistent with the very low prevalence and concentrations of M. tuberculosis in these paediatric specimens.i Due to finite resources, most children only had a single sputum sample obtained. Although this and other studies show the incremental yield of additional specimens where obtained, analysis of a single specimen is likely to reflect practice in most low resource settings due to the incremental cost of processing additional specimens. l p g p g p In conclusion, our results underline the imperfect sensitivity of all currently available methods for bacteriolog- ical diagnosis of childhood TB. ignificantly, a combination of culture and Xpert MTB/RIF increased bacteriological yield without compromising pecificity.ii The choice of method in any particular setting depends on several factors includ- ing prevalence of TB and drug resistance; resources available to risk stratify patients for testing; test sensitivity, cost35, time to detection; and the availability of trained staff and laboratory biosafety facilities. Our data confirm the superior sensitivity of MGIT compared with MODS and Xpert MTB/RIF. Although MODS may have a role in some settings, the operationally simpler Xpert MTB/RIF assay was as sensitive as MODS, demonstrated equiv- alent specificity to MGIT, and combined with MGIT it optimized bacteriological yield. Together these results strengthen the evidence base for inclusion of Xpert MTB/RIF in the reference standard for bacteriologically confirmed childhood TB in both WHO clinical guidelines35 and research. Role of the funding source. The study was supported by the Wellcome Trust in the form of research fel- lowships to AJB (081697) and JAGS (098532), and a core grant to the KEMRI-Wellcome Trust research pro- gramme (077092); and by a project grant from the Pneumonia Etiology Research for Child Health (PERCH) project funded by the Bill & Melinda Gates Foundation at Johns Hopkins Bloomberg School of Public Health. The funders had no role in data analysis or in the decision to publish. Discussion Th l k f l The paucibacillary nature of childhood TB may have a role, and variation between centres also suggests MODS performance is operator dependent4. In support of this, even after one month residential training with an experienced team, we were able to demonstrate a learning effect with increasing MODS yield and reducing time to detection during the two years of the study. We did not compare MODS with culture on solid media, however other studies have shown MODS to be superior to Lowenstein Jensen media for diagnosis of childhood TB6,7,26. g Against a reference standard of mycobacterial culture, Xpert MTB/RIF sensitivity was similar to other paedi- atric studies11, and comparable to MODS - in keeping with the only other study to compare the two methods29. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 6 www.nature.com/scientificreports/ No. MTBC positive specimens No. Xpert MTB/ RIF positive Xpert MTB/RIF Sensitivity, % (95% CI) p value Specimen type sputum specimens 96 75 78.1 (68.5 to 85.9) 0.062 non-sputum specimens 8 4 50.0 (15.7 to 84.3) Specimen smear status smear positive 33 33 100.0 (89.4 to 100.0) <0.001 smear negative 71 46 64.8 (52.5 to 75.8) Patient age 0–4 years 54 39 72.2 (58.4 to 83.5) 0.461 5–14 years 50 40 80.0 (66.3 to 90.0) Patient HIV status HIV positive 19 15 78.9 (54.4 to 93.9) 0.725 HIV negative 85 64 75.3 (64.7 to 84.0) All MTBC positive specimens 104 79 76.0 (66.6–83.8) — Table 6. Sensitivity of the Xpert MTB/RIF assay against the composite reference standard. Table 6. Sensitivity of the Xpert MTB/RIF assay against the composite reference standard. Significantly, a combination of culture and Xpert MTB/RIF increased bacteriological yield without compromising specificity.ii 2. World Health Organisation. Guidance for national tuberculosis programmes on the management of tuberculosis in children. Second edition edn, (World Health Organisation, 2014). 3 World Health Organization A Roadmap for Ensuring Quality Tuberculosis Diagnostics Services within National Laboratory References 1. World Health Organization & Stop TB Partnership. The Global Plan to Stop TB 2011–2015: Transforming the fight towards elimination of tuberculosis (WHO Press, 2010) 1. World Health Organization & Stop TB Partnership. The Global Plan to Stop TB 2011–2015: Transforming the fight towards elimination of tuberculosis. (WHO Press, 2010). 2. World Health Organisation. Guidance for national tuberculosis programmes on the management of tuberculosis in children. Second edition edn, (World Health Organisation, 2014). 3. World Health Organization. A Roadmap for Ensuring Quality Tuberculosis Diagnostics Services within National Laboratory Strategic Plans (World Health Organization, Geneva, 2010). of tuberculosis. (WHO Press, 2010). 2. World Health Organisation. Guidance for national tuberculosis programmes on the management of tuberculosis in children. Second edition edn, (World Health Organisation, 2014). 3. World Health Organization. A Roadmap for Ensuring Quality Tuberculosis Diagnostics Services within National Laboratory Strategic Plans (World Health Organization, Geneva, 2010). www.nature.com/scientificreports/ Meeting Report., (World Health Organization, Geneva, 2016). References SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 7 www.nature.com/scientificreports/ MODS: A user guide. Microscopic observation drug susceptibility assay (2 16. Coronel, J., Roper, M., Caviedes, L. & Moore, D. A. MODS: A user guide. Microscopic observation drug susceptibility assay (2008). 17. Chisti, M. J. et al. A prospective study of the prevalence of tuberculosis and bacteraemia in Bangladeshi children with severe malnutrition and pneumonia including an evaluation of Xpert MTB/RIF assay. PloS one 9, e93776, https://doi.org/10.1371/journal. pone.0093776 (2014). 18. LaCourse, S. M. et al. Use of Xpert for the diagnosis of pulmonary tuberculosis in severely malnourished hospitalized Malawian children. The Pediatric infectious disease journal 33, 1200–1202, https://doi.org/10.1097/INF.0000000000000384 (2014).i 19. Ntinginya, E. N. et al. Performance of the Xpert(R) MTB/RIF assay in an active case-finding strategy: a pilot study from Tanzania. Int J Tuberc Lung Dis 16, 1468–1470, https://doi.org/10.5588/ijtld.12.0127 (2012). ung Dis 16, 1468–1470, https://doi.org/10.5588/ijtld.12.0127 (2012) 20. Pang, Y. et al. Evaluation of the Xpert MTB/RIF assay in gastric lavage aspirates for diagnosis of smear-negative childhood pulmonary tuberculosis. The Pediatric infectious disease journal 33, 1047–1051, https://doi.org/10.1097/INF.0000000000000403 (2014). ( ) 21. Walters, E., Goussard, P., Bosch, C., Hesseling, A. C. & Gie, R. P. GeneXpert MTB/RIF on bronchoalveolar lavage samples in children with suspected complicated intrathoracic tuberculosis: a pilot study. Pediatric pulmonology 49, 1133–1137, https://doi.org/10.1002/ ppul.22970 (2014). 22. Sekadde, M. P. et al. Evaluation of the Xpert MTB/RIF test for the diagnosis of childhood pulmonary tuberculosis in Ugan cross-sectional diagnostic study. BMC infectious diseases 13, 133, https://doi.org/10.1186/1471-2334-13-133 (2013). g y f p g 23. Bates, M. et al. Assessment of the Xpert MTB/RIF assay for diagnosis of tuberculosis with gastric lavage aspirates in children in sub- Saharan Africa: a prospective descriptive study. The Lancet. Infectious diseases 13, 36–42, https://doi.org/10.1016/S1473- 3099(12)70245-1 (2013). ( ) ( ) 24. Zar, H. J. et al. Rapid molecular diagnosis of pulmonary tuberculosis in children using nasopharyngeal specimens. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 55, 1088–1095, https://doi.org/10.1093/cid/cis598 (2012).f 24. Zar, H. J. et al. Rapid molecular diagnosis of pulmonary tuberculosis in children using nasopharyngeal specimens. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 55, 1088–1095, https://doi.org/10.1093/cid/cis598 (2012). 25. Raizada, N. et al. Enhancing TB case detection: experience in offering upfront Xpert MTB/RIF testing to pediatric presumptive TB and DR TB cases for early rapid diagnosis of drug sensitive and drug resistant TB. PloS one 9, e105346, https://doi.org/10.1371/ j l 0105346 (2014) 24. Zar, H. J. www.nature.com/scientificreports/ et al. Rapid molecular diagnosis of pulmonary tuberculosis in children using nasopharyngeal specimens. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 55, 1088–1095, https://doi.org/10.1093/cid/cis598 (2012). 25. Raizada, N. et al. Enhancing TB case detection: experience in offering upfront Xpert MTB/RIF testing to pediatric presumptive TB ffi p f f y f p g ( ) 25. Raizada, N. et al. Enhancing TB case detection: experience in offering upfront Xpert MTB/RIF testing to pediatric presumptive TB and DR TB cases for early rapid diagnosis of drug sensitive and drug resistant TB. PloS one 9, e105346, https://doi.org/10.1371/ journal.pone.0105346 (2014). j p ( ) 26. Tran, S. T. et al. Diagnostic accuracy of microscopic Observation Drug Susceptibility (MODS) assay for pediatric tuberculo Hanoi, Vietnam. PloS one 8, e72100, https://doi.org/10.1371/journal.pone.0072100 (2013). 27. Hatherill, M. et al. Induced sputum or gastric lavage for community-based diagnosis of childhood pulmonary tuberculosis? Arc Child 94, 195–201, https://doi.org/10.1136/adc.2007.136929 (2009).hfi p g 28. Marais, B. J. et al. The bacteriologic yield in children with intrathoracic tuberculosis. Clinical infectious diseases: an official public of the Infectious Diseases Society of America 42, e69–71 (2006). f f y f 9. Nhu, N. T. et al. Evaluation of Xpert MTB/RIF and MODS assay for the diagnosis of pediatric tuberculosis. BMC infectious disease 13, 31, https://doi.org/10.1186/1471-2334-13-31 (2013). 30. Nicol, M. P. et al. Accuracy of the Xpert MTB/RIF test for the diagnosis of pulmonary tuberculosis in children admitted to hospital in Cape Town, South Africa: a descriptive study. The Lancet. Infectious diseases 11, 819–824, https://doi.org/10.1016/S1473- 3099(11)70167-0 (2011). 31. Marais, B. J. et al. The natural history of childhood intra-thoracic tuberculosis: a critical review of literature from the pre- chemotherapy era. Int J Tuberc Lung Dis 8, 392–402 (2004). k b l l f f d d d h f h d py g 2. Naaktgeboren, C. A. et al. Value of composite reference standards in diagnostic research. BMJ 347, f5605, https://doi.org/10.1136 bmj.f5605 (2013). j 3. Alonzo, T. A. & Pepe, M. S. Using a combination of reference tests to assess the accuracy of a new diagnostic test. Statistics in medicine 18, 2987–3003 (1999). 4. Togun, T. O. et al. Contribution of Xpert(R) MTB/RIF to the diagnosis of pulmonary tuberculosis among TB-exposed children in The Gambia. Int J Tuberc Lung Dis 19, 1091–1097, i–ii, https://doi.org/10.5588/ijtld.15.0228 (2015). h 35. World Health Organization. Xpert MTB/RIF assay for the diagnosis of TB. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4. Leung, E., Minion, J., Benedetti, A., Pai, M. & Menzies, D. Microcolony culture techniques for tuberculosis diagnosis: a systematic review. Int J Tuberc Lung Dis 16, 16–23, i–iii, https://doi.org/10.5588/ijtld.10.0065 (2012). 5. Ha, D. T. et al. Microscopic observation drug susceptibility assay (MODS) for early diagnosis of tuberculosis in children. PloS o e8341 (2009). ( ) 6. Oberhelman, R. A. et al. Improved recovery of Mycobacterium tuberculosis from children using the microscopic observation drug susceptibility method. Pediatrics 118, e100–106 (2006).f 7. Oberhelman, R. A. et al. Diagnostic approaches for paediatric tuberculosis by use of different specimen types, culture methods, and PCR: a prospective case-control study. The Lancet. Infectious diseases 10, 612–620, https://doi.org/10.1016/S1473-3099(10)70141-9 (2010). ( ) 8. Zar, H. J., Hanslo, D., Apolles, P., Swingler, G. & Hussey, G. Induced sputum versus gastric lavage for micorbiological confirmation of pulmonary tuberculosis in infants and young children: a prospective study. Lancet 2005, 130–134 (2005). p y y g p p y , ( ) 9. Boehme, C. C. et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 363, 1005–1015, https://doi. org/10 1056/NEJMoa0907847 (2010) p y y g p p y 9. Boehme, C. C. et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 363, 1005–1015, https://doi. org/10.1056/NEJMoa0907847 (2010).f g 0. Boehme, C. C. et al. Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosi of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet 377, 1495–1505 (2011). g p y ( ) 11. Detjen, A. K. et al. Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children: a systematic review and m analysis. The Lancet. Respiratory medicine. https://doi.org/10.1016/S2213-2600(15)00095-8 (2015). h 2. World Health Organisation. Pocket book of hospital care for children: guidelines for the management of common illnesses with limted resources. (World Health Organisation, 2005). 3. Liebeschuetz, S. et al. Diagnosis of tuberculosis in South African children with a T-cell-based assay: a prospective cohort study Lancet 364, 2196–2203 (2004). 14. Rachow, A. et al. Increased and Expedited Case Detection by Xpert MTB/RIF Assay in Childhood Tuberculosis: A Prospective Cohort Study. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, https://doi.org/10.1093/ cid/cis190 (2012). 15. Siddiqi, S. H. & Ruesch-Gerdes, S. MGIT Procedure Manual. (Foundation for Innovative New Diagnostics (FIND), 2006). 16. Coronel, J., Roper, M., Caviedes, L. & Moore, D. A. Acknowledgements g We would like to thank Dr. David Moore, Luz Caviedes and the team at Universidad Peruana Cayetano Heredia in Lima, Peru, for their helpful advice and practical support in MODS training and implementation; and the clinical, laboratory and administrative staff at Kilifi County Hospital and Coast Provincial General Hospital for their support of the study. We would also like to thank the following members of the Kilifi Improving Diagnosis & Surveillance of Childhood TB (KIDS TB) Study Group who contributed to patient recruitment, investigation and management: Victor Bandika, Jay Berkley, Joyce Langat, Kath Maitland, Carole Mulunda, Charles Newton, John Paul Odhiambo, Hemed Twahir, Joshua Wambua. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 8 www.nature.com/scientificreports/ Author Contributions A.J.B., J.A.G.S. and M.L. designed the study with input from D.M., R.M., A.M. and S.C.M. R.M. received residential training in MODS at Universidad Peruana Cayetano Heredia in Lima, Peru, following which he trained A.J.B., D.M. and A.M. in the technique. Laboratory work was performed by D.M., R.M., A.M., S.C.M. and A.J.B. A.J.B. had full access to all the data in the study and final responsibility for the decision to submit for publication. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 Additional Information dd t o a o at o Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-11969-5. Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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/. © The Author(s) 2017 SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 9
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Kun Hwang Ethics Editor, Archives of Plastic Surgery Department of Plastic Surgery, Inha University College of Medicine, Incheon, Korea “In the morning, my hair was black like silk bundles. In the eve­ ning, it became white like snow ( ).”–Li Bai ( , 701–762 AD, a Chinese poet) case now. The only difference relates to filial duty, as a good son was expected to avoid looking older than his parents. It is notable that the author wrote, “It is not sad to die, but it is sad to grow old. People hate aging because they are getting clos­ er to death,” which expresses a remarkably modern sentiment, but was written 200 years ago. With the snapping of smartphone cameras, photographs are everywhere these days. In our visual society, everybody’s insecu­ rities are stamped in time in the annals of the internet. There­ fore, troublesome physical features prompt both men and wom­ en to undergo plastic surgery. In our times, elderly men dye their gray hair rather than pluck­ ing it out. Cosmetic surgery for men is becoming increasingly common throughout the world. If the author lived in Seoul to­ day, he might have written the “Chronicle of Mr. #15 Blade” in­ stead of the “Chronicle of Mr. Tweezer.” Recently I read a book written in Korea 200 years ago (during the Joseon period). The writer, Lee Ok ( , 1760–1815), wrote Kangnoseonsaeng-Jeon ( , Chronicle of Mr. Tweezer) at the age of 50. This book personifies a tweezer as a narrator, and is structured as a set of questions and answers that express psychological anxiety about aging. The author was sen­ sitively aware of aging and described the aging process of the body in detail. The descriptions of changes in hair color and lo­ cations are especially impressive [1]. The author would like to delay aging—if not the aging process itself, then at least aging of one’s external appearance—by plucking out gray hairs (Fig. 1). Fig. 1. An old writer is tweezing gray hairs. Illustration by Hye Won Hu, MFA, Division of Biomedical Art, Incheon Catholic University Graduate School (Incheon, Korea). Why do elderly men desire youth instead of aging? An analysis of Kangnoseonsaeng-Jeon (却老先生傳) by Lee Ok (李鈺) Why do elderly men desire youth instead of aging? An analysis of Kangnoseonsaeng-Jeon (却老先生傳) by Lee Ok (李鈺) Editorial Copyright © 2021 The Korean Society of Plastic and Reconstructive Surgeons py g y g This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/ licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. www.e-aps.org This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons. censes/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is prop Article published online: 2022-05-22 Article published online: 2022-05-22 Article published online: 2022-05-22 Copyright © 2021 The Korean Society of Plastic and Reconstructive Surgeons This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/ licenses/by-nc/4 0/) which permits unrestricted non-commercial use distribution and reproduction in any medium provided the original work is properly cited www.e-aps.org buted under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/ unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. www.e-aps.o y g y g is an Open Access article distributed under the terms of the Creative Com py g y g This is an Open Access article distributed under the terms of the Creative Commons Attribution N licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproductio Correspondence: Kun Hwang Department of Plastic Surgery, Inha University College of Medicine, 27 Inhang-ro, Jung-gu, Incheon 22332, Korea Tel: +82-32-890-3514, Fax: +82-32-890-2918, E-mail: jokerhg@inha.ac.kr This study was supported by a grant from National Research Foundation of Korea (NRF-2020R1I1A2054761). Received: August 5, 2021 • Revised: August 22, 2021 • Accepted: August 23, 2021 pISSN: 2234-6163 • eISSN: 2234-6171 https://doi.org/10.5999/aps.2021.01578 • Arch Plast Surg 2021;48:575-576 Kun Hwang In this book, the following reasons for plucking out gray hairs are given: (1) in order to serve in a governmental post, men must avoid looking old; (2) if one’s parents are still alive, their son should not look older than his parents; (3) if a man marries a young wife, the husband’s old appearance must be avoided; or (4) society treats elderly men poorly. From this book, we realize that 200 years ago in Korea, aristo­ crats ( ) desired to look young for reasons related to their jobs, social treatment, and marital relationships, just as is the Fig. 1. An old writer is tweezing gray hairs. Illustration by Hye Won Hu, MFA, Division of Biomedical Art, Incheon Catholic University Graduate School (Incheon, Korea). Fig. 1. An old writer is tweezing gray hairs. Illustration by Hye Won Hu, MFA, Division of Biomedical Art, Incheon Catholic University Graduate School (Incheon, Korea). 575 Hwang K  Why do elderly men desire youth Conflict of interest 1. Ahn SH. A study on Kangnoseonsaeng-Jeon of Lee Ok: fo­ cusing on the aging and autobiography. J Korean Literature Chin 2016;45:253-76. Kun Hwang is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or deci­ sion process of this article. No other potential conflicts of inter­ est relevant to this article were reported. ORCID https://orcid.org/0000-0002-1994-2538 https://orcid.org/0000-0002-1994-2538 Kun Hwang 576
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Supplementary Fig. S1 from Dual targeting of Raf and VEGF receptor 2 reduces growth and metastasis of pancreatic cancer through direct effects on tumor cells, endothelial cells, and pericytes
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250.0 300.0 24h 48h D. 150.0 200.0 48h change in O.D * * * 0 0 50.0 100.0 %-c * * 0.0 Control 0.25µM 0.5µM 1µM 2µM 5µM Suppl. Figure 1: Effect of NVP-AAL881 on endothelial cell proliferation. Antiproliferative effects were evaluated by MTT analysis, demonstrating that NVP- AAL881 significantly reduced cell proliferation after 48 hours in full serum medium AAL881 significantly reduced cell proliferation after 48 hours in full serum medium (*P<0.01). 250.0 300.0 24h 48h D. 150.0 200.0 48h change in O.D * * * 0 0 50.0 100.0 %-c * * 0.0 Control 0.25µM 0.5µM 1µM 2µM 5µM Suppl. Figure 1: Effect of NVP-AAL881 on endothelial cell proliferation. Antiproliferative effects were evaluated by MTT analysis, demonstrating that NVP- AAL881 significantly reduced cell proliferation after 48 hours in full serum medium AAL881 significantly reduced cell proliferation after 48 hours in full serum medium (*P<0.01). 250.0 300.0 24h 48h D. 150.0 200.0 48h change in O.D * * * 0 0 50.0 100.0 %-c * * 0.0 Control 0.25µM 0.5µM 1µM 2µM 5µM Suppl. Figure 1: Effect of NVP-AAL881 on endothelial cell proliferation. Antiproliferative effects were evaluated by MTT analysis, demonstrating that NVP- AAL881 significantly reduced cell proliferation after 48 hours in full serum medium AAL881 significantly reduced cell proliferation after 48 hours in full serum medium (*P<0.01).
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https://europepmc.org/articles/pmc2979596?pdf=render
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5-Methyl-3,6,7,8a-tetrahydro-2<i>H</i>-diimidazo[1,2-<i>c</i>:1′,2′-<i>e</i>]pyrido[1,2-<i>a</i>][1,3,5]triazin-5-ium iodide
Acta crystallographica. Section E
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organic compounds Experimental Crystal data C12H16N5 +I Mr = 357.20 Monoclinic, P21=n a = 7.6299 (2) A˚ b = 15.3939 (4) A˚ c = 11.4503 (3) A˚  = 92.204 (2) V = 1343.89 (6) A˚ 3 Z = 4 Mo K radiation  = 2.37 mm1 T = 100 K 0.2  0.2  0.1 mm Data collection Oxford Diffraction Xcalibur-E CCD diffractometer Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) Tmin = 0.496, Tmax = 0.789 25955 measured reflections 4406 independent reflections 4018 reflections with I > 2(I) Rint = 0.026 Refinement R[F 2 > 2(F 2)] = 0.029 wR(F 2) = 0.070 S = 1.20 4406 reflections 165 parameters H-atom parameters constrained max = 1.74 e A˚ 3 min = 1.24 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 V = 1343.89 (6) A˚ 3 Z = 4 Mo K radiation  = 2.37 mm1 T = 100 K 0.2  0.2  0.1 mm Table 1 Table 1 Hydrogen-bond geometry (A˚ , ). D—H  A D—H H  A D  A D—H  A C3—H3B  I1i 0.99 3.15 4.007 (3) 145 C6—H6A  I1ii 0.99 3.05 4.010 (3) 163 C9—H9  I1iii 0.95 3.08 4.025 (3) 172 C12—H12  I1iv 0.95 3.14 3.887 (3) 137 C17—H17C  I1i 0.98 3.16 4.025 (3) 148 Symmetry codes: (i) x þ 1 2; y þ 1 2; z  1 2; (ii) x þ 1; y; z; (iii) x þ 1 2; y þ 1 2; z þ 1 2; (iv) x; y þ 1; z þ 1. The structure of the title compound, C12H16N5 +I, shows that the methylation reaction with CH3I occurred at the imine N atom at position 5 of the 3,6,7,8a-tetrahydro-2H-diimidazo- [1,2-c:10,20-e]pyrido[1,2-a][1,3,5]triazine system. In the cation, the sp3-hybridized C atom belonging to the fused dihydro- pyrine and dihydro-1,3,5-triazine rings deviates by 0.514 (3) A˚ from the best plane defined by the remaining cationic non-H atoms. The fused dihydropyridine and dihydro-1,3,5-triazine rings are each in a half-chair conformation with the sp3- hybridized C atom as a flap. The iodide anion is 3.573 (2) A˚ from the methylated N atom and exhibits five short C—H  I contacts with distances less than 3.16 A˚ . The structure has been determined from a non-merohedral twin with twin law [1 0 0 0  1 0 0.115 0 1], minor domain = 0.1559 (12). Data collection: CrysAlis PRO (Oxford Diffraction, 2009); cell refinement: CrysAlis PRO; data reduction: CrysAlis PRO; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008) and WinGX (Farrugia, 1999); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: SHELXL97. Related literature For the synthesis and data reported earlier for the title compound, see: Sa˛czewski & Foks (1981). For the programs used to derive the twin law, see: Cooper et al.(2002); Farrugia (1999). Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: TK2676). 5-Methyl-3,6,7,8a-tetrahydro-2H- diimidazo[1,2-c:1000,2000-e]pyrido[1,2-a]- [1,3,5]triazin-5-ium iodide 5-Methyl-3,6,7,8a-tetrahydro-2H- diimidazo[1,2-c:1000,2000-e]pyrido[1,2-a]- [1,3,5]triazin-5-ium iodide Data collection Oxford Diffraction Xcalibur-E CCD diffractometer Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) Tmin = 0.496, Tmax = 0.789 25955 measured reflections 4406 independent reflections 4018 reflections with I > 2(I) Rint = 0.026 Aleksandra Wasilewska,a Franciszek Sa˛czewskia and Maria Gdaniecb* Cooper, R. I., Gould, R. O., Parsons, S. & Watkin, D. J. (2002). J. Appl. Cryst. 35, 168–174. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Farrugia, L. J. (1999). J. Appl. Cryst. 32, 837–838. Oxford Diffraction (2009). CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, England. Sa˛czewski, F. & Foks, H. (1981). Synthesis, pp. 151–152. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Aleksandra Wasilewska,a Franciszek Sa˛czewskia and Maria Gdaniecb* aDepartment of Chemical Technology of Drugs, Medical University of Gdan´sk, 80-416 Gdan´sk, Poland, and bFaculty of Chemistry, Adam Mickiewicz University, 60-780 Poznan´, Poland Correspondence e-mail: magdan@amu.edu.pl aDepartment of Chemical Technology of Drugs, Medical University of Gdan´sk, 80-416 Gdan´sk, Poland, and bFaculty of Chemistry, Adam Mickiewicz University, 60-780 Poznan´, Poland Correspondence e-mail: magdan@amu.edu.pl Refinement R[F 2 > 2(F 2)] = 0.029 wR(F 2) = 0.070 S = 1.20 4406 reflections 165 parameters H-atom parameters constrained max = 1.74 e A˚ 3 min = 1.24 e A˚ 3 165 parameters H-atom parameters constrained max = 1.74 e A˚ 3 min = 1.24 e A˚ 3 Received 11 May 2010; accepted 24 May 2010 Received 11 May 2010; accepted 24 May 2010 Key indicators: single-crystal X-ray study; T = 100 K; mean (C–C) = 0.004 A˚; R factor = 0.029; wR factor = 0.070; data-to-parameter ratio = 26.7. Table 1 Hydrogen-bond geometry (A˚ , ). References Cooper, R. I., Gould, R. O., Parsons, S. & Watkin, D. J. (2002). J. Appl. Cryst. 35, 168–174. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Farrugia, L. J. (1999). J. Appl. Cryst. 32, 837–838. Oxford Diffraction (2009). CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, England. Sa˛czewski, F. & Foks, H. (1981). Synthesis, pp. 151–152. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. o1486 Wasilewska et al. Acta Cryst. (2010). E66, o1486 doi:10.1107/S1600536810019495 Comment Biguanide derivatives are known to possess diverse biological activities, including antidiabetic, antibacterial, germicidic, antiviral and antimalarial. On the other hand, quaternary ammonium salts constitute a well known class of bacteriostatic agents. Therefore, we have decided to synthesize some N-alkylated cyclic biguanide derivatives for biological testing, based on the previously described procedure (Sączewski & Foks, 1981) which consists in the reaction of 2,3,6,7,8a,13-hexahy- dropyrido[1,2-a]diimidazo[1',2'-c:1'',2''-e]-1,3,5-triazine (1) with an alkyl halide. As shown in Fig. 1, the course of the reac- tion of 1 with methyl iodide has not been established and two products, 2 or 3, arising from either N1 or N5 alkylation have been proposed. In this work, based on X-ray structure analysis (Fig. 2) and hetero-correlation NMR experiments (HSQC and HMBC), the structure of the title compound (2) is determined unambiguously. Regioselectivity of N-alkylation of the cyclic biguanide derivative 1 could not be predicted on the basis of calculated electrostatic potential and charge distribution. The structure of the N5 alkylated product 2 was also confirmed by 2D NMR spectroscopic data. Thus, assignment of signals observed in 1H and 13C-NMR spectra was possible using HSQC spectrum (see numbering scheme in Fig. 3). The crucial signals of quaternary carbon atoms C13a and C4a were found at 145.7 and 152.6 p.p.m., respectively. 3-Bond correlation from the latter carbon to a singlet of three protons at 3.26 p.p.m. observed in the HMBC spectrum (Fig. 4) indicated the placement of methyl group at the N5 nitrogen atom. 5-Methyl-3,6,7,8a-tetrahydro-2H-diimidazo[1,2-c:1',2'-e]pyrido[1,2-a][1,3,5]triazin-5-ium iodide A. Wasilewska, F. Saczewski and M. Gdaniec A. Wasilewska, F. Saczewski and M. Gdaniec Experimental 1D and 2D NMR spectra were recorded on a Varian Unity 500 spectrometer. The title compound was prepared according to the previously described procedure (Sączewski & Foks, 1981); m.p. 492–494 K (decomp.); 1H NMR (500 MHz, DMSO-d6, see Fig. 3 for numbering scheme) δH 6.94 (1H, d, J = 7.2 Hz, H12), 6.22–6.19 (1H, ddd, J=9.8, 6.8, ~1 Hz, H10), 5.86 (1H, t, J = 1 Hz, H8a), 5.67 (1H, dd, J = 9.8, ~1 Hz, H9), 5.41–5.38 (1H, dd, J = 7.2, 6.8 Hz, H11), 4.42 (1H, dt, J = 9.3, 6.8 Hz, H2), 4.20 (1H, dt, J = 9.3, 6.8 Hz, H2), 4.01–3.83 (4H, m, H7, H6, 2xH3),3.78–3.71 (1H, m, H6), 3.49–3.43 (1H, m, H7), 3.26 (3H, s, CH3); 13C NMR (125 MHz, DMSO-d6, see Fig. 3 for numbering scheme) δC 152.6 (C4a), 145.7 (C13a), 125.4 (C12), 124.8 (C10), 113.6 (C9), 103.4 (C11), 67.0 (C8a), 51.7 (C3), 50.7 (C6), 46.6 (C2), 43.3 (C7), 33.7 (C14); IR (KBr, cm–1): 3090, 3079, 3025, 2936, 2880, 1684, 1661, 1550, 1429, 1321, 1301, 1186, 677. 3.26 (3H, s, CH3); 13C NMR (125 MHz, DMSO-d6, see Fig. 3 for numbering scheme) δC 152.6 (C4a), 145.7 (C13a), 125.4 (C12), 124.8 (C10), 113.6 (C9), 103.4 (C11), 67.0 (C8a), 51.7 (C3), 50.7 (C6), 46.6 (C2), 43.3 (C7), 33.7 (C14); IR (KBr, cm–1): 3090, 3079, 3025, 2936, 2880, 1684, 1661, 1550, 1429, 1321, 1301, 1186, 677. supplementary materials supplementary materials Acta Cryst. (2010). E66, o1486 [ doi:10.1107/S1600536810019495 ] Acta Cryst. (2010). E66, o1486 [ doi:10.1107/S1600536810019495 ] Acta Cryst. (2010). E66, o1486 [ doi:10.1107/S1600536810019495 ] Refinement The twin matrix, -1 0 0/0 - 1 0/0.115 0 1, corresponding to 180° rotation about [0 0 1] direct lattice direction has been determined with the program ROTAX (Cooper et al., 2002). For the refinement with the SHELXL97 program (Sheldrick, 2008), the reflection data file was prepared in the HKLF 5 format using the 'Make HKLF5' function of the WinGX program (Farrugia, 1999). The overlapping reflections and those belonging to only one twin domain are used in the refinement (HKLF 5 format of SHELXL97). Those which were excluded, 132 reflections, are partial overlaps which could not be integrated properly at the data processing stage. The BASF parameter refined at 0.1559 (12). The H atoms bonded to C atoms were sup-1 supplementary materials HMBC spectrum of the title compound 2 Crystal data C12H16N5 +·I− F(000) = 704 Mr = 357.20 Dx = 1.765 Mg m−3 Monoclinic, P21/n Mo Kα radiation, λ = 0.71073 Å Hall symbol: -P 2yn Cell parameters from 19213 reflections a = 7.6299 (2) Å θ = 2.7–32.3° b = 15.3939 (4) Å µ = 2.37 mm−1 c = 11.4503 (3) Å T = 100 K β = 92.204 (2)° Block, colourless V = 1343.89 (6) Å3 0.2 × 0.2 × 0.1 mm Z = 4 sup-2 supplementary materials Data collection Oxford Diffraction Xcalibur-E CCD diffractometer 4406 independent reflections Radiation source: fine-focus sealed tube 4018 reflections with I > 2σ(I) graphite Rint = 0.026 ω scan θmax = 32.4°, θmin = 4.1° Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) h = −9→11 Tmin = 0.496, Tmax = 0.789 k = −23→23 25955 measured reflections l = 0→16 Data collection Oxford Diffraction Xcalibur-E CCD diffractometer 4406 independent reflections Radiation source: fine-focus sealed tube 4018 reflections with I > 2σ(I) graphite Rint = 0.026 ω scan θmax = 32.4°, θmin = 4.1° Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) h = −9→11 Tmin = 0.496, Tmax = 0.789 k = −23→23 25955 measured reflections l = 0→16 Data collection Oxford Diffraction Xcalibur-E CCD diffractometer Radiation source: fine-focus sealed tube graphite ω scan Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) Tmin = 0.496, Tmax = 0.789 25955 measured reflections 4406 independent reflections 4018 reflections with I > 2σ(I) Rint = 0.026 θmax = 32.4°, θmin = 4.1° h = −9→11 k = −23→23 l = 0→16 4406 independent reflections Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.029 wR(F2) = 0.070 S = 1.20 4406 reflections 165 parameters 0 restraints Primary atom site location: structure-invariant direct methods Primary atom site location: structure-invariant direct methods Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.029 wR(F2) = 0.070 S = 1.20 4406 reflections 165 parameters 0 restraints Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.0196P)2 + 3.1405P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 1.74 e Å−3 Δρmin = −1.24 e Å−3 supplementary materials placed at calculated positions, with C—H = 0.95–1.00 Å, and refined as riding on their parent atoms, with Uiso(H) = x Ueq(C), where x = 1.5 for the H atoms from the methyl group and x = 1.2 for the remaining H atoms. The maximum and minimum residual electron-density peaks of 1.74 and -1.24 eÅ-3 were located 0.72 Å and 1.84 Å from H6B and I1 atoms, respectively. Fig. 1. Reaction of 2,3,6,7,8a,13-hexahydropyrido[1,2-a]diimidazo[1',2',-c:1",2"-e]-1,3,5- triazine (1) with methyl iodide. Fig. 2. Asymmetric unit for the title salt with displacement ellipsoids shown at the 50% prob- ability level. Fig. 3. HSQC spectrum of the title compound 2 Fig. 4. supplementary materials pp y sup-4 H3A 0.1869 0.3569 0.2173 0.025* H3B 0.3846 0.3898 0.1973 0.025* N4 0.3083 (3) 0.40681 (15) 0.36610 (19) 0.0155 (4) N5 0.4690 (3) 0.27675 (15) 0.4159 (2) 0.0162 (4) C6 0.5326 (4) 0.23510 (18) 0.5250 (2) 0.0189 (5) H6A 0.6623 0.2316 0.5289 0.023* H6B 0.4837 0.1759 0.5322 0.023* C7 0.4659 (4) 0.29506 (18) 0.6205 (2) 0.0201 (5) H7A 0.3677 0.2679 0.6613 0.024* H7B 0.5609 0.3097 0.6785 0.024* N8 0.4068 (3) 0.37245 (14) 0.55467 (19) 0.0147 (4) C9 0.3144 (4) 0.4560 (2) 0.7238 (2) 0.0202 (5) H9 0.3752 0.4175 0.7759 0.024* C10 0.2718 (4) 0.5354 (2) 0.7605 (3) 0.0237 (6) H10 0.2938 0.5510 0.8400 0.028* C11 0.1922 (4) 0.59810 (19) 0.6798 (3) 0.0237 (6) H11 0.1462 0.6510 0.7082 0.028* C12 0.1842 (4) 0.58081 (18) 0.5651 (3) 0.0197 (5) H12 0.1363 0.6226 0.5118 0.024* N13 0.2459 (3) 0.50171 (14) 0.5233 (2) 0.0162 (4) C14 0.2675 (3) 0.42666 (17) 0.6013 (2) 0.0151 (4) H14 0.1556 0.3928 0.6010 0.018* C15 0.3916 (3) 0.35088 (16) 0.4392 (2) 0.0136 (4) C16 0.2403 (3) 0.48697 (17) 0.4045 (2) 0.0156 (5) C17 0.4863 (4) 0.2302 (2) 0.3064 (3) 0.0242 (6) H17A 0.3854 0.1916 0.2934 0.036* H17B 0.5944 0.1957 0.3102 0.036* H17C 0.4909 0.2719 0.2420 0.036* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 I1 0.01712 (8) 0.01865 (8) 0.01406 (7) 0.00055 (6) 0.00212 (5) −0.00050 (6) N1 0.0239 (12) 0.0181 (11) 0.0207 (11) 0.0010 (9) −0.0007 (9) 0.0040 (9) C2 0.0251 (14) 0.0244 (14) 0.0170 (12) 0.0007 (11) −0.0009 (10) 0.0062 (11) C3 0.0271 (15) 0.0233 (14) 0.0114 (11) 0.0015 (11) −0.0001 (9) 0.0007 (10) N4 0.0198 (11) 0.0150 (10) 0.0117 (9) 0.0004 (8) 0.0010 (7) 0.0016 (8) N5 0.0174 (11) 0.0158 (9) 0.0155 (9) 0.0029 (8) 0.0033 (8) 0.0009 (8) C6 0.0189 (13) 0.0187 (12) 0.0191 (12) 0.0056 (10) 0.0012 (9) 0.0035 (9) C7 0.0245 (13) 0.0194 (12) 0.0161 (11) 0.0044 (10) −0.0008 (9) 0.0045 (9) N8 0.0157 (10) 0.0150 (9) 0.0134 (9) 0.0007 (8) −0.0001 (7) 0.0017 (7) C9 0.0213 (13) 0.0242 (13) 0.0152 (11) −0.0016 (10) 0.0003 (9) −0.0024 (10) C10 0.0223 (14) 0.0297 (15) 0.0191 (13) −0.0052 (11) 0.0023 (10) −0.0092 (11) C11 0.0241 (15) 0.0162 (12) 0.0312 (15) −0.0029 (10) 0.0048 (11) −0.0088 (11) C12 0.0178 (13) 0.0126 (11) 0.0289 (14) −0.0021 (9) 0.0045 (10) −0.0026 (10) N13 0.0212 (11) 0.0111 (9) 0.0164 (10) 0.0001 (8) 0.0001 (8) −0.0002 (8) C14 0.0166 (12) 0.0152 (11) 0.0135 (11) −0.0006 (9) 0.0001 (8) −0.0001 (9) C15 0.0113 (11) 0.0147 (11) 0.0148 (10) −0.0024 (8) 0.0022 (8) 0.0012 (8) Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. actional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq I1 0.04305 (2) 0.194494 (11) 0.473423 (14) 0.01657 (5) N1 0.1806 (3) 0.53774 (16) 0.3251 (2) 0.0209 (5) C2 0.2057 (4) 0.4944 (2) 0.2120 (2) 0.0222 (5) H2A 0.2910 0.5272 0.1660 0.027* H2B 0.0931 0.4914 0.1663 0.027* C3 0.2755 (4) 0.40179 (19) 0.2385 (2) 0.0206 (5) sup-3 supplementary materials Atomic displacement parameters (Å2) sup-4 supplementary materials supplementary mate C16 0.0157 (12) 0.0130 (11) 0.0181 (11) −0.0021 (9) 0.0012 (9) 0.0011 (9 C17 0.0316 (16) 0.0213 (13) 0.0201 (12) 0.0047 (11) 0.0075 (11) −0.0047 ( Geometric parameters (Å, °) N1—C16 1.270 (3) C7—H7B 0.9900 N1—C2 1.476 (4) N8—C15 1.364 (3) C2—C3 1.547 (4) N8—C14 1.468 (3) C2—H2A 0.9900 C9—C10 1.336 (4) C2—H2B 0.9900 C9—C14 1.504 (4) C3—N4 1.475 (3) C9—H9 0.9500 C3—H3A 0.9900 C10—C11 1.453 (5) C3—H3B 0.9900 C10—H10 0.9500 N4—C15 1.344 (3) C11—C12 1.340 (4) N4—C16 1.415 (3) C11—H11 0.9500 N5—C15 1.317 (3) C12—N13 1.397 (3) N5—C17 1.455 (4) C12—H12 0.9500 N5—C6 1.470 (3) N13—C16 1.378 (3) C6—C7 1.533 (4) N13—C14 1.466 (3) C6—H6A 0.9900 C14—H14 1.0000 C6—H6B 0.9900 C17—H17A 0.9800 C7—N8 1.471 (3) C17—H17B 0.9800 C7—H7A 0.9900 C17—H17C 0.9800 C16—N1—C2 107.1 (2) C10—C9—C14 121.1 (3) N1—C2—C3 107.4 (2) C10—C9—H9 119.4 N1—C2—H2A 110.2 C14—C9—H9 119.4 C3—C2—H2A 110.2 C9—C10—C11 120.5 (3) N1—C2—H2B 110.2 C9—C10—H10 119.7 C3—C2—H2B 110.2 C11—C10—H10 119.7 H2A—C2—H2B 108.5 C12—C11—C10 119.6 (3) N4—C3—C2 101.0 (2) C12—C11—H11 120.2 N4—C3—H3A 111.6 C10—C11—H11 120.2 C2—C3—H3A 111.6 C11—C12—N13 120.4 (3) N4—C3—H3B 111.6 C11—C12—H12 119.8 C2—C3—H3B 111.6 N13—C12—H12 119.8 H3A—C3—H3B 109.4 C16—N13—C12 118.9 (2) C15—N4—C16 122.4 (2) C16—N13—C14 118.1 (2) C15—N4—C3 129.9 (2) C12—N13—C14 120.6 (2) C16—N4—C3 107.6 (2) N13—C14—N8 107.0 (2) C15—N5—C17 131.1 (2) N13—C14—C9 110.4 (2) C15—N5—C6 110.0 (2) N8—C14—C9 111.1 (2) C17—N5—C6 118.7 (2) N13—C14—H14 109.4 N5—C6—C7 103.6 (2) N8—C14—H14 109.4 N5—C6—H6A 111.0 C9—C14—H14 109.4 C7—C6—H6A 111.0 N5—C15—N4 129.4 (2) N5—C6—H6B 111.0 N5—C15—N8 112.7 (2) C7—C6—H6B 111.0 N4—C15—N8 117.9 (2) H6A—C6—H6B 109.0 N1—C16—N13 127.0 (3) N8—C7—C6 103.0 (2) N1—C16—N4 116.1 (2) sup-5 supplementary materials supplementary materials N8—C7—H7A 111.2 N13—C16—N4 116.9 (2) C6—C7—H7A 111.2 N5—C17—H17A 109.5 N8—C7—H7B 111.2 N5—C17—H17B 109.5 C6—C7—H7B 111.2 H17A—C17—H17B 109.5 H7A—C7—H7B 109.1 N5—C17—H17C 109.5 C15—N8—C14 117.1 (2) H17A—C17—H17C 109.5 C15—N8—C7 108.2 (2) H17B—C17—H17C 109.5 C14—N8—C7 119.1 (2) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A C3—H3B···I1i 0.99 3.15 4.007 (3) 145 C6—H6A···I1ii 0.99 3.05 4.010 (3) 163 C9—H9···I1iii 0.95 3.08 4.025 (3) 172 C12—H12···I1iv 0.95 3.14 3.887 (3) 137 C17—H17C···I1i 0.98 3.16 4.025 (3) 148 Symmetry codes: (i) x+1/2, −y+1/2, z−1/2; (ii) x+1, y, z; (iii) x+1/2, −y+1/2, z+1/2; (iv) −x, −y+1, −z+1. sup-6 supplementary materials Fig 1 Fig. 1 Fig. 1 sup-7 supplementary materials Fig. 2 sup-8 supplementary materials Fig. 3 sup-9 supplementary materials Fig. 4 Fig. 4 sup-10
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Professional Negligence, Mismanagement and Malpractice. Polish Companies in the Light of Supreme Audit Office Materials in the Years 1976-1980
Studia Historiae Oeconomicae
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cc-by-sa
7,170
Krzysztof L es iako w s ki (The University of Łódź, Łódź) Krzysztof L es iako w s ki (The University of Łódź, Łódź) Krzysztof L es iako w s ki (The University of Łódź, Łódź) PROFESSIONAL NEGLIGENCE, MISMANAGEMENT AND MALPRACTICE. POLISH COMPANIES IN THE LIGHT OF SUPREME AUDIT OFFICE MATERIALS IN THE YEARS 1976-1980 The birth of the “Solidarity” Independent Self-governing Trade Union in 1980 was accompanied by a strong revisionist movement. The Polish society expected punishment for the previous admin- istration, led by Edward Gierek, as it was their policy up to that point that resulted with enormous external debt and ubiquitous shortages of consumer goods. A lot was said about wastefulness, cor- ruption and professional negligence, both in the state administration and in the managements of Polish companies. And these opinions were somehow justified, as evidenced by the results of audits performed by the Supreme Audit Office (Najwyższa Izba Kontroli, NIK) in the years 1976–1980. Information recorded in the audit documentation was in line with the social attitudes. Due to for- mal and legal conditions back then, NIK auditors could not inspect the most important companies, however they managed to prove that the scale of illegal actions committed by directors and manag- ers was large and increased year by year. Keywords: Poland 1976-1980, state control, national companies, management. doi:10.1515/sho-2016-0009 doi:10.1515/sho-2016-0009 Studia historiae oeconomicae UAM Vol.  34 Poznań  2016 zhg.amu.edu.pl/sho Studia historiae oeconomicae UAM Vol. 34 Poznań  2016 zhg.amu.edu.pl/sho INTRODUCTION The Supreme Audit Office (NIK) was the key supervisory institution in Poland, therefore, materials prepared by its auditors are very useful when investigating the problem of financial crime. During their statuto- ry auditory activities, the inspectors detected various negative incidents, which were evidence that some aspects of operation of Polish companies, cooperatives and budgetary institutions were of a criminal or quasi-crim- inal nature. Many of these problems were the fault of the management – though dysfunctions in public institutions and the abnormal behavior of Krzysztof Lesiakowski 150 the authorities, although were nothing new, they achieved a whole new level at that time [Jarosz M. 2004: 42]. This was evidenced by a particularly strong revisionary movement in the first sixteen months of the existence of “Solidarity”, in the years 1980-1981, when the society sought revenge for frauds and private criminal activities that people in broadly understood “positions of power” were involved in. This circumstance begs the ques- tion – what had actually happened before that time? Did NIK inspectors, during audits, detect the fact that managerial staff abused their positions and were guilty of mismanagement? It must be noted at this point that from the year 1976, NIK was subordinated to the Prime Minister, which significantly limited its authority, planning capabilities and the course of the audits performed [Sierpowska I. 2004: 55-56; Sylwestrzak A. 2006: 90; Konaszyc A. 1978: 96-100]. Furthermore, the fact that this problem was not noticed in literature on financial crime in Poland is an additional encour- agement to investigate it [Jarosz M. 2004: 236]. This subject was either ig- nored or belittled [Górniok O. 1986: 101]. NEGLIGENCE OF PROFESSIONAL DUTIES Polish companies in the years 1976-1980 functioned in the conditions of state-controlled economy, whose priority was the implementation of the economic plan. In the course of this implementation, many illegal acts were committed, including excessive tolerance towards negligence of professional responsibilities and false reporting. The gap between the official policy of fighting with these problems and the actual impunity of those who committed similar acts meant that the problem kept escalating [Karklins R. 2009: 134-137]. According to NIK, one of the main reasons why the audited companies incurred losses was the fact that the system of professional supervision was poor and the enforcement of responsi- bility for mistakes in company management – inconsistent. This result- ed from the fact that in state-controlled economy, the division of respon- sibilities in particular units of the organization was not clearly defined, particularly in smaller plants [ANIK (Archive of Supreme Audit Office), GP (President’s Office), 294/1, f. 2]. Furthermore, the managers did not use their authority to punish those guilty of negligence due to general problems with employment. On the other hand, on the level of minis- tries, associations or similar units, unreliable reports and information on Professional Negligence 151 the alleged economic achievements of companies were very often accept- ed indiscriminately. Weaker supervision in this respect results from the fact that also the superior units wanted to present economic results as better than they actually were [ANIK, GP. 1627/7l, ff. 248-249]. Improper wage bill management was one of the aspects of manage- rial negligence frequently pointed out by NIK auditors. For example, in 1979, the “Organika-Azot” Chemical Plant in Jaworzno overstated the ex- ecution of the production plan and paid workers undue bonuses in the amount of 6.4 million PLN. The “Stomil” Rubber Company in Wolbrom – made similar undue payments totaling 3.2 million PLN. The director of the Light Industry Construction Company in Dzierżoniów forged doc- uments concerning the works performed, the overtime worked etc. The money he made this way was split with other conspirators operating in the company [ANIK, GP, 148/14, f. 25; 294/1, f. 12]. Although formally this behavior was heavily punished: e.g. NEGLIGENCE OF PROFESSIONAL DUTIES with immediate termination of employment, termination of employment with notice, transfer into a dif- ferent position with different working conditions and salary, a fine, re- ferring the matter to a penal and administrative court, and, in exception- al circumstances, reporting the matter to law enforcement agencies, the problem was recurring. i The audits showed that insufficient professional supervision or even abnormalities in its functioning in terms of wage bill management (pay- ing undue base salaries, benefits, bonuses and awards) was also the prob- lem in state-controlled agricultural farms. Particularly shocking arbitrar- iness and violations were observed in 1979 in the Agriculture and Food Cooperative in Szczekociny (Częstochowa region). Manipulations in the number of daily rates in the period from July 1978 until December 1979 meant that this company paid 18 people 10.7 million PLN. Notably, the cooperative did not turn a profit in 1979, and thus obtained a 21.5 million PLN subsidy from the state budget. The director of the company single- handedly made decisions concerning bonuses and awards, without the approval of the general meeting. He was also the main recipient of the money distributed this way. Another problem was the fact that manageri- al staff were paid undue compensation for using their private cars for pro- fessional purposes. In the whole of 1979, it was 600,000 PLN for 9 people, out of which 360,000 PLN went into the director’s pocket. In the follow- ing year (from January until September), the aforementioned director also Krzysztof Lesiakowski 152 granted himself another 211,000 PLN by way of such compensation de- spite being on sick leave at that time [AAN (Archive of New Files), PZPR (Polish United Workers’ Party), 4014, ff. 133-134]. According to NIK, improper professional supervision was the weak- est link in company management in Poland. This malfunction entailed further adverse consequences: poor work discipline (drinking alcohol at work, thefts, bad performance), fictional turnover, substituting allegedly used machines with operational ones while decommissioning them, lack of supervision with regards to materials used in production (in food in- dustry this meant creating surpluses of e.g. coffee or meat which were not recorded anywhere). The latter problem was related to illegal transactions in fluid fuels. How could it be any different, though, if e.g. NEGLIGENCE OF PROFESSIONAL DUTIES in the “Wega” Wool Company in Bielsko-Biała an audit of drivers’ logs performed by NIK inspectors showed that they reported different distances covered on the same Bielsko-Biała – Straconka route: 20, 23, 38, 48, 50, and even 61 km, while the actual distance is 12 km [ANIK, GP, 1569/10, f. 417]? This was not an isolated case of course. NIK assessed that the worst situation in terms of fuel turnover was observed in agriculture. Tests showed that, officially, farmers bought only 10–15% of the fuel needed, and “presuma- bly obtained the rest of the fuel needed from illegal sources” [ANIK, GP, 1569/12, f. 56]. A clear proof of the terrible state of management in Polish companies was the frequency of sham reporting. In any case it had to be at least toler- ated , and quite probably, even inspired by directors, managers, and pres- idents. In the 4th quarter of 1977, and in the 1st and 2nd quarters of 1978, out of 323 companies audited in this respect, in 234 (72%) unreliable and in- accurate reports were found. The problem was therefore much more se- rious that the Central Statistical Office reported – according to them, 10% of reports contained data inconsistent with the records found in source documentation [ANIK, GP, 1627/4, f. 16]. It must be added that the prob- lem of false reporting concerned all companies: small and large, new and long established, from agricultural cooperatives to coal mines. By manip- ulating reports, the management usually tried to conceal problems with implementing the production plan or achieving the planned level of in- come. Large scale manipulations were reported in national agricultural com- panies and farmers’ cooperatives. Data regarding the number of livestock were falsified, its value was manipulated, also fictional renovations and irrigation works were reported [ANIK, GP, 294/3, f. 27]. Some mines hid Professional Negligence 153 their problems with executing coal extraction plans in the same way. This was the case for example in the “Generał Zawadzki” mine, which in just 5 months of 1980 issued 223 fictional invoices, claiming to have sold 189,000 tons of coal worth 41.5 million PLN. Other mines hid the fact that their yield was lower than expected by overestimating own usage of coal and its alleged stock in heaps [AAN, PZPR, 4014, f. 16]. NEGLIGENCE OF PROFESSIONAL DUTIES The record in this re- spect was probably held by the Jelcz Car Company, which unjustly in- cluded 479 cars worth 333 million PLN in their production figures for the year 1976, while some of the cars were sold as late as April 1977 [ANIK, GP, 1627/3, f. 367]. Dismissal of three vice presidents of the Jelcz compa- ny and of several employees responsible for the incorrect reports proved that NIK findings were correct. NIK also made a request to reconsider the employment of the chief director of the company because, clearly, he ne- glected his professional duties. For similar reasons, based on NIK findings in 1979, the prosecutor’s office arrested the chief director of the “Organika Azot” Chemical Company in Jaworzno [ANIK, GP, 148/14, f. 27]. ABUSES One of the most prevalent problems in Polish companies was the abuse of managerial positions for private purposes. In this respect, the behavior of the management of the Radio and Television Committee was particular- ly telling. The audit in the Committee began in April 1980, because previ- ous proceedings initiated in 1977 and 1979 had been suspended upon the request of the Prime Minister. NIK accused the management of this insti- tution of diminishing currency-related income to the state budget [AAN, PZPR, 4014, f. 147] and incurring losses as a result of unreasonable cur- rency purchases, including those made for personal benefit [Lesiakowski K. 1998: 418]. In particular this involved the creation of two companies abroad by the management of the Committee. Foreign cooperators made payments due for broadcasts (for example from the Pope’s visit in Poland) and for commissioned films and television programs to the accounts of these companies. From May 1979 until September 1980, these payments amounted to USD 1.6 million, which the Committee’s management had at their free disposal. NIK also questioned the purchase of equipment and personal items from the so-called 2nd payment area, without an in-depth analysis of needs and justification of the purchase: 154 Krzysztof Lesiakowski As a result, a large part of purchased items made was either not used or used very lit- tle. Some were handed over to the management or other employees of the Committee for their personal use [AAN. PZPR, 4014, f. 149]. As a result, a large part of purchased items made was either not used or used very lit- tle. Some were handed over to the management or other employees of the Committee for their personal use [AAN. PZPR, 4014, f. 149]. This included sports equipment, radio and television equipment, cassette recorders or calculators. Based on the results of the audit, NIK made a re- quest to the prosecutor’s office to initiate penal proceedings against the management of the Committee [Komisja 2013: 309]. NIK also reported massive abuses by and privileging of the people in power in the housing construction industry. The situation in this sector was difficult – in the late 1976 in Poland there were 700,000 members of housing cooperatives waiting for flats [Jarosz D. 2010: 258] – which gave rise to abuses in institutions making decisions on flat allocation. ABUSES The people responsible for allocating flats were found guilty of non-compliance with the transparency of allocation procedures and of excluding some flats from social su- pervision [ANIK. GP, 1569/6, f. 228]. For example, the construction of 13 single-family detached houses by the Housing Cooperative in Rzeszów for selected employees of the Agri­ cultural Construction Association in Rzeszów and associated companies had criminal features. Furthermore, interested workers of the Association, including the director and his two deputies, led to a situation where the executive companies set the house prices clearly below the actual costs of construction [ANIK, GP, 1627/3 f. 75]. l Similar problems, including allocating flats to ineligible people or priv- ileging people in positions of power, occurred during allocation of flats from the so-called company pool. In 1978, NIK inspectors established that the “Stomil” Automotive Rubber Company in Dębica allocated a dozen or so flats to workers who had already received their own cooperative flats. Even more shocking evidence for privileging the management was found in the Industrial Construction Company No. 1 in Wrocław. The construction of 14 single-family homes for the managerial staff of the company and oth- er associated entities (including the Construction Association) consumed 77% of the total number of working hours in all sites, despite the fact that this investment comprised only 9% of the total housing construction plan of the company until the end of 1976. As a result, in the 1st quarter of 1977, the construction of the 14 houses was finished, but none of the 214 flats in multi-family buildings for the employees of the company were built until the middle of the year, even though, according to the plan, 90 flats should Professional Negligence 155 had been finished a year earlier, that is by July 30, 1976. Notably, the hous- es for directors were built in a much better standard than stipulated in the design and pricing documentation [ANIK, GP, 1627/3, f. 331-332]. The problems indicated in NIK reports also included irregularities in allocating construction and recreational plots. After an audit conducted in 1977 in the Land Management and Environment Protection Department at the Regional Administration Office in Szczecin, the director of this in- stitution was dismissed. Also in Ełk the scale of abnormalities in sales of plots for building single-family homes was so large that the head of the city had to be replaced [ANIK, GP, 294/3, f. ABUSES 31; 294/4, k. 26]. NIK inspec- tors also noticed quite a commotion regarding allocating plots in Piastów, near Warsaw. These were allocated to people whose housing needs had already been satisfied elsewhere. Also the order of examining the requests was not observed, and the value of the property or plants located in plots was underestimated [ANIK, GO, 294/5, f. 47]. Later, it turned out that the problem escalated, and that people from the highest party and state ad- ministration positions took part in this despicable criminal activity. p p p y According to NIK materials, another consequence of the directors’ or managers’ acquisition of plots was subsequent use of the production capa- bilities of their mother companies and their materials for building private residential and recreational houses. This was done, for example, by the di- rector and the main engineer of the “Orzeł Biały” Mining and Foundry Company in Piekary and the director of the “Lenko” Linen Company in Bielsko-Biała [ANIK, GP, 294/1, f. 13, 16; 294/4, f. 17]. In this situation, it is hardly surprising that companies which fundamentally sponsored the construction investments of the “elites” generated massive losses. For ex- ample, the “Insbud” Construction Cooperative in Suwałki lost 18 million PLN in similar circumstances in the year 1978 and the 6 months of the year 1979. Interestingly, when the company’s main accountant, probably un- willing to permanently endorse giving away construction materials to in- dividuals for free or at wholesale prices and underestimating payments for the single-family homes built, informed her superiors of this situation, she lost her job and was dismissed from the PZPR. Only after NIK had been alarmed and had performed a new audit, the scale of abnormalities in “Insbud” was confirmed, leading to the dismissal of the cooperative’s director and several other people [ANIK, GP, 294/5, ff. 48-49]. How could it have been any different, though, if the Minister of Construction and Construction Materials Industry himself, Adam Glazur, was such a bad role model? When building his own recreational house, he used materi- Krzysztof Lesiakowski 156 als and workers from various companies worth 1.7 million PLN without any authorization. In October 1972, he was sentenced to 7 years in pris- on and the house was confiscated by the Treasury [Seidler B.1988: 380, 389]. ABUSES In the case of the management of the Residential Construction Company in Kielce, which, for two years, Professional Negligence 157 organized trips to Bulgaria for privileged employees, the “only” problem was the fact that these trips were settled from the social fund as domes- tic trips. In 1977, the management of the Poultry Company in Prochów fi- nanced foreign trips – and to be more precise, a voyage on the “Stefan Batory” cruise ship for 13 white-collar workers, including 9 people in managerial positions – from the company’s current assets. NIK also noticed the problem of bribery. In its classic form, it oc- curred when state institutions met private business. This was evidenced by convicting, in 1979, two deputy presidents of the “Lech” Leather Goods Company in Poznań, to 1.5 and 1 year imprisonment and fines of 20,000 and 80,000 PLN, for having accepted a 180,000 PLN bribe for selling acryl- ic imported from France to private manufacturers [ANIK, GP, 148/14, f. 28].i A specific form of bribery involved “gifts” from directors and manag- ers to their superiors and more important cooperators. The fact that those who were receiving those gifts due to their positions treated them as their private property and not the property of the company they represented was even more telling: The court you had was the court you could afford. The higher you are, the larger your court, and the more substantial the privilege at your disposal [Seidler B. 1988: 412]. The court you had was the court you could afford. The higher you are, the larger your court, and the more substantial the privilege at your disposal [Seidler B. 1988: 412]. The court you had was the court you could afford. The higher you are, the larger your court, and the more substantial the privilege at your disposal [Seidler B. 1988: 412]. This criminal business thrived in particular – as it was revealed by an au- dit in 1980 – in the Radio and Television Committee. The management of the Committee presented selected people with gold and silver watches and pens and valuable works of art, while – by means of comparison – the “Sintur” Invalid Cooperative in Turek in 1977 gave away only tapestried bed covers, rugs and decorative throws. ABUSES Edward Gierek, until September 1980 the First Secretary of the Polish United Worker’s Party Central Committee, managed to avoid convic- tion, though. Based on NIK materials, the prosecutor’s office accused him of spending money from the funds of the Regional Entertainment Park Construction Committee in Chorzów on his residential house in Katowice [Gajdziński P. 2014: 343]. [ j ] Investigation of the large-scale use of state company resources for building private residential and recreational houses of prominent people was started by NIK in accordance with the resolution of the 6th Plenary Session of PUWP Central Committee of October 6, 1980, which obliged NIK and the state financial supervision institutions to verify the financing sources of all dubious private construction investments executed in the 1970s – and in particular all single-family homes (apart from those in agri- cultural farms) and recreational houses. For this purpose, the president of NIK, Mieczysław Moczar, appointed a special team headed by Władysław Pilatowski, a member of the Chamber’s council. The team operated un- til June 30, 1981. 7149 investments were investigated, out of which 2819 (39.4%) belonged to people in managerial or administrative positions, in- cluding the First Secretary, 2 Central Committee secretaries, 23 first secre- taries of Regional Committees, 34 secretaries of Regional Committees, 7 deputy prime ministers, 18 ministers, 31 deputy ministers, 21 governors and 31 deputy governors [Smith H., Smolar A., Wosleński M. 1983: 19]. 3422 people were found guilty (47.9%), including 2245 (79.6%) in promi- nent positions [Korupcja 1983: 148–206; Mac J. S. 1981: 5; ANIK, GP, 148/1, ff. 14-14v; 148/2, f. 8]. This meant that the scale of abuses by prominent people in the area of private construction was particularly large. Abuse of authority and positions for personal purposes went beyond matters related to the construction of residential and recreational houses. It also involved the use of company cars, tools, typewriters, recorders, loud- speakers or portable television sets for private purposes. Another exam- ple of abuse was the use of managerial positions to arrange foreign trips. In 1979, the aforementioned agriculture and food company in Szczekociny financed a trip to Canary Islands for three people who were not employ- ees of the company, including the daughter and the son-in-law of the com- pany president [AAN, PZPR, 4014, f. 134]. 1 Thus, Edward Gierek was wrong when he said many years later that during his ad- ministration, only licenses that had been implemented for production were bought. ABUSES This indicates that the mechanism of giving and accepting “gifts” was common, and the value of those items depended on the company’s capabilities, but surely this phenomenon can- not be justified by the Polish society’s fairly tolerant attitude towards sim- ilar behaviors at the time [Kutyłowski A., Rzepliński A. 1981: 152-154]. On a similar basis, the “Silesian Insurgents” Coking Plant in Zdzieszowice (as revealed during an audit in 1978) gave away crystals and paintings that had been bought earlier, and the management of the Copper Mines and Foundries Company in Lubin, which had more resources, gifted sil- ver items worth almost 500,000 PLN that had been specially manufactured in the company’s prototype workshop [AAN, PZPR, 4008, f. 9; ANIK, GP, 294/4, ff. 4-5; 294/3, ff. 16-17, 23]. 158 Krzysztof Lesiakowski MISMANAGEMENT MISMANAGEMENT The scale of the problem is ev- idenced by the fact that during the NIK council sitting on March 14, 1979 it was concluded that the cases of professional negligence “can be qual- ified as economic sabotage”. Every case of such negligence led to tangi- ble financial losses. For example the “Minex” export and import company paid its Brazilian supplier for goods (magnesite) based on the weight de- clared by the exporter and not the actual weight confirmed in the Polish port once the products had been unloaded. As a result, they paid USD 273,000 more than needed for the delivery of 1.6 t of magnesite in the years 1977-1978; the money could not be recovered. Furthermore, it turned out that the bosses of trade companies sent different goods than stipulated in contracts, which also meant losses – they had to provide discounts or cover return costs. For example, the “Textilimpex-Tricot” company lost USD 150,000 after having sent to Libya 60,000 men’s coats made of a dif- ferent fabric than stipulated in the contract (discount). Polish companies also incurred losses by carelessly delivering products to unreliable com- panies. Although in 1976 the “Skórimpex” leather company had not re- ceived payment of SEK 92,400 for shoes delivered to a Swedish compa- ny, the following year they sent another batch of shoes to the same buyer [ANIK, GP, 1569/8, ff. 465-568]. Negotiation of prices for exported goods was also often neglected. In the years 1977–1978, the “Unitra” Foreign Trade Company exported silver powder at a price that did not cover the raw material and production costs. The conduct of the “Ciech-Siarkopol” sulfur company was also despicable – not only was the research of foreign market limited to information provided in the bi-monthly trade magazine “Sulphur”, but also, despite actual increase in sulfur prices in 1978, the company sold it at lower prices than in the previous year [Rurarz Z.1990; f. 59; ANIK, GP, 1569/8, ff. 467-468]. What is more, NIK also pointed out serious irregularities with regard to organizing foreign business trips. These trips were planned in a partic- ularly extravagant way. MISMANAGEMENT The information materials drafted by NIK in January 1977 reported that the actual losses resulting from mismanagement and wastefulness were much higher than what the Prosecutor’s General Office had found, amounting to around 750 million PLN a year. NIK admitted, though, that a large part of the losses was hard to establish, because the system of reg- istration, settlements and supervision in production and sales made it possible to include them into operational costs of the companies. As a re- sult, the manufacturing or trading costs in companies grew, but the in- crease did not affect the assessment of their activity in the form of gener- al financial results, because many of them achieved or even exceeded the results planned. However, this was not due to rational management or decreasing prime costs, but, for example, to price manipulation and falsi- fying the quality and types of resources and materials [ANIK, GP, 1627/3, ff. 60-61]. The most frequently encountered examples of mismanagement, very often punishable by law, included: making irrational and unnecessary pur- chases of machines and tools (including imports); insufficient protection of property; not issuing the required documents for materials and resourc- es sold and dispatched from warehouses; unjustified settling of materials at underestimated prices; using improper technologies in production (e.g. during roadworks); improper storage or processing of agricultural prod- ucts; neglect of livestock in cooperative and state agricultural farms. When it comes to buying equipment for foreign currency it must be noted that the “Pioma” Mining Equipment Company in Piotrków Try­ bunalski was particularly careless. An audit in 1976 showed that the com- pany bought in West Germany three complete welding stations for manu- facturing automated machines whose production had been canceled even before the stations were imported [ANIK, GP, 2941, f. 12]. In 1979, the “Bumar” and “Energopol” Foreign Trade Companies paid in foreign cur- rency for the import from Belgium of two completely useless prototypes of extracting machines [Rolicki J. 1990: 96]1, which were supposed to be used in Gdańsk during the construction of a refinery [ANIK, GP, 148/14, f. 27]. In this case, the NIK audit led to charging the people responsible. 159 Professional Negligence NIK made many mismanagement accusations towards the bosses of foreign trade centers who were responsible for negotiating prices and en- forcing payments for the exported goods. MISMANAGEMENT Some were completely unnecessary, as evidenced by the actions of the “Kolmex” foreign trade company, which in July, August and September of 1976 sent three consecutive teams to Turkey in order to prepare a tender proposal for delivering Diesel locomotives for this country – even though it had been known from the very beginning that the Polish party could not enter the tender because they were not able to offer engines as powerful as the Turks required. Another problem was Krzysztof Lesiakowski 160 the fact that, as found during audits, teams were too large and included unnecessary employees, for example, too many members of the manage- ment. In 1977, a NIK report stated that in some cases, directors spent over 100 days abroad! [AAN, PZPR, 3852, ff. 44-45; ANIK, GP, 1569/12, ff. 564, 568]. If we add that some of these trips were organized at the expense of foreign companies, but prior to signing the contract, then the situation must have risen suspicion of corruption [Madej K. 2003: 262]. PERSONAL RESPONSIBILITY IN THE LIGHT OF NIK AUDITS Identifying those personally responsible for the detected cases of neg- ligence, abuses and mismanagement was very difficult for auditors. It was much easier to say that the situation is abnormal and define the conse- quences than to specify who should be held responsible (professionally, legally and financially). Difficulties in this respect resulted mainly from poor organization and functioning of companies, a complicated (collec- tive) decision-making processes and the weakness of professional super- vision. In many cases obstacles were created by informal, local groups of a crim- inal nature, the so-called cliques [Górniok O. 1986: 65-66]. This is evidenced by the case of punishing those guilty of delivering bad meat products in the Nisk Meat Company (including return of exported canned ham worth 2.7 million PLN). The district unit of NIK in Rzeszów made requests to pun- ish the company’s management twice, in 1978 and 1979. The first time, the prosecutor’s office discontinued proceedings, and the second time, no ver- dict was given due to the amnesty. Only the third attempt proved effec- tive. The vice director of the company for production was sentenced to two years in prison (verdict suspended for 3 years) and a 30,000 PLN fine, and the export production manager – to 1 year and 6 months in prison (verdict suspended for 3 years) and a 20,000 PLN fine. However, it was impossible for NIK to lead to the dismissal of the manager in the “Wasiułki” brick com- pany (the worst unit of the Construction Ceramics Company in Olsztyn), which, as a result of numerous acts of negligence, for a long time intro- duced bricks of very bad quality to the market. The manager was defended by the Polish United Workers’ Party committee in Nidzica and the Workers Council [ANIK, GP, 148/14, f. 27]. NIK also failed to enforce professional Professional Negligence 161 punishment (not to mention any other consequences) for people from the so-called research and extraction coal mine in the Lublin region who al- lowed for shaft deepening works to be conducted contrary to the applicable technological and geotechnical procedures, which resulted in a temporary flooding of the mine in October 1979 [AAN, PZPR, 4014, f. 18]. Despite similar obstacles, NIK managed to enforce personal responsi- bility in many cases. PERSONAL RESPONSIBILITY IN THE LIGHT OF NIK AUDITS If the shortage caused by mismanagement or waste- fulness was qualified as “significant”, then the case was transferred to law enforcement agencies [ANIK, GP, 1586/6, f. 5].2 In 1979, NIK audited 7769 units. 5396 people were sanctioned for abuses and mismanagement proved during audits. Professional sanctions were applied against 4624 people, including 803 directors, deputy directors and main accountants. 58 people were dismissed immediately (including 24 directors and deputy directors), 350 were dismissed with notice, 312 people (including 78 direc- tors and deputy directors) were transferred to different positions with dif- ferent salaries. Furthermore, 85 cases were transferred to prosecutor’s of- fice as qualifying for a criminal investigation. NIK also imposed 772 fines in the total amount of 3.2 million PLN. In 1980, NIK also imposed professional sanctions against 4872 people, out of whom 777 were in managerial positions. In total, 460 people were dismissed, out of whom 100 without notice. Fines in the total amount of 3.4 million PLN were administered towards 807 people found guilty, in- cluding 397 in managerial positions. Furthermore, 180 cases were trans- ferred to the prosecutor’s office [AAN, PZPR, 4014, f. 9]. 2 Shortage was referred to as “significant” on the basis of comparison between its size and the overall stock of the goods that the perpetrator supervised or managed. CONCLUSIONS The aforementioned data show that NIK had proven criminal incli- nation among part of the Polish managerial staff in the second half of the 1970s, before the problem was publicized by “Solidarity”. The number of people in managerial positions who were punished professionally, finan- cially, by dismissal or by request for launch of a criminal investigation by the prosecutor’s office gradually increased. Auditors concentrated mainly on lower ranking managers, though – at company, cooperative or commu- Krzysztof Lesiakowski 162 nal levels. Managers in higher positions – in associations and ministries – guilty of professional negligence, abuses and mismanagement on a much larger scale than those lower in the hierarchy, were beyond the reach of NIK auditors due to formal considerations (as NIK reported to the Prime Minister). Even more important is the fact that the government and in- dividual ministries did not draw any conclusions from this information. Therefore, NIK audits were not an effective preventive measure and did not lead to any significant decrease in criminal practices among the direc- tors and managers of Polish companies. ARCHIVE MATERIALS 1569/6, Information for NIK council on some important housing prob- lems in the light of analyses and audits performed by NIK, May 1977, f. 228. g y y y ANIK – GP, sign. 1627/3, A note to the President of the Council of Ministers regarding some conclusions from the audit, October 8, 1977, f. 75. ANIK – GP, sign. 1627/3, Information on the condition of collective property protection against abuses, wastefulness and mismanagement, January 1977, ff. 331-332. ANIK – GP, sign. 294/3, Information on the mode of examining and handling complaints and requests sent to NIK in 1977, February 1978, f. 31; ibidem, sign. 294/4, Report on complaints handled in 1978 r., February 1979, f. 26. p y ANIK – GP, sign. 294/5, Report on complaints and requests handled in the year 1979, [1980], f. 47. ANIK – GP, sign. 294/1, Information on the mode of examining and handling complaints and requests sent to NIK in 1976, February 1977, f. 13, 16; ibidem, sign. 294/4, sign. 294/4, Report on complaints handled in 1978, February 1979, f. 17. y ANIK – GP, sign. 294/5, Report on complaints and requests handled in the year 1979, [1980], f. 48-49. ANIK – GP, sign. 148/1, Decision no. 1/90 of the president of NIK, October 21, 1980, f. 14- 14v; ibidem, sign. 148/2, Decision no. 11 of the president of NIK, June 30, 1981, f. 8. g p AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 134. g y ANIK – GP, sign. 294/3, Information on the mode of examining and handling complaints and requests sent to NIK in 1977, February 1978, f. 3, 16. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor General and cooperation between NIK and law enforcement agencies in 1979, March 1980, f. 28. AAN – PZPR, sign. 4008, A note to the president of NIK on the audit in the Radio and Television Committee [1980], f. 9; ANIK – GP, sign. 294/4, Report on complaints han- dled in 1978, February 1979, ff. 4-5; ibidem, sign. 294/3, Information on the mode of examining and handling complaints and requests sent to NIK in 1977, February 1978, ff. 16-17, 23. ANIK – GP, sign. 1627/3, A note to the President of the Council of Ministers regarding some conclusions from the audit, October 8, 1977, ff. 60-61. ANIK – GP, sign. ARCHIVE MATERIALS Archiwum Akt Nowych (Archive of New Files, AAN)  – Polska Zjednoczona Partia Robotnicza (Polish United Workers’ Party, PZPR), sign. 3852, 4008, 4014 i Archiwum Najwyższej Izby Kontroli (Archive of Supreme Audit Office, ANIK) – Gabinet Prezesa (Chairman’s Office, GP), sign. 148/1, 148/2, 148/14, 294/1, 294/3, 294/4, 294/5, 1569/6, 1569/8, 1569/10, 1569/12, 1586/6, 1627/3, 1627/4, 1627/7, 2941 ANIK – GP, sign. 294/1, Information on the mode of examining and handling complaints and requests sent to NIK in 1976, February 1977, f. 22. q y ANIK – GP, sign. 1627/7, Responsibility in the light of NIK audits, September 7, 1978, ff. 248-249. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor General and NIK cooperation with law enforcement agencies in 1979, March 1980, f. 25; ibidem, sign. 294/1, Information of the mode of examining and handling com- plaints and requests sent to NIK in 1976, February 1977, f. 12. AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], ff. 133-13 ANIK – GP, sign. 1569/10, Information on the results of personal management audit, June 1978, f. 417. ANIK – GP, sign. 1569/12, Information of NIK council on the results of the audit on ex- panding and efficiency of operation of liquid fuels and oil distribution network in the country, October 4, 1978, f. 56. ANIK – GP, sign. 1627/4, Information on the results of the audit of accuracy of statistical reports, June 1978, f. 16. ANIK – GP, sign. 294/3, Information on the mode of examining and handling complaints and requests sent to NIK in 1977, February 1978, f. 27. q y AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 16. ANIK – GP, sign. 1627/3, A note to the President of the Council of Ministers regarding some conclusions from the audit, October 8, 1977, f. 367. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor General and cooperation between NIK and law enforcement agencies in 1979, March 1980, f. 27. AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 147. NIK operation in the year 1980 [1981], f. 14 AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 149. Professional Negligence 163 ANIK – GP, sign. ARCHIVE MATERIALS 2941, Information on the mode of examining and handling complaints and requests sent to NIK in 1976, February 1977, f. 12. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor General and cooperation between NIK and law enforcement agencies in 1979, March 1980, f. 27. ANIK – GP, sign. 1569/8, Minutes of the sitting of NIK council on March 14, 1979, f. 436; ibi- dem, Information on the results of the audit of the efficiency of foreign trade, February 1979, ff. 465-468.i ANIK – GP, sign. 1569/8, Information on the results of the audit of the efficiency of foreign trade, February 1979, ff. 467-468. AAN – PZPR, sign. 3852, A report from NIK operation in the year 1977 [1978], ff. 44-45; ANIK – GP, sign. 1569/12, Information the conclusions from the audit on foreign busi- ness trips [1977], ff. 564, 568. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor General and cooperation between NIK and law enforcement agencies in 1979, March 1980, f. 27. AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 18. ANIK – GP, sign., 1586/6, A bulletin from NIK president, January 6, 1977, f. 5. g p y AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 9. Krzysztof Lesiakowski 164 LITERATURE Gajdziński P. (2014), Gierek. Człowiek z węgla [Gierek. A  man of coal], Wydawnictwo Poznańskie, Poznań. dziński P. (2014), Gierek. Człowiek z węgla [Gierek. A  man of coal], Wydawnictwo Poznańskie, Poznań. rniok O. (1986), Przestępczość gospodarcza: wybrane przejawy i uwarunkowania [Financia crime: selected symptoms and conditions], Uniwersytet Śląski, Katowice. Jarosz D. (2010), Mieszkanie się należy... Studium z peerelowskich praktyk społecznych [A house is your right... A study of social practices in the Polish People’s Republic], Oficyna Wydawnicza Aspra-JR, Warsaw. y p Jarosz M. (2004), Władza, przywileje, korupcja [Power, privilege, corruption], Wydawnictwo Naukowe PWN, Warszawa. Karklins R. (2009), Wszystkiemu winien system. Korupcja w krajach postkomunistycznych [The system is to blame. Corruption in post-communist countries], Wydawnictwo Sic!, Warsaw. Komisja Tadeusza Grabskiego 1981 [Tadeusz Grabski Commission 1981] (2013), Jabłonowski M., Janowski W. (ed.), Faculty of Journalism and Political Science, University of Warsaw, Warsaw. Konaszyc A. (1978), Kontrola parlamentarna w PRL, Zakład Narodowy im. Ossolińskich [Parliamentary control in the Polish People’s Republic], Wrocław-Warszaw-Kraków. Korupcja polskiej „nomenklatury”. ARCHIVE MATERIALS Z tajnych akt Najwyższej Izby Kontroli PRL [Corruption of the Polish “nomenklatura”. From the secret files of Supreme Audit Office] (1983), “Zeszyty Historyczne” (Paris), no. 64, p. 148-206. Kutyłowski A., Rzepliński A. (1981), Opinie na temat łapownictwa jako przestępstwa i zjawis- ka społecznego [Opinions on bribes as a crime and a social problem], [in:] “Opinia pub- liczna i środki masowego przekazu a ujemne zjawiska społeczne” [Public opinion and mass media and negative social phenomena], Wydawnictwo Prawnicze, Warszawa, p. 142-156. p Lesiakowski K. (1998), Mieczysław Moczar „Mietek”. Biografia polityczna [Mieczysław Moczar, “Mietek”. A political biography], Oficyna Wydawnicza „Rytm”, Warszawa. p g p yi y y y Mac J. S. (1981), Duże pranie [Big laundry], “Prawo i Życie”, no. 28, p. 5. Madej K. (2003), Siermiężna i dolarowa – korupcja w PRL w latach 1956-1980 [Poverty and dol- lars – corruption in socialist Poland in the years 1956-1980], [in:] Stola D., Zaremba M. (ed.), “PRL. Trwanie i zmiana” [Socialist Poland. Stagnation and change] , Wydawnictwo Wyższej Szkoły Przedsiębiorczości i Zarzadzania im. Leona Koźmińskiego, Warsaw, p. 249-280. p Rolicki J. (1990), Edward Gierek. Replika (wywiad rzeka) [Edward Gierek. A replica (an inter- view)], Polska Oficyna Wydawnicza ”BGW”, Warsaw. i )i y y Rurarz Z. (1990), Byłem doradcą Gierka [I used to be Gierek’s advisor], Andy Grafik, Warszawa. Seidler B. (1988), Ludzie i paragrafy [People and law], Wydawnictwo Literackie, Kraków. Sierpowska I. (2003), Funkcje kontroli państwowej. Studium prawno-porównawcze [Function of state controls. A legal and comparative study], Kolonia Limited , Wrocław. Smith H., Smolar A., Wosleński M. (1983), Władza i przywileje [Power and privileges], Oficyna WE, Warsaw Sylwestrzak A. (2006), Najwyższa Izba Kontroli. Studium prawnoustrojowe [Supreme Audit Office. Systemic and legal study], Wydawnictwo Sejmowe, Warszawa. Professional Negligence 165 Krzysztof Lesiakowski  – PhD, Profesor, who works at the Polish and World History Faculty at the University of Łódź. His main research interests include the relationship between authorities and society in Poland after the Second World War, Polish People’s Republic, policy towards the young generation, Polish biographies in the 20th century. His publications include: Mieczysław Moczar „Mietek”. Biografia polityczna [Mieczysław Moczar, “Mietek”. A  political biography] (Warszawa 1998); Jarocin w obiektywie bezpieki [Jarocin Festival through the lenses of the Security Services] (Warszawa 2004) (co-written with two authors); Powszechna Organizacja “Służba Polsce” 1948–1955 – powstanie, działalność, likwidac- ja”, [The Common Organization “Service for Poland” (1948–1955): origin, activity, liqui- dation] vol. ARCHIVE MATERIALS 1–2 (Łódź 2008); Strajki robotnicze w Łodzi 1945–1976 [Workers’ strikes in Łódź 1945–1976] (Łódź 2008). He is also the author of numerous articles and the editor of sever- al collected works, including Łódzki strajk studencki styczeń-luty 1981. Spojrzenie po latach” [Students’ strikes in Łódź January–February 1981. A perspective after the years] (Łódź- Warsaw 2014).
https://openalex.org/W3110626367
https://sajs.co.za/article/download/7688/10642
English
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Accomplishments in wheat rust research in South Africa
South African journal of science
2,020
cc-by
8,548
FUNDING: FUNDING: Biotechnology and Biological Sciences Research Council / Department for International Development (BBSRC-DfID; BB/ F004125/1); Sustainable Crop Production Research for International Development, Biotechnology and Biological Sciences Research Council (BBSRC-SCPRID; BB/J011525/1) The objective of this review is to provide a summary of recent accomplishments in wheat rust research in South Africa. EMAIL: pretorza@ufs.ac.za • Collaboration among plant pathologists, geneticists and breeders has provided momentum in rust research and control in South Africa in recent years. • Collaboration among plant pathologists, geneticists and breeders has provided momentum in rust research and control in South Africa in recent years. DATES: Received: 29 Nov. 2019 Revised: 16 Mar. 2020 Accepted: 18 Mar. 2020 Published: 26 Nov. 2020 Accomplishments in wheat rust research in South Africa AUTHORS: Zacharias A. Pretorius1 Renée Prins2,3 Elsabet Wessels2 Cornel M. Bender1 Botma Visser1 Willem H.P. Boshoff1 AFFILIATIONS: 1Department of Plant Sciences, University of the Free State, Bloemfontein, South Africa 2CenGen (Pty) Ltd., Worcester, South Africa 3Department of Genetics, Stellenbosch University, Stellenbosch, South Africa Rust diseases, although seasonal, have been severe constraints in wheat production in South Africa for almost 300 years. Rust research gained momentum with the institution of annual surveys in the 1980s, followed by race identification, an understanding of rust epidemiology, and eventually a focused collaboration amongst pathologists, breeders and geneticists. Diversity in South African populations of Puccinia triticina, P. graminis f. sp. tritici and P. striiformis f. sp. tritici has been described and isolates are available to accurately phenotype wheat germplasm and study pathogen populations at national, regional and global levels. Sources of resistance have been, and still are, methodically analysed and molecular marker systems were developed to incorporate, stack and verify complex resistance gene combinations in breeding lines and cultivars. Vigilance, capacity, new technologies, collaboration and sustained funding are critical for maintaining and improving the current research impetus for future management of these important diseases. © 2020. The Author(s). Published under a Creative Commons Attribution Licence. ARTICLE INCLUDES: ☒ Peer review ☐ Supplementary material No mention is made of rust during the foundational years of cereal production in South Africa but, according to Theal3, a critical shortage of wheat in 1727 was ascribed in the previous year to rust – a disease known in South Africa only on rye at the time. The regular occurrence of rust led Neethling2 to conclude: ‘There is no doubt that rust, owing to the severe damage caused, is the most important factor which caused the extinction and origin of varieties in South Africa’. Nhemachena and Kirsten4 gave a detailed account of wheat cultivar development in South Africa, Smit et al.5 summarised wheat research between 1983 and 2008, and overviews of wheat rust research in South Africa were provided by De Jager6, Lombard7 and Pretorius et al.8 Early milestones were interspecies crosses to transfer stem rust resistance genes to bread wheat (Triticum aestivum L.) in 1912 followed by pathotyping isolates of Puccinia graminis f. sp. tritici Erikss. & E. Henn. (Pgt) and P. triticina Erikss. (Pt) in the 1920s and 1930s.8 The establishment of a centre for dedicated small grains research at Bethlehem in 1976, currently named Agricultural Research Council – Small Grain (ARC-SG), resulted in appropriate training in rust methodologies, surveillance, race analysis and germplasm evaluation. These initiatives were expanded with the formation of a rust laboratory at the University of the Free State in 1989. DATA AVAILABILITY: ☐ Open data set ☐ All data included ☒ On request from author(s) ☐ Not available ☐ Not applicable EDITORS: Teresa Coutinho Salmina Mokgehle KEYWORDS: Puccinia, resistance, rust, surveillance, wheat In recent decades, notable events and initiatives in South African wheat rust research include annual surveys8, Sr24 virulence9, the appearance of stripe rust (caused by P. striiformis Westend. f. sp. tritici, Pst)10, Ug99 stem rust studies11-15, the mapping of quantitative resistance loci16,17, genetic characterisation of Puccinia isolates18-20, comprehensive phenotyping of wheat germplasm, and establishment of a marker service laboratory with a particular focus on rust resistance genes (https://www.cengen.co.za). Background The sowing of small grain cereals in South Africa occurred within 2 months after the United (Dutch) East India Company set foot on land in present-day Cape Town in 1652.1,2 Crop failures, in particular due to damage caused by heavy rains, wind storms and unadapted cultivars, were common occurrences. Nonetheless, efforts to successfully grow wheat continued and systematically included new production areas, different sowing times, new cultivars – not only from Europe but also from India, and exports when grain supplies allowed. Varietal assessments during the early years provided evidence for the first selection of higher-yielding types in South Africa.2 The pioneering wheat cultivars are not well documented, but reference is made of ‘white’ wheat in 1659, ‘Sarut’ from India in 1673, ‘Roode’ and ‘Grijse Winter’ in 1677, until names based on phenotype (e.g. ‘Bloukoring’, ‘Kleinkoring’, ‘Baardkoring’, ‘Zwartbaard’, ‘Vroeëbaard’), origin (e.g. ‘Ciciliaans’, ‘Bengaalsch’), or growers (e.g. ‘Du Toits’, ‘Niewoudts’, ‘Tautes’) became customary.2 HOW TO CITE: Pretorius ZA, Prins R, Wessels E, Bender CM, Visser B, Boshoff WHP. Accomplishments in wheat rust research in South Africa. S Afr J Sci. 2020;116(11/12), Art. #7688, 8 pages. https://doi.org/10.17159/ sajs.2020/7688 CORRESPONDENCE TO: Zacharias Pretorius • Rust diseases threaten wheat crops worldwide, including in South Africa. • Management of rusts includes regular surveillance, pathogen diversity studies, rigorous screening of wheat germplasm, and efficient breeding and selection for resistance. Rust surveillance and phenotypic analysis Surveillance and race typing are routinely conducted by the ARC-SG to determine rust distribution, impact and pathogenicity in the major wheat-producing areas of South Africa. Recent reports of similarities in races between southern African countries have also emphasised the importance of regional sampling.21-23 © 2020. The Author(s). Published under a Creative Commons Attribution Licence. Handling obligate rust fungi in controlled experiments such as race typing or host plant screening requires specific infrastructure. In addition to facilities for plant growth, inoculation and incubation, equipment for collection Volume 116| Number 11/12 November/December 2020 Review Article https://doi.org/10.17159/sajs.2020/7688 Volume 116| Number 11/12 November/December 2020 Volume 116| Number 11/12 November/December 2020 Review Article https://doi.org/10.17159/sajs.2020/7688 1 Wheat rusts in South Africa Page 2 of 8 Thew (Lr20) and Agent (Lr24), all Lr genes occur in a Thatcher wheat background. New races are further characterised on an additional set containing 23 Lr genes.27 Infection types on the lines RL6011 (Lr12), CT263 (Lr13), RL6044 (Lr22a), RL6058 (Lr34), RL6082 (Lr35) and Thatcher control (Lr22b) are determined on flag leaves of adult plants. Thew (Lr20) and Agent (Lr24), all Lr genes occur in a Thatcher wheat background. New races are further characterised on an additional set containing 23 Lr genes.27 Infection types on the lines RL6011 (Lr12), CT263 (Lr13), RL6044 (Lr22a), RL6058 (Lr34), RL6082 (Lr35) and Thatcher control (Lr22b) are determined on flag leaves of adult plants. and application of small amounts of urediniospores is essential. Because these specialised items are not commercially available, Pretorius et al.24 developed an additive manufacturing process to assemble spore collectors and atomisers through 3D printing. Using these devices, traditional race analysis is done by infecting seedlings of a predetermined (differential) set of wheat host lines with a rust isolate. An appropriate experimental set-up and experience in achieving accurate seedling infection types are essential for reliable phenotyping. Examples of infection types are shown in Figure 1. (differential) set of wheat host lines with a rust isolate. An appropriate experimental set-up and experience in achieving accurate seedling infection types are essential for reliable phenotyping. Examples of infection types are shown in Figure 1. Figure 1: Seedling (top, left to right: wheat leaf rust, stem rust and stripe rust) and adult plant phenotypes commonly encountered for leaf rust (second from top), stem rust (third from top) and stripe rust (bottom). Rust surveillance and phenotypic analysis Its broad virulence and subsequent specialisation in 13 pathotypes have raised serious concerns about sustained wheat production in many regions of the world.15 Stem rust race 2SA106 (TTKSP North American race code) detected in 2007, 2SA107 (PTKST, 2009), 2SA88+ (TTKSF+, 2010) and 2SA42 (PTKSK, 2017) all show phenotypic similarities to race 2SA88 (TTKSF, 2000), which was the first stem rust race in the Ug99 lineage detected in South Africa.12,13,18,21,32,35 These races are phenotypically characterised by differences in virulence for Sr9h, Sr21, Sr24 and Sr31.13,32 Sr24 and Sr31 have been reported to occur in South African wheat germplasm36 and virulence was not unexpected. Likewise, the virulence adaptation of TTKSF+ was recently confirmed by the endorsement of Sr9h in the wheat cultivar Matlabas.37 Despite being less virulent compared to the more recently detected Ug99 races, TTKSF remains the dominant variant.8,31,32,38 Stem rust races TTKSF (2009), TTKSF+ (2010) and PTKST (2010) were also identified in samples collected in Zimbabwe and PTKST was confirmed in Mozambique.15,21 The most significant change in the Pgt population since 2005 was the regular appearance of new races in the Ug99 lineage. African race Ug99, named after the country of first detection (Uganda) and year of description (1999)11, was the first race with virulence for the widely used Sr31 resistance gene. Its broad virulence and subsequent specialisation in 13 pathotypes have raised serious concerns about sustained wheat production in many regions of the world.15 Stem rust race 2SA106 (TTKSP North American race code) detected in 2007, 2SA107 (PTKST, 2009), 2SA88+ (TTKSF+, 2010) and 2SA42 (PTKSK, 2017) all show phenotypic similarities to race 2SA88 (TTKSF, 2000), which was the first stem rust race in the Ug99 lineage detected in South Africa.12,13,18,21,32,35 These races are phenotypically characterised by differences in virulence for Sr9h, Sr21, Sr24 and Sr31.13,32 Sr24 and Sr31 have been reported to occur in South African wheat germplasm36 and virulence was not unexpected. Review Article https://doi.org/10.17159/sajs.2020/7688 Rust surveillance and phenotypic analysis No new Pt races were detected between 1988 and 2008 in South Africa.8,28 This situation changed with the report of race 3SA145 (CCPS North American race code) in 2009, followed by races 3SA146 (MCDS, 2010), 3SA147 (FBPT, 2010), 3SA115 (CBPS, 2012), 3SA10 (CFPS, 2016), 3SA38 (CDPS, 2016) and 3SA248 (CFPS, 2016).27- 30 The frequency of Pt races with virulence to Lr3, Lr12, Lr13, Lr15, Lr26 and Lr37 is high and varied between 79% and 98% during recent surveys.27 The Pt population was dominated for many years by race 3SA133 (PDRS) which initially was common on winter wheat in the Free State. This changed significantly with the appearance of races 3SA145, 3SA146 and 3SA115 which accounted for >80% of isolates typed during the 2012–2016 surveys.27 The more recently described races 3SA38 and 3SA10 are increasing in prevalence and comprised more than 50% of the isolates typed from the 2018 growing season.31 Pt race MCDS was common in Zimbabwe and Zambia with FBPT and SCDS detected in Zimbabwe and Malawi.23 Twenty differential wheat lines are used for stem rust pathotyping. Although the resistance genes are similar to the proposal of Jin et al.26, Acme (Sr9g), Renown (Sr17) and Trident (Sr38) have replaced CnSr9g, Combination VII and VPM1, respectively. Additional differentials include Barleta Benvenuto (Sr8b), the triticales Coorong (Sr27), Kiewiet (SrKw) and Satu (SrSatu), and either LcSrWst-2Wst (Sr9h) or Matlabas (Sr9h).32 New races are further characterised on an extended set of tester lines.33 Although differential lines grown in the field can provide an indication of prevailing Pgt races, Boshoff et al.34 showed that certain resistance genes are not well expressed in adult plants whereas other lines contain resistance in addition to that observed in seedling assays. The most significant change in the Pgt population since 2005 was the regular appearance of new races in the Ug99 lineage. African race Ug99, named after the country of first detection (Uganda) and year of description (1999)11, was the first race with virulence for the widely used Sr31 resistance gene. Genetic analysis of Puccinia isolates In a recent study, Li et al.48 provided genomic evidence of somatic hybridisation in Pgt, shedding light on the origin of Ug99 through the exchange of nuclei between standard race 21 and an unknown race. This is an important discovery to understand the formation of new diversity in the absence of sexual recombination. The Ug99 lineage on the other hand, first detected in South Africa in 2000 with the description of TTKSF12, has expanded into five variants13,14,21,35. I t t t th U 99 li ll fi S th Af i i t d Stem rust assessments for local germplasm are summarised in Figure 2. Only cultivars with seedling infection types <2 (0 to 4 scale)53, and a coefficient of infection54 <20, were considered to carry true ASR. Some cultivars regarded as resistant as seedlings showed an intermediate stem rust reaction in the field and were thus not classified as displaying true ASR. Inoculum loads in the Greytown field nursery are extremely high and not all ASR genes provide complete rust protection under such conditions. In most cases it is assumed that these cultivars will be acceptable in commercial fields where inoculum pressure is lower. The opposite was also observed where some cultivars were classified as intermediate in the seedling stage but stem rust resistant in the field. The effect of using race PTKST in the field from 2011 onwards is clear from the initial decrease in resistance before a gradual improvement in resistant entries as breeders adapted their selection and breeding strategies. Collectively such information, also for leaf and stripe rust, adds to an understanding of disease risk and management at production level. To support field data, protocols for accelerated and reliable greenhouse assays have been developed for stripe rust16,55,56, leaf rust57 and stem rust58. The current South African Pt population consists of two primary genetic lineages20, but at least five were evident according to Pt isolates collected during the previous century49. Rust surveillance and phenotypic analysis However, it is not yet known if Pst will successfully establish in Zimbabwe and, as anticipated, migrate to South Africa. throughout the lifespan of the plant whereas adult plant resistance (APR), often polygenic and partial in manifestation, becomes effective at more mature growth stages.51 Phenotypes commonly encountered on adult plants are shown in Figure 1. As some APR genes are considered durable, this resistance type is frequently preferred in breeding and selection. Wheat cultivars carrying the pleiotropic race non-specific APR genes Sr2/Yr30, Lr34/Yr18/Sr57, Lr46/Yr29/Sr58 and Lr67/Yr46/Sr55 have maintained moderate levels of rust resistance under epidemic field trial conditions in South Africa and might not provide adequate protection when deployed singly under high disease pressure. Soko et al.52 recorded grain yield losses due to stem rust of between 10.1% and 19.5% for APR cultivars as opposed to a 6.4% loss in an ASR line. Previously, Pretorius et al.8 mentioned losses as high as 65% for susceptible wheat cultivars infected with stripe rust and a 56% yield gain was obtained when leaf rust was controlled by fungicide application on a susceptible cultivar. Breeders are therefore encouraged to either combine APR sources or stack them with ASR genes, the latter especially in areas prone to early- season infection. It has been suggested that wheat cultivated at a higher elevation in Lesotho during summer serves as a source of Pst inoculum for winter- grown crops in South Africa.8 Although not customary, some hectares may also be sown to wheat in the Free State during summer. The impact of these formal off-season productions on the epidemiology of the rusts has, however, not been studied in detail. Volunteer wheat has generally been assumed to provide a green bridge for the survival of these biotrophic pathogens between seasons. Genetic analysis of Puccinia isolates Three of these appear to be extinct while only one lineage is expanding.27,28,30 Similar to Pgt, these new races probably represent exotic introductions as races with similar phenotypes and genotypes were found in countries to the north of South Africa.23 Globally, the South African Pt races grouped significantly with isolates from the Middle East, Pakistan and New Zealand.50 Based on microsatellite analysis, the four Pst races described in South Africa represent a single, clonal lineage.19 As opposed to these races, the recently identified Pst race in Zimbabwe was genetically very similar to two Kenyan isolates45, indicating a southerly expansion of stripe rust diversity in Africa. Genetic studies of host resistance provide information on the monogenic or polygenic nature of genes involved, their identity and chromosome location, association with known genes or quantitative trait loci (QTL), and molecular markers for tracking the resistance. Together this knowledge contributes to assumptions of durability and targeted attempts to achieve long-lasting resistance. Ramburan et al.16 were the first to map rust resistance in a South African wheat cultivar. They identified three major stripe rust resistance loci in the spring wheat cultivar Kariega and paved the way for fine mapping and marker development for QYr.sgi-2B.1 and QYr.sgi-4A.1, and confirmation of the pleiotropic resistance gene Lr34/ Yr18/Sr57.57,59 In a similar approach, the durable stripe rust resistance of the European wheat cultivar Cappelle Desprez was mapped17 with subsequent identification of the major effect QTL QYr.ufs-2A along with three QTL of smaller effect, QYr.ufs-2D, QYr.ufs-5B and QYr.ufs-6D. Using histological techniques, Maree et al.60,61 investigated fungal behaviour in lines containing different combinations of the stripe rust resistance Due to the unique ability of markers to distinguish genotypes independently of their associated phenotypes, genetic screening of field isolates can detect variants before a new phenotype becomes evident. While Pt races 3SA38, 3SA10 and 3SA248 were first detected as phenotypic variants in 201627, their unique genotypes were already abundant in field isolates collected in 201549. These markers also indicated that within each phenotype, significant genetic variation was present, making genetic markers an effective supplementary tool to race phenotyping. Genetic analysis of Puccinia isolates Analysis of Pt, Pgt and Pst with microsatellite markers has contributed to explaining genetic diversity within the three populations. In the absence of functional alternate hosts for wheat rust pathogens in South Africa, wind dispersal, human activities, mutation and possibly somatic recombination are considered as drivers of variation. The South African Pgt population consists of two highly diverse genetic lineages.18 In the absence of viable historical samples in South Africa, the close genetic similarity of members of the non-Ug99 genetic lineage with Australian standard races 21-0 collected in 1954, and 326 and 194 collected in 1969, respectively, suggested that this lineage represents the original South African population.46 Included in this lineage are races that are specific for both wheat and triticale. The acquisition of virulence within this group appears to be the result of step-wise mutations.32,33 On a global scale, this lineage grouped closest with Pgt samples from Pakistan, Czech Republic47,48 and Australia due to the proposed movement of urediniospores on high-altitude westerly winds46. The damage potential of wheat rusts is a reality, and it remains important to verify the resistance status of local germplasm and embark on appropriate breeding and selection programmes. As part of risk assessment and compilation of production guidelines, all commercially recommended wheat cultivars in South Africa, as well as leading breeding lines, are tested annually against a panel of rust races. These tests comprise seedling assays for ASR and field tests under high inoculum pressure in carefully managed rust nurseries. The University of the Free State has implemented rust nurseries with great success at the research facilities of Corteva AgriscienceTM at Greytown in KwaZulu-Natal since the early 1990s. The Greytown environment is highly conducive to the vigorous development of both spring and winter wheat types as well as rust development. In a typical year, stripe rust would be first to establish during the cooler months of August and September, followed by leaf rust in October and finally stem rust, which peaks at the end of the season. The Ug99 lineage on the other hand, first detected in South Africa in 2000 with the description of TTKSF12, has expanded into five variants13,14,21,35. In contrast to the non-Ug99 lineage, all five South African variants and the original TTKSK11 shared more than 85% genetic similarity and fall within the bigger Ug99 race group from east Africa15. Rust surveillance and phenotypic analysis Likewise, the virulence adaptation of TTKSF+ was recently confirmed by the endorsement of Sr9h in the wheat cultivar Matlabas.37 Despite being less virulent compared to the more recently detected Ug99 races, TTKSF remains the dominant variant.8,31,32,38 Stem rust races TTKSF (2009), TTKSF+ (2010) and PTKST (2010) were also identified in samples collected in Zimbabwe and PTKST was confirmed in Mozambique.15,21 Seedling infection types produced on the World and European differential sets39,40, followed by an A+ or A- suffix to describe virulence or avirulence for the YrA gene in Avocet R41, are used for Pst race designations in South Africa. Near-isogenic lines with Avocet S as the recurrent parent are used as additional tester lines for race characterisation and in field plots.42 Following the detection of Pst race 6E16A- in 199610, proposed to be a foreign introduction from Central or Western Asia either by wind or human intervention43,44, there is strong evidence that adaptation to the host genes Yr25 (race 6E22A-, cultivar Hugenoot, 1998) and YrA (6E22A+, PAN 3195, 2005) resulted from selection pressure10,19. The Pst population has remained relatively stable since the detection of race 6E22A+ on winter wheat in the eastern Free State in 200519 with 6E22A+ persisting as the most dominant race, comprising 58% of isolates in 201831. Figure 1: Seedling (top, left to right: wheat leaf rust, stem rust and stripe rust) and adult plant phenotypes commonly encountered for leaf rust (second from top), stem rust (third from top) and stripe rust (bottom). Figure 1: Based on the pathogenicity of an isolate on entries in the differential set, a race (pathotype) name is allocated. Apart from an alpha-numerical code to name leaf and stem rust races in South Africa8, the North American system of nomenclature25,26 is used to place races in an international context. The standard South African differential set for determining seedling infection types to Pt isolates contains 20 entries.27 Except for Volume 116| Number 11/12 November/December 2020 Review Article https://doi.org/10.17159/sajs.2020/7688 Volume 116| Number 11/12 November/December 2020 2 Wheat rusts in South Africa Page 3 of 8 Wheat rusts in South Africa Page 3 of 8 The outbreak of stripe rust on irrigated spring wheat in 2018 represented the first report of the disease in Zimbabwe.45 Showing virulence to Yr3a, Yr4a, Yr9 and Yr27, race 30E142A+ was distinctly more virulent on South African wheat cultivars than 6E22A+ and poses a potential threat to the local industry. Breeding and selection Table 1: Yield performance of selected marker-assisted selection (MAS) lines of Corteva AgriscienceTM compared to commercial cultivars Table 1: Yield performance of selected marker-assisted selection (MAS) lines of Corteva AgriscienceTM compared to commercial cultivars Entry Relative yield (%)a Genes incorporated through MAS Cultivar 01 102 Confidentialb Cultivar 02 97 Confidential Cultivar 03 94 Confidential Cultivar 04 100 Confidential Cultivar 05 106 Confidential Cultivar 06 106 Confidential MAS Line 01 99 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 02 96 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 03 98 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 04 99 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 05 96 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 06 102 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 07 95 Sr2/Yr30; Lr34/Yr18/Sr57/Pm38 aYields measured in tons/ha are expressed relative to Cultivar 04 which was taken as the benchmark (100%). bDeveloped through traditional breeding without MAS. Marker-assisted selection (MAS) is widely accepted as a key strategy to pyramid resistance genes into wheat genotypes, in particular, genes that do not exhibit easily distinguishable phenotypes.72 In South Africa, large-scale MAS was not implemented by breeding companies in the early 2000s8, although it was routinely used to select for several traits in countries such as Australia, Mexico, USA, the UK and India72. In 2011, a proposal by CenGen (Pty) Ltd. titled ‘Establishment of a molecular marker service laboratory for routine application of marker-assisted selection in South African wheat breeding programs’ (WCT/W/2009/02), was approved for funding by the Winter Cereal Trust. The capital expense of establishing a MAS laboratory and routine maintenance justified a central facility at CenGen for all wheat breeding programmes. The project is based on (1) purity testing of donor lines and confirmation of the target trait, (2) planning of breeding schemes and crosses to transfer the new trait, and (3) tracking the trait in subsequent filial generations. Host resistance Resistance phenotypes in wheat are typically growth stage mediated. All- stage resistance (ASR), conferred by major genes, is clearly expressed Volume 116| Number 11/12 November/December 2020 Review Article https://doi.org/10.17159/sajs.2020/7688 3 Wheat rusts in South Africa Page 4 of 8 Wheat rusts in South Africa Page 4 of 8 QTL characterised in Kariega and Cappelle Desprez, respectively. These studies confirmed the value of gene stacking and careful selection of lines with the best ability to mitigate fungal invasion. or commercial cultivars and from the F1-generation doubled haploid lines are developed to integrate the genes/QTL into better adapted backgrounds. This approach has proven to be successful in pyramiding rust resistance genes/QTL (Figure 3). They have managed to develop a line containing multiple genes for resistance to all three rust pathogens, which is now used as a key donor line to incorporate complex resistance into existing cultivars. Figure 2: The frequency of South African wheat varieties expressing a low seedling response, adult plant resistance (APR) and true all-stage resistance (ASR) to stem rust over 8 years. In 2009 and 2010, entries were tested with Puccinia graminis f. sp. tritici pathotype UVPgt59 (TTKSP), and since 2011 with the more virulent UVPgt60 (PTKST) pathotype. Number of lines containing at least 2 target genes/QTL Figure 3: Complement of rust resistances incorporated in the Sensako doubled haploid marker-assisted selection programme. Numbers in brackets indicate the maximum number of genes/quantitative trait loci (QTL) screened for in the specific combination. For each combination, lines containing at least two target genes/QTL were selected for field evaluation in 2019. For all combinations, at least one line was recovered containing all targets. Number of lines containing at least 2 target genes/QTL Figure 2: The frequency of South African wheat varieties expressing a low seedling response, adult plant resistance (APR) and true all-stage resistance (ASR) to stem rust over 8 years. In 2009 and 2010, entries were tested with Puccinia graminis f. sp. tritici pathotype UVPgt59 (TTKSP), and since 2011 with the more virulent UVPgt60 (PTKST) pathotype. Complement of rust resistances incorporated in the Sensako doubled haploid marker-assisted selection programme. Numbers in brackets indicate the maximum number of genes/quantitative trait loci (QTL) screened for in the specific combination. For each combination, lines containing at least two target genes/QTL were selected for field evaluation in 2019. For all combinations, at least one line was recovered containing all targets. Host resistance Figure 3: Prins et al.62 assessed stem rust response in an African wheat collection and identified several marker-trait associations in a genome-wide study. Two lines with exceptional APR were identified and biparental mapping populations developed. Marker-trait associations on chromosomes 6AS and 3BS and the Lr34/Yr18/Sr57 resistance locus were confirmed, along with stem rust resistance QTL not detected in the association study, one of which was the significant QTL QSr.ufs-4D. This emphasises the value of applying multiple approaches to unravel host resistance, particularly in cases where marker coverage in certain chromosomal areas is too low to detect QTL. The South African wheat breeding programme of Corteva AgriscienceTM follows a more traditional approach of gradually incorporating multiple genes/QTL into their breeding lines. Gene enrichment is done at the F2- generation, and the presence of the target genes is confirmed in the F5-generation after three cycles of selection for agronomic traits. Pure lines containing the target genes are then either used to generate new resistance gene combinations or, if within the tolerance levels set for the different milling and baking quality criteria, are considered for commercial release. This programme has been successful in combining APR genes for stem, stripe and leaf rust resistance into elite breeding material that performs similarly to current commercial cultivars in yield trials (Table 1). The availability of Pgt races with virulence attributes appropriate for targeting certain sources of resistance has contributed to several studies. These projects addressed phenotyping and genetics of resistance to Ug99 races63-66, resistance characterisation of triticale67 and lines with genes transferred from Aegilops sharonensis68 and Thinopyrum ponticum69,70. Furthermore, Pretorius et al.71 demonstrated the application of remote sensing and the normalised difference vegetation index in reliably phenotyping wheat stripe rust response in the field. Review Article https://doi.org/10.17159/sajs.2020/7688 We declare that there are no competing interests. Figure 5: Number of data points generated since inception of the marker- assisted selection programme. Acknowledgements Substantial funding by BBSRC-DfID for the project ‘Assessment of genetic biodiversity of durable disease resistance in African wheat genotypes, leading to the development of novel marker systems for wheat breeding’ (BB/F004125/1) and by BBSRC-SCPRID for ‘Implementing effective marker technologies into disease resistance wheat breeding programmes within Africa’ (BB/J011525/1) is gratefully acknowledged. Additional support by the Winter Cereal Trust, University of the Free State, National Research Foundation, Cornell University (DRRW project) and many local and international colleagues is also acknowledged. Conclusions The relatively frequent introduction of new rust races into South Africa strongly suggests the possibility of further incursions. Stem rust and stripe rust, in particular, are extremely damaging diseases and the description of highly virulent and aggressive Pst and Pgt races in other wheat regions77,78 emphasises continued vigilance. The introduction of such races could impact severely on cultivar response with a consequent increase in production risk and cost. The survival of rust on off-season wheat crops and ancillary hosts such as wild rye (Secale strictum subsp. africanum) in the Roggeveld Mountains of the southwestern Karoo79, requires further attention. Although samples collected from wild rye revealed Pst, the stem and leaf rust forms were those of cultivated rye and not bread wheat.79 Wild rye is, however, moderately susceptible to Pgt and could serve as an inoculum source. The occurrence of both Pt and Pgt on a summer wheat crop in the eastern Free State in January 2020 (WHP Boshoff, unpublished) is of concern and supports the expansion of surveys to this period. Scientists should thus continue with surveillance, studies of pathogen variability, characterisation of cultivars, genetic analyses, resistance discovery, focused breeding and selection, and communication of research outcomes to producers. Overarching activities include international, regional and national collaboration; capacity building and training; embracing of new technologies; resistance gene stewardship; and sourcing sustained funding. Figure 4: Targets of rust resistance genes screened for in the marker- assisted selection programme at CenGen. Molecular markers for target genes/QTL are obtained from the public domain and research articles, or from in-house mapping projects by CenGen and collaborators. These include simple sequence repeat (SSR), sequence-tagged site (STS), cleaved amplified polymorphic site (CAPS) and single nucleotide polymorphism (SNP) markers. Since 2013, the implementation and upgrade of KASPTM SNPLlineTM instruments (LGC, UK) at CenGen greatly enhanced high-throughput capacity. The number of data points (calculated as the number of samples x number of markers tested per sample) that are generated annually continues to increase (Figure 5) despite a decrease in industry funding. Figure 5: Number of data points generated since inception of the marker- assisted selection programme. Breeding and selection Entry Relative yield (%)a Genes incorporated through MAS Cultivar 01 102 Confidentialb Cultivar 02 97 Confidential Cultivar 03 94 Confidential Cultivar 04 100 Confidential Cultivar 05 106 Confidential Cultivar 06 106 Confidential MAS Line 01 99 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 02 96 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 03 98 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 04 99 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 05 96 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 06 102 Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A MAS Line 07 95 Sr2/Yr30; Lr34/Yr18/Sr57/Pm38 South African seed companies use different strategies to breed for rust resistance, dependent on their approach, resources and location. Yet there is a collective focus on pyramiding rust resistance genes, in particular those that confer durable APR, to uphold the international drive of gene stewardship. Sensako (Pty) Ltd., a private breeding company with headquarters in Bethlehem (Free State, South Africa), follows a strategy in which they combine target genes/QTL in doubled haploid donor lines. This is followed by a top cross with their elite lines Volume 116| Number 11/12 November/December 2020 Volume 116| Number 11/12 November/December 2020 4 Wheat rusts in South Africa Page 5 of 8 Wheat rusts in South Africa Page 5 of 8 The MAS programme commenced in 2011 with the capacity to screen for 19 genes/QTL, of which 13 were related to rust resistance. These targets included the popular APR genes Lr34/Yr18/Sr57/Pm38 (Pm is the notation for powdery mildew resistance genes) and Sr2/Yr30, the leaf rust resistance gene Lr1973 as well as QTL previously identified for stripe rust resistance in the cultivars Kariega16 and Cappelle-Desprez17. Since its inception, the programme has grown to include 63 genes/ QTL of which 29 are associated with rust resistance (Figure 4). These are obtained by breeders through international collaboration with organisations such as CIMMYT, or are newly identified sources from ongoing local research projects.62 by-sequencing in wheat were published, creating yet another avenue for genomics-assisted breeding (Figure 6).76 The challenge remains for South African breeders and geneticists to follow international trends in genomics-assisted breeding and sensibly implement selection strategies for multi-locus traits. MAS, marker-assisted selection; GS, genomic selection; GBS, genotyping by sequencing Figure 4: Targets of rust resistance genes screened for in the marker- assisted selection programme at CenGen. MAS, marker-assisted selection; GS, genomic selection; GBS, genotyping by sequencing Figure 6: Timeline of MAS in South Africa (bottom) compared to international programmes (top). Competing interests We declare that there are no competing interests. 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De Jager JNW. Authors’ contributions Z.A.P. developed the outline, wrote the Abstract, Background, ‘Host resistance’ section and Conclusions; R.P. and E.W. wrote the ‘Breeding and selection’ section including table and figures; C.M.B. provided long- term cultivar data; B.V. wrote ‘Genetic analysis of Puccinia isolates’; W.H.P.B. wrote ‘Rust surveillance and phenotypic analysis’. All authors contributed to editing of the final manuscript. Z.A.P. and W.H.P.B. provided photographs of rust phenotypes. Notwithstanding the success of the implementation and application of the MAS programme for single locus traits such as rust resistance, the status of MAS in South Africa trails behind that of international programmes, which are exploring an integrated genomics-assisted breeding approach.74 In 2010, crop geneticists started to investigate genomic selection in wheat to select for complex, multi-locus traits.75 By 2012, reports of the value of genomic selection using genotyping- Volume 116| Number 11/12 November/December 2020 Volume 116| Number 11/12 November/December 2020 Review Article https://doi.org/10.17159/sajs.2020/7688 5 Wheat rusts in South Africa Page 6 of 8 Wheat rusts in South Africa Page 6 of 8 19. Visser B, Herselman L, Pretorius ZA. Microsatellite characterization of South African Puccinia striiformis races. S Afr J Plant Soil. 2016;33(3):161–166. http://www.tandfonline.com/doi/full/10.1080/02571862.2015.1125957 References Genetic comparison of Ug99 with selected South African races of Puccinia graminis f. sp. tritici. Mol Plant Pathol. 2009;10(2):213–222. https://doi.org/10.1111/j.1364-3703.2008.00525.x Review Article https://doi.org/10.17159/sajs.2020/7688 6 Volume 116| Number 11/12 November/December 2020 Wheat rusts in South Africa Page 7 of 8 Wheat rusts in South Africa Page 7 of 8 37. Wessels E, Prins R, Boshoff WHP, Zurn JD, Acevedo M, Pretorius ZA. Mapping a resistance gene to Puccinia graminis f. sp. tritici in the bread wheat cultivar Matlabas. Plant Dis. 2019;103(9):2337–2344. https://doi.org/10.1094/PDIS- 10-18-1731-RE 57. Prins R, Pretorius ZA, Bender CM, Lehmensiek A. QTL mapping of stripe, leaf and stem rust resistance genes in a Kariega x Avocet S doubled haploid wheat population. Mol Breed. 2011;27(2):259–270. https://doi.org/10.1007/ s11032-010-9428-y 38. Terefe TG, Pretorius ZA, Paul I, Mebalo J, Meyer L, Naicker K. Occurrence and pathogenicity of Puccinia graminis f. sp. tritici on wheat in South Africa during 2007 and 2008. S Afr J Plant Soil. 2010;27(2):163–167. 58. Bender CM, Prins R, Pretorius ZA. Development of a greenhouse screening method for adult plant resistance in wheat to stem rust. Plant Dis. 2016;100(8):1627–1633. https://doi.org/10.1094/PDIS-02-16-0163-RE 39. Johnson R, Stubbs RW, Fuchs E. Nomenclature for physiological races of Puccinia striiformis infecting wheat. Trans Brit Mycol Soc. 1972;58:475–480. https://doi.org/10.1016/S0007-1536(72)80096-2 59. Agenbag GM, Pretorius ZA, Boyd LA, Bender CM, MacCormack R, Prins R. High-resolution mapping and new marker development for adult plant stripe rust resistance QTL in the wheat cultivar Kariega. Mol Breed. 2014;34(4):2005–2020. https://doi.org/10.1007/s11032-014-0158-4 40. Johnson R, Taylor AJ. Yellow rust of wheat. In: Annual report 1976. Cambridge: Plant Breeding Institute;1976. p 106–109. 60. Maree GJ, Prins R, Bender CM, Boshoff WHP, Negussie TG, Pretorius ZA. Phenotyping Kariega x Avocet S doubled haploid lines containing individual and combined adult plant stripe rust resistance loci. Plant Pathol. 2019;68(4):659–668. https://doi.org/10.1111/ppa.12985 41. Wellings CR, McIntosh RA. Puccinia striiformis f. sp. tritici in Australasia: Pathogenic changes during the first 10 years. Plant Pathol. 1990;39(2):316– 325. https://doi.org/10.1111/j.1365-3059.1990.tb02509.x 61. Maree GJ, Prins R, Boyd L, Castelyn HD, Bender CM, Boshoff WHP, et al. Assessing the individual and combined effects of QTL for adult plant stripe rust resistance derived from Cappelle Desprez. Agron. 2019;9(3):154. https://doi.org/10.3390/agronomy9030154 42. Wellings CR, Singh RP, McIntosh RA, Pretorius ZA. The development and application of near isogenic lines for stripe (yellow) rust pathosystem. Paper presented at: 11th Cereal Rusts and Powdery Mildews Conference; 2004 August 23–26; Norwich, UK. p. A1.39. 43. Hovmøller MS, Yahyaoui AH, Milus EA, Justesen AF. References Labuschagne R. Microsatellite analysis of the Puccinia triticina population in South Africa [MSc dissertation]. Bloemfontein: University of the Free State; 2019. 50. Kolmer JA, Ordoñez ME, German S, Morgounov A, Pretorius Z, Visser B, et al. Multilocus genotypes of the wheat leaf rust fungus Puccinia triticina in worldwide regions indicate past and current long distance migration. Phytopathology. 2019;109(8):1453–1463. https://doi.org/10.1094/PHYTO- 10-18-0411-R 68. Millet E, Steffenson B, Sela H, Allen A, Pretorius ZA. Genome targeted introgression of resistance to African stem rust from Aegilops sharonensis into bread wheat. The Plant Genome. 2017;10(3):1–11. https://doi. org/10.3835/plantgenome2017.07.0061 69. Li H, Zheng Q, Pretorius ZA, Li B, Tang D, Li Z. Establishment and characterization of new wheat – Thinopyrum ponticum addition and translocation lines with resistance to Ug99 races. J Gen Genomics. 2016;43(9):573–575. https:// doi.org/10.1016/j.jgg.2016.07.004 51. Pretorius ZA, Ayliffe M, Bowden RL, Boyd LA, DePauw RM, Jin Y, et al. Advances in control of wheat rusts. In: Langridge P, editor. Achieving sustainable cultivation of wheat. Volume 1: Breeding, quality traits, pests and diseases. Cambridge: Burleigh Dodds Science Publishing; 2017. p. 295–343. 52. Soko T, Bender CM, Prins R, Pretorius ZA. Yield loss associated with different levels of stem rust resistance in bread wheat. Plant Dis. 2018;102(12):2531– 2538. https://doi.org/10.1094/PDIS-02-18-0307-RE 70. Li H, Boshoff WHP, Pretorius ZA, Zheng Q, Li B, Li Z. Establishment of wheat – Thinopyrum ponticum translocation lines with resistance to Puccinia graminis f. sp. tritici race Ug99. J Gen Genomics. 2019;46(8):405–407. https://doi. org/10.1016/j.jgg.2019.07.005 53. McIntosh RA, Wellings CR, Park RF. Wheat rusts: An atlas of resistance genes. Dordrecht: Kluwer Academic Publishers; 1995. 71. Pretorius ZA, Lan CX, Prins R, Knight V, McLaren NW, Singh RP, et al. Application of remote sensing to identify adult plant resistance loci to stripe rust in two bread wheat mapping populations. Prec Agric. 2017;18(4):411– 428. https://doi.org/10.1007/s11119-016-9461-x 54. Roelfs AP, Singh RP, Saari EE. Rust diseases of wheat: Concepts and methods of disease management. Mexico DF: CIMMYT; 1992. 55. Pretorius ZA, Pienaar L, Prins R. Greenhouse and field assessment of adult plant resistance to stripe rust of wheat. Austr Plant Pathol. 2007;36(6):552– 559. https://doi.org/10.1071/AP07058 72. Gupta P, Langridge P, Mir R. Marker-assisted wheat breeding: Present status and future possibilities. Mol Breed. 2010;26(2):145–161. https://doi. org/10.1007/s11032-009-9359-7 56. Boshoff WHP, Prins R, De Klerk C, Krattinger SG, Bender CM, Maree GJ, et al. Point inoculation method for measuring adult plant response of wheat to stripe rust infection. Plant Dis. 2019;103(6):1228–1233. https://doi. org/10.1094/PDIS-08-18-1312-RE 73. References Rapid global spread of two aggressive strains of a wheat rust fungus. Mol Ecol. 2008;17(17):3818– 3826. https://doi.org/10.1111/j.1365-294X.2008.03886.x 62. Prins R, Dreisigacker S, Pretorius Z, Van Schalkwyk H, Wessels E, Smit C, et al. Stem rust resistance in a geographically diverse collection of spring wheat lines collected from across Africa. Front Plant Sci. 2016;7:973. https://doi. org/10.3389/fpls.2016.00973 44. Ali S, Gladieux P, Leconte M, Gautier A, Justesen AF, Hovmoller MS, et al. Origin, migration routes and worldwide population genetic structure of the wheat yellow rust pathogen Puccinia striiformis f.sp. tritici. PLoS Pathol. 2014;10(1), e1003903. https://doi.org/10.1371/journal.ppat.1003903 63. Rouse M, Nirmala J, Jin Y, Anderson JA, Chao S, Fetch TG, et al. Characterization of Sr9h, a wheat stem rust resistance allele effective to Ug99. Theor Appl Genet. 2014;127(8):1681–1688. https://doi.org/10.1007/ s00122-014-2330-y 45. Boshoff WHP, Visser B, Lewis CM, Adams TM, Saunders DGO, Terefe T, et al. First report of Puccinia striiformis on wheat in Zimbabwe. Plant Dis. 2020;104(1):290. https://doi.org/10.1094/PDIS-07-19-1395-PDN 64. Pretorius ZA, Prins R, Malaker PK, Barma MCD, Hakim MA, Thapa D, et al. Assessing the vulnerability of wheat germplasm from Bangladesh and Nepal to African stem rust. Phytoparasitica. 2015;43(5):637–645. https://doi. org/10.1007/s12600-015-0487-9 46. Visser B, Meyer M, Park R, Gilligan C, Burgin LE, Hort MC, et al. Microsatellite analysis and urediniospore dispersal simulations support the movement of Puccinia graminis f. sp. tritici from southern Africa to Australia. Phytopathology. 2019;109(1):133–144 https://doi.org/10.1094/PHYTO-04-18-0110-R 65. Babiker EM, Gordon TC, Chao S, Rouse MN, Wanyera R, Newcomb, M, et al. Genetic mapping of resistance to the Ug99 race group of Puccinia graminis f. sp tritici in a spring wheat landrace CItr 4311. Theor Appl Genet. 2016;129(11):2161–2170. https://doi.org/10.1007/s00122-016-2764-5 47. Lewis CM, Persoons A, Bebber DP, Kigathi RN, Maintz J, Findlay K, et al. Potential for re-emergence of wheat stem rust in the UK. Comm Biol. 2018;1:13 https://doi.org/10.1038/s42003-018-0013-y 66. Zurn J, Rouse M, Chao S, Aoun M, Macharia G, Hiebert C, et al. Dissection of the multigenic wheat stem rust resistance present in the Montenegrin spring wheat accession PI 362698. BMC Genomics. 2018;19:67. https://doi. org/10.1186/s12864-018-4438-y 48. Li F, Upadhyaya NM, Sperschneider J, Matny O, Nguyen-Phuc H, Mago R, et al. Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation. Nat Comm. 2019;10:5068. https://doi.org/10.1038/ s41467-019-12927-7 67. Olivera PD, Pretorius ZA, Badebo A, Jin Y. Identification of resistance to races of Puccinia graminis f. sp. tritici with broad virulence in triticale (X Triticosecale). Plant Dis. 2013;97(4):479–484. https://doi.org/10.1094/ PDIS-05-12-0459-RE 49. Review Article https://doi.org/10.17159/sajs.2020/7688 79. Pretorius ZA, Bender CM, Visser B. The rusts of wild rye in South Africa. S Afr J Bot. 2015;96(1):94–98. https://doi.org/10.1016/j.sajb.2014.10.005 References Prins R, Groenewald JZ, Marais GF, Snape JW, Koebner RMD. AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat. Theor Appl Genet. 2001;103(4):618–624. https://doi.org/10.1007/PL00002918 7 Review Article https://doi.org/10.17159/sajs.2020/7688 Volume 116| Number 11/12 November/December 2020 Wheat rusts in South Africa Page 8 of 8 Wheat rusts in South Africa Page 8 of 8 74. Leng P, Lübberstedt T, Xu M. Genomics-assisted breeding – A revolutionary strategy for crop improvement. J Integr Agric. 2017;16:2674–2685. https:// doi.org/10.1016/S2095-3119(17)61813-6 77. Ali S, Rodriguez-Algaba J, Thach T, Sørensen CK, Hansen JG, Lassen P, et al. Yellow rust epidemics worldwide were caused by pathogen races from divergent genetic lineages. Front Plant Sci. 2017;8:1057. https://doi. org/10.3389/fpls.2017.01057 75. Crossa J, De Los Campos G, Pérez P, Gianola D, Burgueño J, Araus JL, et al. Prediction of genetic values of quantitative traits in plant breeding using pedigree and molecular markers. Genetics. 2010;186(2):713–724. https:// doi.org/10.1534/genetics.110.118521 78. Olivera PD, Sikharulidze Z, Dumbadze R, Szabo LJ, Newcomb M, Natsarishvili K, et al. Presence of a sexual population of Puccinia graminis f. sp. tritici in Georgia provides a hotspot for genotypic and phenotypic diversity. Phytopathology. 2019;109(12):2152–2160. https://doi.org/10.1094/PHYTO- 06-19-0186-R 78. Olivera PD, Sikharulidze Z, Dumbadze R, Szabo LJ, Newcomb M, Natsarishvili K, et al. Presence of a sexual population of Puccinia graminis f. sp. tritici in Georgia provides a hotspot for genotypic and phenotypic diversity. Phytopathology. 2019;109(12):2152–2160. https://doi.org/10.1094/PHYTO- 06-19-0186-R 78. Olivera PD, Sikharulidze Z, Dumbadze R, Szabo LJ, Newcomb M, Natsarishvili K, et al. Presence of a sexual population of Puccinia graminis f. sp. tritici in Georgia provides a hotspot for genotypic and phenotypic diversity. Phytopathology. 2019;109(12):2152–2160. https://doi.org/10.1094/PHYTO- 06-19-0186-R 76. Poland J, Endelman J, Dawson J, Rutkoski J, Wu S, Manes Y, et al. Genomic selection in wheat breeding using genotyping-by-sequencing. The Plant Genome. 2012;5(3):103–113. https://doi.org/10.3835/plantgenome2012.06.0006 79. Pretorius ZA, Bender CM, Visser B. The rusts of wild rye in South Africa. S Afr J Bot. 2015;96(1):94–98. https://doi.org/10.1016/j.sajb.2014.10.005 79. Pretorius ZA, Bender CM, Visser B. The rusts of wild rye in South Africa. S Afr J Bot. 2015;96(1):94–98. https://doi.org/10.1016/j.sajb.2014.10.005 79. Pretorius ZA, Bender CM, Visser B. The rusts of wild rye in South Africa. S Afr J Bot. 2015;96(1):94–98. https://doi.org/10.1016/j.sajb.2014.10.005 Volume 116| Number 11/12 November/December 2020 8 Volume 116| Number 11/12 November/December 2020 Review Article https://doi.org/10.17159/sajs.2020/7688 Review Article https://doi.org/10.17159/sajs.2020/7688 Review Article https://doi.org/10.17159/sajs.2020/7688
W2323523459.txt
https://zenodo.org/records/2030262/files/article.pdf
de
Kolloidchemische Studien über die Keimwirkung
Colloid and polymer science/Colloid & polymer science
1,921
public-domain
3,319
254 war durch die konstatierte Beziehung zwischen der Stiirke der Siiuren und ihren • inbezug auf Wolle der Beweis erbracht, d a fi A dsorptionen fiberhaupt durch chemische Affinitiiten bedingt werden. Dieser ScbluB hat soweit ailgemeii~e Gfiltigkeit, insolange nicht etwa gefunden werden sollte, dab die SOrption von Siiuren dutch Wolle kein Sorptions- (bzw. Adsorptions-) vorgang ist a). Lundelius sagt weiteres auf So 150" ,Untersuchungen i i b e r die Abhlingigkeit der Adsorption v o n d e r . Natt.,r des festen St0ffes h a b e n gezeigl, dab sie nur gering ist. Stoffe wie: Kohle, Wolle, Seide, Baumwolle zeigen wohl eine absolut gerechnet sehr verschiedime Adsorption, wahrend die relative Menge immer gleich istl" Die Unrichtigkeit dieser Regel ist Ftirbereiehemikern schon lange bekannL Sie gilt aber auch nicht fiir die Aufnahme yon farblosen S~uren durch Wolle und Kohle, worauf ich schon in meiner ersten Abhandlung d e r . Studien fiber Adsorption in L6sungen~0/ in Ge~neinschaft mit A. P o l l a k hingewiesen hatte. Auf S. 149 spricbt ferner L u n d e l i u s die Vermutung aus, daft die adsorbierten Mengen eines Stoffes yon der Temperatur nur wenig abhgmgig sein dfirftenl Auch diese Annahme ist insofern nieht ganz ricbtig, weft sie in m~nchen Fallen nicht-zut~ifft, Es.zeigt sich dies namenflieh in dem Verhalten yon Farbstoffen gegen Wolle undSeideT), wobei die Regel gilt, dat~ bei niedriger Temperatur die Affinitiit f/Jr Seide grSfier a|s f/Jr Wolle ist, wahrend bei hSherer Temperatur der Farbstoff haupts~tchlich yon der Wollfaser aufgenommen wird. Abet-au, ch bei Kohle zeigt sich in ihrer mannigfaltigen ArtWendung zum Entfiirben yon L6sungen eine deutliche Abh~ingigkeit der Sorption yon der Temperatur. Endlich sei noch konstatiert, dab Sorption und LSslichkeit sicher nicht immer ~im umgekehrten Verh~itnis zueinander stehen" ( L u n d e l i u s S. 147). Denn- meine Fiirbeve~suche mit den Azofarbstoffen aus ~-Naphthol und den a Naphthylaminsulfosiiurena) hatten ergeben, dag die aus 1,4 und 1,5 Naphthylaminsulfos[iuren erzeugten Farbstoffe, welche sowohl im Wasser wie auch in verdtinnter Essigsiiure leichter 16s. Itch als ihre lsomeren sind, yon Wolle (aus essigsaurem Bad) in viel grSt~eren Mengen als diese aufgenommen werden. 5) Siehe diesbezfiglich auch Koll:Zeitsehr. 14, 7) Ioc. r 191i, 479. 8) G. O e o r g i e v i c s u. L. S p r i n g e r , Monatsh. f. Chem. 1900, 626. 72]73 (1914). ~) loc. cit. 1911, 478'79. Prag (BOhmen). Deutsche tedmische Hochschule. Kolloidchemische Studien iiber die Reimwirkung. Yorl[iufige Von Mitteilung. F. S e k e r a Die AuslSsung iibers~ttigter LSsungen, unterkfihlter Schmetzen und dergleichen durch Keimwirkung wurde vielfach in den Rahmen katalytischer Betrachtungen einbezogen. Erst in letzter Zeit, da man einen, wenn auch vorderhand nur oberflfichlichen Einblick in den Mechanismus der heterogenen Katalyse gewinnen konnte, zeig~en sich ~in vieler Hinsicht Verschiedenheiten zwischen den beiden Klassen von Erscheinungen, so dafl sch!iet~lich das Problem der Keimwirkung als ,,terra incognita" yore Gesichtsfeld der katalytischen Forschung abgetrennt wurde. Die Frage, ob es sich hier um katalytische Erscheinungen handelt oder nieht, muB wohl so lange often bleiben so lange der Mechanismus. der Keimwirkung unaufgekl/irt ist. (W~en). (~ing~ng~n a,n t. Vet,r,a~r l ~ t . ) Es schien nun ein aussichtsreiches Beginnen dem Problem der Keimwirkung vom Standpunkt der Kolloidchemie hither zu treten, um so mehr, als sich damit gleichzeitig die Gelegenheit erg a b , in die Natur des Orenzgebietes kolloider und molekularer Dimensionen tiefer einzudringen ~). I. U e b e r s i i t t i g t e LSsungon. Zuwiederholten Malen 2) wurde der Zustand der Ueber............ 1) Die folgenden Dar!egungen sind das TeilergebhiS ether noch im Oange befindlichen Arbeit fiber ,,Die M o r p h o l o g i e der N i e d e r s c h l a g s f o r m e n". Die experimentellen Daten werden im l~ahmen dieser Arbeit verOffentlicht werden; hier beschr~nke ich reich darauf, meine Anschauungen fiber die Keimwirkun~ zur Diskussiou zu stellen. t) ~3iehe insbesondere die zahlreichen Arbeiten P. P. v. Wet m arn's in dieser Zeitschrift. 255 s~ttigung einer L0sung Ms der Uebergang vom molekular- zum koltoiddlspersen System gekennzeichnet. Schon in konzentrierten L6sungen beginnen die L6sungsnlolekiile zu'Komp|exen zusammenzutreten, die an Or6ge um so mehr zunehmen, je .hS-her die Lfsungskonzentrationen sind. Nach den Anschauungen, zu denen uns die Laue'schen Photogramme geliihrt haben, diirften auch schon diese em.bryonalen Formen einer stoffllchen Aggregation an gewisse Oesetzm.~fligkeiten gebunden sr Das Raumgitter, das im festen K6rper zu voUkommener Starrheit entwickelt ist, wird .man sich auch bet den Molek/ilkomplexen einer konzentrierten LOsung vorstellen kOnnen, nur mit dem Unterschied, da~ die Orientierung noch nicht voUk0mmen is[, und auch die Starrheit noch nicht [enen Grad erreicht hat, wie sic sich im Raumgitter des festen Teilehens erketinen liiBt. Der Austausch der L/$sungsmolekfile an der enorm entwickelten spezifischeri Oberfliiche dieser Molekfilkomplexe~ sowie das auf dieselbe Oberfl~iche einwirkende Bombardement der LSsungsmolekfile lassen diese Vorstellungen berechtigt erscheinen. Je gr~Ber die Mo!ekfilkomplexe werden (d. h. ie konzentrierter die LOsung.ist), urn so widerstandsfiihiger wird ihre Oberfliiche den Einwirkungen yon au6en, um so ungestSrter und starter wird das"Raumgitter, his end[ich, sobald kolloide Dimensionen erreicht sird, die. Raumgitterstrukturexperimentel.I eJ-kennbar wird. An' der Hand dieser Vorstellungen dfirfte die Kontinuit~t zwischen Molekii! und Kotloid am klarsten sein. Die eben betrachtete TeilchenvergrOgerung erleidet nun, wie es nach den bisher fiblichen Anschauungen-~tiber die LOslichkeit der Stoffe den Anschein hatte, irgendeine unvermittelte St~rung. Sobald der ,LOslichkeitsgrad ~ des vorliegenden Stoffes erreicht ist, fiihrt iede weitere KonzentrationserhtShungzur Bildun g ether zweiten Phase, die wohl auch den Weg der Dispersit~,tsverminderung ei.nschliigt, aber in ganz anderer Weise, als er bis zumL6stichkeitspunkt geffihrt hat. Inwieweit und w.arum dieser Knick in der Dispersitiitsverminderung eintritt, soil sptiter erOrtert werden. Hier interessieren wir uns besonders daffir, da6 es unter Wahrung besonderer VorsichtsmaBregeln gelingt fiber diesen kritischen Punkt hinwegzukommen und die Verringerung des Dispersitiitsgrades weiterzutreiben, ohne die Bildung ether zweitenPhase zu beobachten. Man erreicht dann den Zustand der ,Uebers.;ittigung" und lernt diesen Zustand als einen unstabilen Zustand kenrien Tats/ichlich geniigen auch die bescheidensten Unregelm[it3igkeiten an den GefiiBwiinden, Staubteilchen usw, um das Zustandekommen einer Uebersiittigung zu verhindern. Besonderes Interesse aber land das Phiinomen der Ausl6sung einer Uebersiittlgung dutch die Berfihrung mit einem festen Keim des gel6steu Stoffes. Unwiigbare a) Mengen desselben genfigen, um eine Uebers~tiigung aufzuheben und eine dem L6slichkeitsgrad entsprechende Trennung in gesattigte L~sung und Bodenk0rper herbeizuffihren. Fiir das Verst~indnis dieser Erscheinung fehlte bisher iede Orund|age, um so mehr als sich die Vorg~nge einer quantitativen Untersuchung kaum zugiinglich erwiesen. Der diskrete Zusammenhang zwischen Keim und dem durch ihn a~s'gel6sten Vorgang brachte es mit sich, eine katalytische Erscheinung zu vermuten. Erst glaubte man, die Uebers~ttigung, da" sie sich als eine verz0gerte Bildung der heterogenen Phase zeigt, auf einen Widerstand zurfickffihren zu k6nnen, der sich denkristallbauenden Kriiften entgegenstellt. Dieser an und ffir sieh recht subiektive Erkl~irungsversuch versagt abet vo|lkommen bet den fibers~ttigten L0sungen yon Gasen, bet denen an einen derartigen Widerstand nicht gedacht werden kann. Welchen Weg hat nun ein kolloidchemischer Erkl~irungsversuch zu gehen? ~ Zwei Falle sind zu berficksichtigen: Die Aufhebung ether Uebersiittigung dutch einen kfinstliChen Keim und die langsame, yon selbst verlaufende Entmischung, die den fibersattigten L6sungen die Eigenschaft der Unbest.~indigkeit aufpragt. S c h o n eingangs wurde eine fibersattigte L~sung als ein.sehr hochdisperses System charakterisiert, desseu disperse Phase aus den blolekfilkomplexen des ge16sten Stoffes besteht. In diese L0sung wird nun ein KristaU des festen Stoffes eingetragen; es besteht dann neben einei aut~erst hochdispersen Phase eine grobdisperse desselben Stoffes. Ein solcher Unterschied des Dispersitatsgrades bedingt naturgernaB entsprechende Unterschiede in den physikalischen Eigenschaften der beiden Phasen. Beim Studium der iibersatJigten LOsungen war es wohl das naheliegendste, die Dispersit|itsfunkti0n der L 6 s I i c h k ei t zu berficksichtigen. Bekanntlich ist der LSslichkeitsdruck fiber e!ner wenig gekriimmten Oberflache geringer als fiber ether stark gekrfimmten ; m. a. W. kieine Teilchen~sind 18slicher als grot~e. Diese Abhangigkeit der LOslichkeit yore Dispersitatsgrad ist im Gebiete grober Dispersitlit kaum merkbar, wird bet fortschreitend a) Siehe Nachtrag. 256 abnehme/ader kolloider TeilchengrSfle immer deutlieher, urn im Grenzgebiet kolloider und molekularer Dimensionen eine Hauptrolle bei allen Vorgiingen, die sich in diesem DimensiOns bereich abspielen, zu ttbernehmen, tn der beigefiigten Skizze "ist diese Funktion schematisch dargestellt. Die L0slichkeitskurve steigt im Bereiche grober Dispersitat nut allm~,hl.ich an u.nd wird:im Gebiete der kolloiden..Dimensionen zunehmend steiler, urn schliefilich asymptotisch zu enden. Uebertragen wir nun dieses Bild auf unsere iibersiittigte L6sung. Das Verhii/tnis der Molekiilkomplexe zurn etngetragenen Keim sei durch die Punkte A und B d~r Kurve .fes'cgel~egt, wobei die Ordinaten dieser Punkte die Menge yon L S s u n g s m o l e k i i l e n vorstelien, mit" denen der Keim einerseits und die Molekiitkomplexe andrerseits im Gleichgewicht stehen. B s Fig. 1 Es ergibt sich nun als selbstverstandlieh, dab zwischen den beiden Phasen mit verschiedener LSsltchkeit, da sic sich im selben LSsungsmittel befinden, ein Ausgleich erfolgen wird. So langr Teflchen vorhanden sind, die mit mehr L 6 s u n g sm o l e k f i l e n im G|eichgewicht stehen als tier Keim, wird letzterer dieses Plus an MOleklilen in sein Raumgitter aufnehmen und damit so lange wachsen, bis die kleinen Teilehen (die Molekiilkomplexe) aufget6st sind. Die Uebersiittigung ist aufgehohen, sobald yon den Mcdekiiien, die friiher mit derr Molekiilkomplexen im Gleichge~vicht gestanden hatten, nur so vieie iibrig sind, als der LOslichkeit des Keimes entspricht 4). 4) !m-Prinzip ist diese Anschauung durehaas nicht neu. Der Vorgang des Waehsens grol~ererTeilchen auf Kosten der kleineren ist als O s t w a I d'scher Reiftmgsprozel3 vielfach beschrieben worden (Z- B. LfappoCramer u:R. E. t.iesegang, P.P.v.Weimarn usw.). Die vorliegende Arbeit gelangte blo6 zu dem Befund. dal~ dasselbe Oesetz auch fiir Molektilkomplexe Oeltung hat. Vielfach ist nun eine beabsichtigte Aufhebung eiher UebersAtt/gung~durch einen kiinstlichen Keim gar nicht nStig. Es geniigen ja die beschetdensten Konzentrationsunterschiede innerhalb des L6sungsvolumens, um einen, wenn auch sehr geringen Unterschied des Assoziationsgrades tiervorzurufen. Da ist die Begrenzung des L6sungsvolumens, insbesondere d'er Meniskusrand und unvermeidliche Unebenheiten der GefliBw~nde der Sitz yon Oberfl~ichenspannungen, die dutch Adsorption lokale. Konzentrationsverschiebungen zur Folge haben. Es bestehen dann gr6flere Molektilkomplexe veben kleineren, yon denen die gr0ileren die Roile des kfinstlichen Keimes you vorhin tragen. Der iangsame Verlauf tines solchen Ausl6sungsvorglinges ist nun durchaus begreiflich, wenn man bedenkt, daft der L6slichkeitsunterschied zwischen den verschieden groflen Motekiilkomplexen ein sehr geringer ist und es sehr lange Zeit braucht, bis der Motekfilaustausch den L6slichkeitsausgleich besorgt hat. Es liegt in dieser Ansch~uung ein scheinbarer Widerspruch : beim L6slichkeitsausgleich zwischen zwei verschieden groflen Molektilkomplexen beteiligen sich. ungleich mehr L6sungsmolekfile als im FaUe eines grobdispersen Keimes; .es miiflte sich also der L~s]ichkeitsausgleich SchneUer votlziehen als i:m letzteren Falle. DieserWiderspruch ~llt abet s0fort weg, wenn mart sich nur vergegenw~irtigt, daB im Verlaufe des Aus.16sungsvorganges der gr6flere Molektilkomptex in der Dispersit~ttsgradserie fortlauferld dem grobdispersen Ende entgegenstrebt und auf diesem ganzen Weg als Keim wirkt. Einem kiinstlichen,-yon Haus aus grobdispersen Keim bleibt dagegen dieser Weg erspart. Schon aus diesen~ Ueberlegungen geht hervor, dab sich das gesamte Problem der Keimwtrkung einzig mit z w e i L 6 s u n g s g l e i c h gewichten zu befassen hat, die durch das Schema " Keim ~ _ C1 L6sungsmolekfile/~. Molekiiikomplex ~ Ca , ~t':~::>tSl dargestellt sind. So lange C~ > C1 ist, werden die Molekiilkomplexe der iibers~itigten L6sung abgebaut, wfihrend das Plus an LOsungsmolekftlen (C_~-C1) yore Keim aufgenommen wird. Welche Bedeutung hat nun der , Ue b e rs iittigun-gsgrad?" dedenfaIls handelt es slch um einen r e l a t i v e n Begriff. Aus dem vorher'ehenden ist der Uebers~ittigungsgrad e.mer vorliegenden L6sung einem grobdispersen Keim teegen/iber gr6ger als einem hochdispersen Keim gegeniiber, da bei gleichem C2 257 this C1 im letzteren Fall gr6fier ist. Jecler Angabe des Uebers~ittigungsgrades sollte also.die Angabe des Dispersit~tsgrades des Keimes beigeschlossen sein. lm allgemeinen verzichtet man wohl auf diese notwendige Erg,~inzung deshalb, well d~e Uebe~'~ttigung in bezug auf die grobdisperse Phase gemein! wird: Weitergehend kommen wit zu dergrunds~ifzlichen Erkenntnis, daft wir eine/~bers~ittigte L6sung als eine ges~Ittigte LOsung anzusehen haben, deren ,feste Phase'. die Molekfilkomplexe sind, und die eben mit einer viel gr6fieren Menge yon L~sungsmo]ekiilen im Qleichgewicht stehen, als eine grobdisperse Phase es rut. Wenn wir also eine ilberS/ittigte L~)sung als ein unstabiles System ansehen, so ist dies unrichtig. K6nnte man die unvermeidliehen Ursachen einet lokalen Konzentrationsverschiedenheit innerhalb des LOsungsvolumens ausschalten, so h~tte man eine ebenso stabile L6sung vor sich, wie es jede ges~ttigte L6sung ist. Zur ~bersiittigten L6sung wird eine solche hochkonzentrierte L~5sung erst durch die Existenz grober disperser und daher weniger 16slicher Teilchen. .Was nun die Versuche betrifft, die zu den hier entvcickelten Anschauungen geffihrt haben und die ausffihrlich an anderer Stel~e ver6ffentlicht werden sol[en, so zeigte sich mit voUster Klarheit die Bedeutung der L6slichkeit des ge16sten Stoffes, wie sie in der Theorie zum Ausdrttck k0mmt. Bei leicht 16slichen Stoffen ist die Keimwirkung spontan, da sich eine groi~e Menge yon L6sungsmolekfile~ am LOslichkeitsausgleich zwischen Keim und den Molekfilkomplexen beteiligt. Bei schwer 16slichen Stoffen dagegen ist die Keimwirkung sehr tr/ige, so daft man Zeit genug hat, um den Vorgang zeitlich zu verfolgen. Mit Hilfe einer geeigneten Apparatur ist es auch gelungen, die Bildung con Niederschlggen und die damit verquickten Fragen der Keimwlrkung zu studieren. von den Uebersilttigungserscbeintmgcgt, die dem Experimentator ja auf Schritt und Tritt begegnen, sei noch der sogenann~e , S i e d e v e r z u g ". erwiihnt, dessen unangenehme Folgen schon oft zum Nachdenken fiber die Ursachen angeregt haben mag, ohne daft eine Erklarung gefund,en werden, konnte. Aut/ empirisehem Wege hat man den Siedeverzag dadurch umgehen k6nnen, indem man entweder.andauernd Luftblasen in die erhitzte Fliissigkeit eintreten I.~fit, oder mit Hilfe eines in das GerM5 ein 2 geschmolzenen Ptatindrahts eine lokal konzentrierte W~irmezufuhr er~eicht. Die Wirk'ungsweise dieser beiden Kunstgriffe wird nun sofort yerst~indlich, wenn man sich vorstellt, daft das beim Verdampfen der.Plfissigkeit gebildete Gas primlir in derselben gelt~st wird und auch da Uebersitttigungen im fiblichen Sinne auftreten k6nnen: Diese Uebersattigungen werden dann durch die als Keime wirksamen eingeleiteten Gasblasen bzw. dutch den infolge der grol~en Ueberhitzung mit tier Dampfhfille umkteideten Platindraht aufgehoben. lI. U n t e r k f i h l t e S c h m e l z e n . Dieselben Gestchtspunkte, die fiir die Erk|tirung der Aus16sung fibersattigter LOsungen mat~gebend warerr, geiten auch f/it u n t - e r k f i h l t e S c h m e l z e n . Da wie dort handelt es sich um hochdisperse Systeme mit Molekillkomplexen als disperse Phase. Die Rolle, die vorhin die LSslichkeit gespielt hat, wird im jetztgen Falle yon. der S c h m elz b a rkeit fibernommen. Die Abh~aagigkeit des Schmelzpunktes yore Dispersitiitsgrad ergibt eine. der L~Sslichkeitsfunktion voltkommen analoge Kurve, so dat~ es kaum schwer fallen wird, sich den Vorgang vorzustellen, der sich zwischen Keim und den Molekfilkomplexen einer unterklihlten Schmelze abspielt. I[I. Die F r a g e d e r K a t a l y s e . Auf Gru~d der angestellten Ueberlegungen fiber den Mechanismus der Keimwirkung kann nun die Frage beantwortet werden, die anfangs often bleiben mutate: Handelt es sich urn katalytisehe Vorgange oder nicht?. Nach dem, was wir gegenwitrtig v o n d e r Katalyse wissen, ist sie vor allem dadurch gekennzeiehnet, dab der Katatysator wohl eine bestehende Reaktion zeitlich zu beeinflussen vermag, abet niemals imstande ist, eine Reakfion hervorzurufen. Welter betitigt sich eirrKatalysator e ~ e r g e t i s e h an der Reaktton, indem er entweder ein Zwischenprodukt aufbaut, das durch seinen Zerfall schneller zum Endprodukt fflhrt als die nicht katalysierte Reaktion (homogene Katalyse) oder unter Zuhilfenahme der Oherflachenenergien auf ahnliche Weise wirkt, wie es bei der heterogenen Katalyse der Fall ist. Rile diese Kennzeiehen fehlen der Keimwirkung. Wit sind 2u der Erkenntnis gelangt; daft fiber~i"ttigte L6sungen nichts anderes sind als gesiittigte LSsungen, deren ,Bodenk6rper" die Molekfilkomplexe sind und die sieher keine Tendenz zeigen wt'irden, ihren Zustand zu verAndern, wean nicht unvermeidliche Keimwirkungen grOt~erer Molekfilkomplexe mitspielen wf|rden. Es ist also gar ketn Vorgang.da, der katalysiert werden k6nnte. Der Keim wirkt welter nicht energetisch, wie die vorhin be- 258 zeichneten F~ille, sondern sehafft dutch sein~ Anwesenhait sozusagen ein Reservoir ffir jenes Plus an "Liivungsmolekiilen, mit denen er ins seiner geringeren LOslichkeit nicht im Gleichgewicbt zu stehen yermag. Es handelt sich einzig um das Zustreben auf ein Li~sungsgleich-gewieht mit einer hinzugekomrr~nen Phase. - Dies sind die Hauptgrfin6e, .die maggebend erscheinenj der Keimwirkung den vermeintlichen Katalysencharakter abzusprechen. Wien. On Dezember 1920. Tedanische Hochsehule. Nachtrag. Dutch etne briefliche Aussprache mit Herrn Prof. Wo. O s t w a l d wurde ieh angeregt, auf folgendes niiher einzugehen. W i I h e I m O s tw a I d 5) konnte experimentell den Beweis ftihren, dag zur Aufltebung einer Ueberstittigung, wenn auch sehr .geringe, so doch gewisse Mengen Keinisubstanz n6tig sind. lch versuchte naturgemag im Rahmen meiner Arbeiten dieses Ergebnis 2u .reprodazieren uhd kam zu dem Resuliat, dab diese Bedingung eines Keimminimums wohl bei L6sungen leicht 15slicher Stoffe und auch dann nur im ,stark fiberstittigten"~ experimentell festgesteltt werd~n kann, nicht aber bei/ibersiittigten LSsungen schwer 16slicher Stoffe oder in allen Fallen bei geringem Uebers~ittigungsgrad. Wenn e8 mix aueh nfcht gelungen ist, diesem Umstand quantitafiv n~iher zu kommen; so m~chte ieh iolgende Deutung in Betracht ziehen. Die Ltistichkeit eines Stoffes ist nicht 5) Wilhelm Ostwatd, Lehrbuch der all~,emeinen Chemie. nur vom Dispersittitsgrad alleiu abhtlngig, sondern auch yon der Form des Ke~mteilchen~. Es kommt sehr viel darauf an, ob ein unbesch~idigter Kristall oder Bruchsgicke eines Kristalles als Keimewirken. Unter derVoraussetzung, dab in beiden F~iilen gleiche Gewichtsmengen Keimsubstanz zur Anwendung gelangen, ist die L6slichkeit der KristaUbruchstiicke wesentlich gr6ger als die des unbeschiidigten Kristalles, da die ersteren vor aUem Trehr und auch schiiffere Kanten besitzen. Und die Kanten sind doch der Sitz der hSheren L0slichkeit. Wi. O s t w a I d hat nun zur Feststellung des Keimminimums offensichtlich Mahiprodukte, also Kristalltr/iramer verwendet. Da ka n n es nun eintreten, daft beim Eintragen eines solchen Bruchstfickes in eine stark fibersiittigte L6sung diese gegeniiber dem Keim gar nicht fibers~ttigt ist. Es steht vielmehr der Keim infolge seiner grogen L6slichkeit mit mehr L~Ssungsmolekiilen im Oleichgewicht als die MolekQlkomplexe. Das Verhiiltnis zwischen Keim und Molekfilkomplexen ist dana gerade umgekehrt als es in der Regel der Fall ist. Die Folge: die Molekiilkomplexe zehren den Keim auf und die hohe LOsungskonzentration bleibt bestehen. In ~weniger i~bersiit~tigten" L6sungen wird man dieses abfiormale Verh~hnis zwisehen Keim und Molekfilkomplexen nichl beobachten k6nnen, well die Motekfilkomplexe immer kleiner sein Werden als ein w~igbarer Keim. In iibers~ittigten L6sungen schwer 16slicher Stoffe ist die Diffusionsbewegung so tr~ige, daft derartige Versuche unmSglich sind.. Wien, im Januar 1921. Technische Hodzschule. (Jeber die Lichtempfindlkhkeit des :Rrsentrisuifidsols, Von H. F r e u n d l i c h a n d A. N a t h a n s o h n . Dem einen yon uns (N.)schien es im Hinblick auf biologische Pragen wichtig ~zu sein, photoehemische Vorg~inge in kollolden Gebilden zu untersuchen..Wir gingen deshalb yon der Beobachtung des anderen (FrX aus, dab das AS2Sa-Sol offenbar stark lichtempfindlich ist; ihm war es schon vor Jahren aufgefalleh, dag sich vielfach ein A% Sa-Sol, alas sich im Dankeln lange klar gehalten hatte, am Tageslicht trtibtel). Es wutde deshalb eine Reihe yon Versuchen derart angestellt, dag das Sol in Reagenz1) $iehe auch H. F r e u n d l i c h u. H Schuch,t, Zelt~r f. ph~ik. Chem, 80, 5,65 (1912). (lgingegaugenam 9. Mlirz |921.) gltisern durch eine 3000-HK-Nitralampe, i n 30 .cm Abstand, unter Dazwischenschaltung eines Wassertroges beleuchtet wurde. S.chon nach etwa zehn Minuten zeigte sich eine Triibung des vorher' im durchfallenden Lichte ganz klaren S01s, und bei ltingerem, stundenlangem Beleuchten schied sich aus dem Sol Sehwefel ats :fein'es, weiBes Pulver ab. D~e zu den Versuchen benutzten Sole wurden f01gendermaflen bereitet : 40 -- 100 ccm eil~er kaltgestittigten L6sung yon Arsenkioxyd w e r d e n ' a u / 2 0 0 ccm verdiinnt and mit 100 ccm einer H~S ' Lssung, die 1 ccm ges~ittigtenSchwefel-
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to the classification and identification of Weinmannia (Cunoniaceae) in Madagascar and the Comoro Islands
Zenodo (CERN European Organization for Nuclear Research)
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MOTS CLÉS Weinmannia, Cunoniaceae, cladistique, classification, clés taxonomiques, inflorescence, Madagascar, Comores. KEY WORDS Weinmannia, Cunoniaceae, cladistics, classification, taxonomic keys, inflorescence, Madagascar, Comoro Islands. RÉSUMÉ Application d’une analyse cladistique à la classification et l’identification des Weinmannia (Cunoniaceae) à Madagascar et aux Comores. g Les espèces malgaches de Weinmannia sont actuellement difficiles à identifier avec les publications disponibles en raison à la fois de la pauvreté des caractères qualificatifs utilisés dans les clés d’identification et d’un nombre élevé d’espèces non décrites. Cependant, des caractères morphologiques nouvellement recon- nus, en particulier ceux de l’inflorescence, peuvent êre utilisés pour les dia- gnoses des sections et des groupes d’espèces malgaches. Ces caractères, employés précédemment pour une analyse cladistique du genre Weinmannia (BRADFORD 1998) sont appliqués ici dans les diagnoses et une clé de détermi- nation des sections et groupes d’espèces de Weinmannia à Madagascar. ADANSONIA, sér. 3 • 2001 • 23 (2) : 237-246 © Publications Scientifiques du Muséum national d’Histoire naturelle, Paris. The application of a cladistic analysis to the classification and identification of Weinmannia (Cunoniaceae) in Madagascar and the Comoro Islands Jason C. BRADFORD Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, U.S.A. bradford@mobot.org ABSTRACT Weinmannia species from Madagascar are currently difficult to identify with available published accounts due to both a paucity of qualitative characters used in identification keys, and a large number of undescribed species. However, newly recognized morphological characters, especially of the inflo- rescence, can be used to diagnose Malagasy sect. and species-groups. These characters, used previously for a comprehensive cladistic analysis of the genus Weinmannia (BRADFORD 1998), are applied here in diagnoses and a key to the resulting sections and species-groups of Weinmannia in Madagascar. ADANSONIA, sér. 3 • 2001 • 23 (2) : 237-246 © P bli ti S i tifi d M é ti l INTRODUCTION BARNES 2001), is represented in Madagascar solely by its largest genus, Weinmannia L., with c. 150 species worldwide. Most Weinmannia species are trees and shrubs of tropical montane BARNES 2001), is represented in Madagascar solely by its largest genus, Weinmannia L., with c. 150 species worldwide. Most Weinmannia species are trees and shrubs of tropical montane Cunoniaceae, a southern hemisphere family with 26 genera and c. 300 species (BRADFORD & 237 Bradford J.C. forests, although tropical lowland species occur in Madagascar, and temperate species in Chile, Argentina, Australia, New Zealand and South Africa. Cunonia L. and Platylophus D. Don, from South Africa, are the only African members of the family. As exemplified by the number of species found during field work at Marojejy (BRADFORD & MILLER 2001), Madagascar is one of the most species-dense areas in the world for Weinmannia, with levels of sympatry similar to that found in Andean forests. Unlike other parts of the world, however, the species richness of Malagasy Weinmannia is accompanied by a diversity of morphological characters that enable relatively rapid identification of species. y Cunoniaceae have been the subject of recent phylogenetic and revisionary investigations, with most attention given thus far to Weinmannia (BRADFORD 1998; HOPKINS 1998a, 1998b, 1998c; HOPKINS & FLORENCE 1998). Based on cladistic studies (discussed below), the infra- generic classification of BERNARDI (1961, 1964) and the placement of some species has been slightly modified to make sections mono- phyletic. BRADFORD (1998) recognized five sec- tions of Weinmannia that circumscribe species from the Americas plus two species from the Mascarenes (sect. Weinmannia), Malesia and Melanesia (sect. Fasciculata Bernardi), the South Pacific (sect. Leiospermum D. Don), and Madagascar and the Comores (sect. Spicata Bernardi and Inspersa Bernardi; see also HOPKINS 1998a). Taxonomic changes affect a few Malagasy species. p p Study of available herbarium specimens and field work indicate that 35-40 Weinmannia species occur in Madagascar, 15-20 more than were known to BERNARDI (1965, 1969). Given the large number of undescribed species and the limited array of qualitative characters used by BERNARDI in his keys, it is difficult to identify Weinmannia species. INTRODUCTION The present paper addresses this problem by: (1) describing and illustrating previously overlooked qualitative character varia- tion among Malagasy Weinmannia, (2) present- ing a synoptic key to the sections and species-groups of Weinmannia in Madagascar and the Comores, (3) providing diagnostic features for each section and species-group, and (4) listing all presently described species in each species- group. Because a number of Weinmannia species remain to be described from Madagascar, it would be premature to write a key to the species level. Instead, the key given below aids species- level identification by reducing the number of species with which one must compare an uniden- tified specimen. This synoptic key has proven useful as the basis for a species-level key of Weinmannia from the Marojejy massif (BRADFORD & MILLER 2001), and could be applied to other regions and eventually the entire genus in Madagascar. Malagasy Weinmannia species were last treated by BERNARDI (1964, 1965, 1969). He estab- lished two endemic sections Spicata and Inspersa, and placed a few species in the more wide- spread sections Weinmannia and Leiospermum (sect. Leiospermum has priority over BERNARDI’s equivalent sect. Racemosa, see HOPKINS 1998a). However, BERNARDI’s assignment of some Malagasy-Comoral species to sections normally found outside of Madagascar has been proven incorrect. Weinmannia baehniana Bernardi and W. comorensis Tul., which BERNARDI placed in sect. Leiospermum, belong instead in sect. Spicata, and the species he included in sect. Weinmannia, W. rutenbergii Engl. and W. venusta Bernardi, belong in sect. Inspersa (see BRADFORD 1998 and below). Both a morphological cladistic analysis of Weinmannia species-groups and sec- tions (BRADFORD 1998) and molecular system- atic work on the genus (BRADFORD 2000; BRADFORD & BARNES 2001) support the mono- phyly of sections Spicata and Inspersa as now cir- cumscribed and provide evidence that they are sister taxa. g g The key and diagnoses are largely based on a recent morphological cladistic study (BRADFORD 1998) that found several characters not discussed by BERNARDI (1964, 1965). In Madagascar, sev- eral groups of species can be recognized that share unique combinations of these qualitative charac- ters. The key and diagnoses for the Malagasy species-groups utilize inflorescence terminology that may not be familiar. For this reason, figures, illustrations and a discussion of important char- acters precede the key. ADANSONIA, sér. 3 • 2001 • 23 (2) 238 Cladistic analysis of Weinmannia (Cunoniaceae) Fig. 1. INTRODUCTION — Cladogram of Weinmannia species-groups (with representative species) based on morphology (from BRADFORD 1998). The main characters used in the key are plotted on the tree. Fig. 1. — Cladogram of Weinmannia species-groups (with representative species) based on morphology (from BRADFORD 1998). The main characters used in the key are plotted on the tree. m of Weinmannia species-groups (with representative species) based on morphology (from BRADFORD 1998). Th sed in the key are plotted on the tree. ADANSONIA, sér. 3 • 2001 • 23 (2) FROM CLADOGRAMS TO KEYS monophyletic. At present, not enough is known about the phyletic relationships of Weinmannia species-groups A-G to give them formal taxo- nomic names and rank, which would violate the principles of phylogenetic systematics and proba- bly lead to taxonomic instability. Nevertheless, a real need exists to communicate group informa- tion on this complex genus, which can be done by informally recognizing species-groups A-G for the purpose of discussion. Such informal tax- onomies have a long history (e.g. BENTHAM & HOOKER 1862-1883) and are commonly used in phylogenetic systematics when cladistic resolu- tion is lacking or clades are poorly supported (e.g. Angiosperm Phylogeny Group 1998). By acknowledging uncertainty, informal classifica- tions may actually communicate information more effectively than do some rigid systems that require all individuals to be part of a formal taxon at each hierarchical rank. Prior to conducting a cladistic analysis of Weinmannia and the related genus Cunonia, specimens representing all known species of these genera were examined to find unique combina- tions of qualitative morphological characters among species (see vouchers in BRADFORD 1998). This process led to the recognition of species- groups, each representing from one to many species, that served as a terminal units in the cladistic analysis. Seven species-groups were rec- ognized in Madagascar and the Comores, five of which have apparent autapomorphies, whereas two lack clearly apomorphic features (see charac- ter states in Fig. 1). Species-groups B, C, D, E, and G (see below) may therefore be mono- phyletic, whereas no characters have been found that support the monophyly of species-groups A and F. Classifications communicate grouping infor- mation, but phylogenetic systematics also requires that formally recognized groups be Cladograms can be applied towards taxonomic identification by including in a standard dichoto- mous key the qualitative characters of the data ADANSONIA, sér. 3 • 2001 • 23 (2) 239 matrix as plotted on a dichotomously branching tree. The key presented below is organized using character states as mapped on the cladogram from more general characters to more specific ones. In other words, synapomorphies of the largest clades are used in the first order couplet, followed by more exclusive synapomorphies in second order couplets, and terminating with apo- morphic or diagnostic characters for a species- group (Fig. 1). often organized into compound structures borne on a peduncle, and these are called Inflorescence Modules (IMs). FROM CLADOGRAMS TO KEYS Among sections or species- groups, IMs may have distinct patterns of organi- zation along the main stem. The Total Inflorescence (TI) is formed by the architectural arrangement of the IMs and Racemes (Fig. 2). Figure 2 does not show the full range of TI varia- tion among Malagasy species, but is sufficient to diagnose species-groups. It may not always be practical to apply the characters used in a cladistic analysis to taxo- nomic keys if, for example, most of them are anatomical and therefore difficult to use with herbarium specimens or on material in the field. The case of Weinmannia provides a good exam- ple. The first couplets use inflorescence and pedi- cel characters that can readily be seen. Some couplets also emphasize structural features of the flower-bearing axis that can be used whether the material is in bud, flower or fruit. By contrast, the key in BERNARDI (1965) emphasizes the floral nectary, the pubescence of the ovary, and the dis- tribution of trichomes on seeds. Most of the species-groups in Madagascar (A, B, F, G; see below) have similar architectural fea- tures, in that IMs consist of a single metamer (i.e. an internode plus a node and associated organs) with an opposite pair of Racemes and a terminal vegetative bud. The number of IMs and their position may vary somewhat among species. For example, the TI may be reduced to a terminal IM or a pair of lateral IMs (Fig. 2). Species-groups C and D can be recognized by a terminal Raceme in the IM, although they are variable and may have the more general IM form (i.e. that found in A, B, F, G). Even when the IMs are of the general form, members of groups C and D can often be recognized by a prolific TI, in which IMs occur at several subdistal nodes along the main stem. Prolific TIs are especially common in W. rutenbergii, which is indicated by the broken lines and arrow in Fig. 2. Species in group D have a branched IM, whereas branching is rare in group C. In both groups C and D, the TI is basitonic (i.e. IMs are larger at basal nodes and smaller at terminal nodes). This paper provides detailed illustrations and discussions of important features in Malagasy species of Weinmannia. FROM CLADOGRAMS TO KEYS BRADFORD (1998) may be referred to for a more general discussion of characters in the genus and cladistic methods used to study them. Figure 1 shows the inferred plesiomorphic and derived conditions for several qualitative traits that differ among Malagasy species-groups. Figures 2-4 illustrate and com- pare these characters (except for seed indumen- tum), which are discussed below. Group E is easily recognized by the absence of IMs. Racemes are borne from leaf axils along the main stem. Architecture The two sections of Weinmannia in Mada- gascar can be distinguished by whether the flow- ers are subtended by a pedicel (sect. Inspersa) or are sessile along the Raceme (sect. Spicata). BERNARDI (1964, 1965) instead emphasized the form of the floral nectary to circumscribe sec- tions, which is why he placed some Malagasy species in sections otherwise from the Malesian- Pacific region. However, nectary form is homo- Weinmannia inflorescences can be described at three architectural levels: the Total Inflorescence, the Inflorescence Module, and the Raceme (Fig. 2) (BRADFORD 1998). The term Raceme (with capital “R”) is used generally to describe the unbranched, ultimate flower-bearing axes in Weinmannia, which have also been called pseudo- racemes or spikes (BERNARDI 1964). Racemes are ADANSONIA, sér. 3 • 2001 • 23 (2) 240 Cladistic analysis of Weinmannia (Cunoniaceae) Cladistic analysis of Weinmannia (Cunoniaceae) Fig. 2. — Inflorescence architecture among Weinmannia species-groups in Madagascar. See main text for the discussion of features and the list of species in each group. Fig. 2. — Inflorescence architecture among Weinmannia species-groups in Madagascar. See main text for the discussion of features and the list of species in each group. plasious and varies within Malagasy sections (BRADFORD 1998), although it can be useful to diagnose Malagasy species-groups. the axil of a bract dissociate during elongation of the flower-bearing axis, the relationship between a flower and its bract can usually be traced by a ridge or differently-colored linear zone between them. Flowers are often clustered in groups of four, and appear to develop from decussate pairs of buds (see Fig. 4, Group E). When floral groups dissociate during axis elongation, the pair of flowers perpendicular to the floral bract is spread apart more so than the pair of flowers borne in the same plane as the bract (see Fig. 4, Group F). The organization of flowers along the Raceme varies in two important ways: (1) whether flow- ers are initiated solitarily or in a group, and (2) whether flowers remain near their point of initi- ation or dissociate from it. Characters of the Raceme are often easiest to see when flowers are in bud, or when mature flowers or fruits are removed to view clearly their positional relation- ships (Figs. 3, 4). Even when flowers initiated in ADANSONIA, sér. 3 • 2001 • 23 (2) 241 Bradford J.C. Fig. 3. Architecture — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Inspersa species-groups: Group B: Weinmannia lowryana with a single flower dissociating from a bract (Humbert 23057, MO 05006602); Group C: Weinmannia rutenbergii for portion shown in bud (Bradford 699, MO 05079575), and for portion in fruit, racemes often in threes, flowers mostly solitary and in the axil of a bract [shown in bud to left] or flowers grouped at the base of the medial raceme [shown in fruit on right] (Bradford 701, MO 05079557); Group D: Weinmannia venusta, with Inflorescence modules usually branched (only one portion shown), and flowers grouped in the axil of a bract (Service Forestier (Capuron) 27632, MO 05006598). Some flowers have been removed to see pedicels clearly. Group B Group D Group C 5 mm 50 mm 5 mm 5 mm 5 mm 5 mm 5 mm 50 mm Group B 5 mm Fig. 3. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Inspersa species-groups: Group B: Weinmannia lowryana with a single flower dissociating from a bract (Humbert 23057, MO 05006602); Group C: Weinmannia rutenbergii for portion shown in bud (Bradford 699, MO 05079575), and for portion in fruit, racemes often in threes, flowers mostly solitary and in the axil of a bract [shown in bud to left] or flowers grouped at the base of the medial raceme [shown in fruit on right] (Bradford 701, MO 05079557); Group D: Weinmannia venusta, with Inflorescence modules usually branched (only one portion shown), and flowers grouped in the axil of a bract (Service Forestier (Capuron) 27632, MO 05006598). Some flowers have been removed to see pedicels clearly. Fig. 3. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Inspersa species-groups: Group B: Weinmannia lowryana with a single flower dissociating from a bract (Humbert 23057, MO 05006602); Group C: Weinmannia rutenbergii for portion shown in bud (Bradford 699, MO 05079575), and for portion in fruit, racemes often in threes, flowers mostly solitary and in the axil of a bract [shown in bud to left] or flowers grouped at the base of the medial raceme [shown in fruit on right] (Bradford 701, MO 05079557); Group D: Weinmannia venusta, with Inflorescence modules usually branched (only one portion shown), and flowers grouped in the axil of a bract (Service Forestier (Capuron) 27632, MO 05006598). ADANSONIA, sér. 3 • 2001 • 23 (2) Architecture Some flowers have been removed to see pedicels clearly. Bracts may fall at floral or fruit maturity, but because bract scars are shaped differently than floral scars the positional and numerical relation- ships between these structures remain clear. Floral scars are round, with the vascular cylinder espe- cially visible in species of sect. Spicata. Bract scars are more difficult to see, but they may be cres- cent-shaped, or small, irregular protrusions along ADANSONIA, sér. 3 • 2001 • 23 (2) 242 Cladistic analysis of Weinmannia (Cunoniaceae) Fig. 4. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Spicata species-groups. A central portion of a spike is shown: Group E: Weinmannia lucens (voucher unknown) with groups of flowers clustered near bract; Group F: Weinmannia hildebrandii, with groups of flowers dissociating from a bract (Service Forestier (Capuron) 24417, MO 05012268); Group G: Weinmannia arguta, with a single flower dissociated from a bract (Miller & Randrianasolo 4659, MO 3759761). Flowers removed to see positional relationships more clearly, leaving a circular scar with a point in the middle. (Illustration of Weinmannia lucens from BERNARDI 1965) Group E Group F Group G 5 mm 5 mm 5 mm 5 mm Group E Group F Group G Fig. 4. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Spicata species-groups. A central portion of a spike is shown: Group E: Weinmannia lucens (voucher unknown) with groups of flowers clustered near bract; Group F: Weinmannia hildebrandii, with groups of flowers dissociating from a bract (Service Forestier (Capuron) 24417, MO 05012268); Group G: Weinmannia arguta, with a single flower dissociated from a bract (Miller & Randrianasolo 4659, MO 3759761). Flowers removed to see positional relationships more clearly, leaving a circular scar with a point in the middle. (Illustration of Weinmannia lucens from BERNARDI 1965) the Raceme. In sect. Inspersa, remaining bases of pedicels show the locations of flowers. Comparing the position of flower clusters in W. rutenbergii to that of lateral Racemes in a close relative, W. venusta, suggests that they are homol- ogous. Flowers along other parts of the Raceme in W. rutenbergii are usually solitary, although pairs of flowers do occur sometimes in many specimens. Comparing the position of flower clusters in W. rutenbergii to that of lateral Racemes in a close relative, W. venusta, suggests that they are homol- ogous. Architecture Flowers along other parts of the Raceme in W. rutenbergii are usually solitary, although pairs of flowers do occur sometimes in many specimens. The structure of the central portion of the flower-bearing axis is most consistent and useful for identification. Often flowers will arise in clus- ters at the very base of a Raceme but will other- wise be solitary (e.g. Fig. 3, Group C illustration on right). Usually, the distal portion of a flower- bearing axis bears solitary flowers no matter what the rest of the axis is like. The key below includes six new species described and one new species-level combina- tion made for Weinmannia from the Marojejy massif (BRADFORD & MILLER 2001). These new taxa represent three of the seven species groups, with four of the novelties found in group G alone. Because group G is so species-rich in general, additional couplets are provided to sub- divide it into more manageable groups, although these are not based on cladistic characters (Fig. 1). Authors of the recognized taxa are given below following the diagnoses of each species-group. One species, W. rutenbergii (Group C), may sometimes key out to species-group D because of variation in the number of flowers per bract (although the IM rarely branches in members of species-group C as it does in those of D, see Fig. 2). Often in W. rutenbergii, at the base of the terminal Raceme a large, stipular bract can be seen that subtends clusters of flowers (Figs. 2, 3). Between this cluster of flowers and other flowers on the axis a sterile gap can be observed. ADANSONIA, sér. 3 • 2001 • 23 (2) 243 Key to sections and species-groups of Malagasy Weinmannia Key to sections and species-groups of Malagasy Weinmannia 1. Pedicels distinct, easily visible and slender; seeds with surfaces more or less evenly covered with trichomes .... .......................................................................................................................................... 2, sect. Inspersa 1’. Pedicels lacking, very short, or thick as if an extension of the receptacle; seeds usually comose at ends only, sometimes more or less covered with trichomes throughout ................................................ 5, sect. Spicata 2. Flowers positioned away from the floral bract at maturity; IMs equal in size; racemes borne in pairs from a short internode with a vegetative bud between them ................................................................................ 3 2’. WEINMANNIA L. Trees and shrubs, sometimes hemiepiphytic and strangling. Populations monoecious and flowers bisexual, dioecious or polygamodioe- cious. Leaves decussate in pairs or rarely whorls, imparipinnate or unifoliolate, rachis alate or not, leaf margins toothed or rarely mostly entire. Inflorescence racemose (including spikes and pseudoracemes); borne solitary in leaf axils or in compound units of various forms, compound units borne axillary or terminal. Flowers initiated solitarily or grouped in the axil of a small bract, remaining near bract or dissociating from it dur- ing elongation of the flower-bearing axis; pedicel- late or sessile; bisexual or unisexual; perianth of sepals and petals, 4-5-merous, hypogynous or perigynous, imbricate, calyx lobes free or barely fused basally, petals persistent or caducous; floral nectary annular or segmented; androecium diplostemonous (rarely one whorl of stamens), filaments slender, equal to or exceeding petals, anthers dorsifixed, bithecal with an apical con- MALAGASY CLADE OF WEINMANNIA (sections Inspersa and Spicata) Flowers mostly pentamerous, hypogynous, bisexual, petals persistent, calyx persistent, endo- carp and exocarp usually separating in old fruits. DESCRIPTION OF WEINMANNIA AND DIAGNOSTIC FEATURES OF MALAGASY CLADE, SECTIONS AND SPECIES-GROUPS nective, longitudinally dehiscent; ovary bicarpel- late, carpels fused to the level of the styles, locules two, each with an axile placenta, ovules 2 to numerous in two rows per locule, styles slender and diverging, stigmas small and terminal. Fruits capsular, carpels diverging and opening along their ventral sutures; calyx persistent or caducous; placenta often remaining upright between sepa- rate carpels; endocarp and exocarp separating in old fruits or remaining adherent; seeds comose or trichomes more or less covering the surface, lack- ing wings. WEINMANNIA L. Architecture Flowers positioned in the axil of the floral bract at maturity; IMs larger basally; racemes borne in threes or at the ends of a decussate branching system with multiple internodes that usually terminate in a raceme, or racemes in pairs with a vegetative bud between them ................................................................................ 4 3. Floral bracts subtending groups of flowers ........................................................................ species-group A 3’. Floral bracts subtending solitary flowers ............................................................................ species-group B 4. IM unbranched; floral bracts subtending solitary flowers or less often groups of flowers .... species-group C 4’. IM branched; floral bracts subtending groups of flowers .................................................. species-group D 5. Spikes borne solitarily in leaf axils; floral nectary composed of more or less distinct segments; flowers distrib- uted near each other and the floral bract, borne in clusters of usually four ........................ species-group E 5’. Spikes borne in pairs from a short internode in leaf axils or terminally; floral nectary usually an entire, membranous disc (rarely segmented); flowers not distributed in clusters, but spread in a line away from the floral bract ................................................................................................................................................ 6 6. Floral bracts subtending multiple flowers; floral nectary sometimes easily broken into segments .................. .......................................................................................................................................... species-group F 6’. Floral bracts subtend a single flower; nectary usually entire and membranous .................... species-group G Acknowledgments BENTHAM G. & HOOKER J.D. 1862-1883. — Genera plantarum: ad exemplaria imprimis in Herberii Kewensibus servata definita. A. Black (v.1, pt. 1); Reeve & Co. (v.1, pt. 2-v. 3), London. For financial support I thank the Mellon Foundation, the Missouri Botanical Garden and Washington University’s Division of Biology and Biomedical Sciences. Most illustrations in Figs. 3 and 4 were kindly done by Kim MARTIN. The Muséum National d’Histoire Naturelle, Laboratoire de Phanérogamie kindly permitted the use of an illustra- tion of W. lucens in Fig. 3 that originally appeared in BERNARDI (1965, fig. 7b). For field work assistance I thank my Malagasy collaborators at the Parc Bota- nique et Zoologique de Tsimbazaza, especially Guy RAFAMANTANANTSOA and Frank RAKOTONASOLO. p BERNARDI L. 1964. — Revisio generis Weinmanniae Pars III: Sect. III-IV-V-VI (verteris orbis). Bot. Jahrb. Syst. 83: 126-184. y BERNARDI L. 1965. — Cunoniacées. Flore de Madagascar et des Comores, in HUMBERT H. (ed.). Muséum National d’Histoire Naturelle, Laboratoire de Phanérogamie, Paris. g BERNARDI L. 1969. — Weinmanniae species nova, isoste- mona, ex Madagascaria boreali. Candollea 24: 85-87. g BRADFORD J.C. 1998. — A cladistic analysis of species-groups in Weinmannia (Cunoniaceae) based on morphology and inflorescence architecture. Ann. Missouri Bot. Gard. 85: 565-593. Weinmannia sect. Inspersa Bernardi Flowers pedicellate, floral nectary usually annular and ribbed; seed surface with dense indu- mentum throughout. ADANSONIA, sér. 3 • 2001 • 23 (2) 244 Cladistic analysis of Weinmannia (Cunoniaceae) Weinmannia sect. Spicata Bernardi Bradford, W. rakotomalazana J.C. Bradford. Spikes borne in IMs, flowers initiated in groups that dissociate from bract, nectary annular and thin or composed of more or less distinct segments. species-group G. — Species are listed within the key given below. Spikes borne in IMs, flowers borne solitarily and usually dissociate from axil of bract, nectary usually annular and thin. 1. Leaves unifoliolate .................................................................................................................................... 2 1’. Leaves imparipinnate ................................................................................................................................ 3 2. Ovaries glabrous ................................................................................................ W. humbertiana Bernardi 2’. Ovaries pubescent ........................................................................................................................................ ......W. bojeriana Tul., W. mammea Bernardi, W. pauciflora, J.C. Bradford, W. integrifolia J.C. Bradford 3. Ovaries glabrous ................................................................................................................ W. decora Tul. 3’. Ovaries pubescent .................................................................................................................................... 4 4. Hairs on seeds restricted to ends ................................................................................ W. humblottii Baill., ...................... W. sanguisugarum Bernardi, W. arguta (Bernardi) J.C. Bradford, W. venosa J.C. Bradford 4’. Hairs on seeds widely distributed .............................................. W. eriocarpa Tul., W. stenostachya Baker 1. Leaves unifoliolate .................................................................................................................................... 2 1’. Leaves imparipinnate ................................................................................................................................ 3 2. Ovaries glabrous ................................................................................................ W. humbertiana Bernardi 2’. Ovaries pubescent ........................................................................................................................................ ......W. bojeriana Tul., W. mammea Bernardi, W. pauciflora, J.C. Bradford, W. integrifolia J.C. Bradford 3. Ovaries glabrous ................................................................................................................ W. decora Tul. 3’. Ovaries pubescent .................................................................................................................................... 4 4. Hairs on seeds restricted to ends ................................................................................ W. humblottii Baill., ...................... W. sanguisugarum Bernardi, W. arguta (Bernardi) J.C. Bradford, W. venosa J.C. Bradford 4’. Hairs on seeds widely distributed .............................................. W. eriocarpa Tul., W. stenostachya Baker Weinmannia sect. Spicata Bernardi species-group A. — Weinmannia henricorum Bernardi, W. madagascariensis DC. ex Ser. species-group A. — Weinmannia henricorum Bernardi, W. madagascariensis DC. ex Ser. TI isotonic, IM terminating in a bud, flowers borne in clusters that dissociate from bract. Flowers sessile; seeds comose, only rarely seed surface with indumentum throughout. species group E Weinmannia lucens Baker y y surface with indumentum throughout. species-group E. — Weinmannia lucens Baker, species-group B. — Weinmannia commersonii Bernardi, W. louveliana Bernardi, W. lowryana J.C. Bradford. TI isotonic, IM terminates in a bud, flowers borne solitarily and dissociate from bract. p g p W. minutiflora Baker, W. comorensis Tul., W. baehniana Bernardi. Spikes borne in leaf axils, flowers borne in clus- ters that remain in axil of bract, nectary com- posed of more or less distinct segments. W. minutiflora Baker, W. comorensis Tul., W. baehniana Bernardi. Spikes borne in leaf axils, flowers borne in clus- ters that remain in axil of bract, nectary com- posed of more or less distinct segments. species-group C. — Weinmannia hepaticarum Bernardi, W. rutenbergii Engl. TI basitonic, IM terminating in a raceme or a bud, IM sometimes branched, flowers borne solitarily or sometimes in small groups and remaining in axil of bract. species-group C. — Weinmannia hepaticarum Bernardi, W. rutenbergii Engl. TI basitonic, IM terminating in a raceme or a bud, IM sometimes branched, flowers borne solitarily or sometimes in small groups and remaining in axil of bract. species-group F. — Weinmannia hildebrandtii Baill., W. icacifolia Bernardi, W. marojejyensis J.S. Mill. & J.C. Bradford, W. rakotomalazana J.C. Bradford. Spikes borne in IMs, flowers initiated in groups that dissociate from bract, nectary annular and thin or composed of more or less distinct segments. species-group G. — Species are listed within the key given below. Spikes borne in IMs, flowers borne solitarily and usually dissociate from axil of bract, nectary usually annular and thin. species-group F. — Weinmannia hildebrandtii Baill., W. icacifolia Bernardi, W. marojejyensis J.S. Mill. & J.C. Bradford, W. rakotomalazana J.C. Bradford. Spikes borne in IMs, flowers initiated in groups that dissociate from bract, nectary annular and thin or composed of more or less distinct segments. species-group G. — Species are listed within the key given below. Spikes borne in IMs, flowers borne solitarily and usually dissociate from axil of bract, nectary usually annular and thin. species-group F. — Weinmannia hildebrandtii Baill., W. icacifolia Bernardi, W. marojejyensis J.S. Mill. & J.C. Manuscript received 14 February 2001; revised version 19 September 2001. ADANSONIA, sér. 3 • 2001 • 23 (2) REFERENCES BRADFORD J.C. 2000. — Phylogenetic systematics of Cunoniaceae (Oxalidales), with an emphasis on species-groups and inflorescence evolution in Weinmannia and related genera. Ph.D. thesis. ANGIOSPERM PHYLOGENY GROUP 1998. — An ordinal classification for the families of Flowering Plants. Ann. Missouri Bot. Gard. 85: 531-553. ADANSONIA, sér. 3 • 2001 • 23 (2) 245 Bradford J.C. HOPKINS H.C.F. 1998b. — A revision of Weinmannia (Cunoniaceae) in Malesia and the Pacific. 2. Sulawesi and the Philippines. Adansonia, sér. 3, 20: 43-66. Washington University, Department of Biology, St. Louis. BRADFORD J.C. & BARNES R.W. 2001. — Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology. Syst. Bot. 26: 354-385. HOPKINS H.C.F. 1998c. — A revision of Weinmannia (Cunoniaceae) in Malesia and the Pacific. 3. New Guinea, Solomon Islands, Vanuatu and Fiji, with notes on the species of Samoa, Rarotonga, New Caledonia and New Zealand. Adansonia, sér. 3, 20: 67-106. p gy y BRADFORD J.C. & MILLER J.S. 2001. — New taxa and nomenclatural notes on the flora of the Marojejy massif, Madagascar. V. Cunoniaceae: Weinmannia. Adansonia, sér. 3, 23: 219-236. HOPKINS H.C.F. 1998a. — A revision of Weinmannia (Cunoniaceae) in Malesia and the Pacific. 1. Introduction and an account of the species of Western Malesia, the Lesser Sundas and the Moluccas. Adansonia, sér. 3, 20: 5-41. HOPKINS H.C.F. & FLORENCE J. 1998. — A revision of Weinmannia (Cunoniaceae) in Malesia and the Pacific. 4. The Society, Marquesas and Austral Islands. Adansonia, sér. 3, 20: 107-130. Manuscript received 14 February 2001; revised version 19 September 2001. ADANSONIA, sér. 3 • 2001 • 23 (2) 246
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Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 80 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata Pelajaran Bahasa Indonesia Bunga Lidinta Putri1*, Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4 1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia 3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar, Indonesia *Korespondesi Penulis: Bunga Lidinta Putri Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia Email: a310170214@student.ums.ac.id doi: 10.56972/jikm.v2i2.44 Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022 Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022 Abstrak Jurnal Ilmiah Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 80 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata Pelajaran Bahasa Indonesia Bunga Lidinta Putri1*, Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4 1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia 3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar, Indonesia *Korespondesi Penulis: Bunga Lidinta Putri Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia Email: a310170214@student.ums.ac.id doi: 10.56972/jikm.v2i2.44 Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022 Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022 Abstrak Jurnal Ilmiah Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 80 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata 80 Jurnal Ilmiah Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata Pelajaran Bahasa Indonesia Bunga Lidinta Putri1*, Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4 1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia 3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar, Indonesia Bunga Lidinta Putri1 , Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4 1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia 3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar, Indonesia *Korespondesi Penulis: Bunga Lidinta Putri Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia Email: a310170214@student.ums.ac.id doi: 10.56972/jikm.v2i2.44 Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022 Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022 Abstrak Kondisi pembelajaran di dunia pendidikan saat ini harus merubah sistem pendidikannya karena me- nyesuaikan dengan situasi dan kondisi selama masa pandemi Covid-19. Pembelajaran sekolah diadakan secara daring karena tidak bisa dipungkiri, dalam proses pembelajaran terjadi kontak antar individu dalam jumlah banyak. Motivasi siswa terhadap kegiatan belajar harus ditingkatkan melalui pembelaja- ran yang menyenangkan. Salah satunya adalah peningkatan kemampuan literasi siswa selama masa pandemi dengan didampingi oleh mahasiswa KKN-DIK Universitas Muhammadiyah Surakarta guna meningkatkan kemampuan literasi menulis siswa SMA Prawira Marta. Metode yang dijalankan dalam KKN-Dikmas ini adalah metode observasi dan metode dokumentasi. Hasil yang diperoleh berupa bukti tugas-tugas yang berkaitan dengan kemampuan literasi menulis siswa yang berbeda-beda. Pentingnya kegiatan peningkatan literasi ini perlu untuk dikuasai agar siswa menjadi seseorang dengan kemam- puan bahasa yang baik. Kata Kunci: literasi menulis, media whatsapp doi: 10.56972/jikm.v2i2.44 Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022 Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022 Abstrak Kondisi pembelajaran di dunia pendidikan saat ini harus merubah sistem pendidikannya karena me- nyesuaikan dengan situasi dan kondisi selama masa pandemi Covid-19. Pembelajaran sekolah diadakan secara daring karena tidak bisa dipungkiri, dalam proses pembelajaran terjadi kontak antar individu dalam jumlah banyak. Motivasi siswa terhadap kegiatan belajar harus ditingkatkan melalui pembelaja- ran yang menyenangkan. Salah satunya adalah peningkatan kemampuan literasi siswa selama masa pandemi dengan didampingi oleh mahasiswa KKN-DIK Universitas Muhammadiyah Surakarta guna meningkatkan kemampuan literasi menulis siswa SMA Prawira Marta. Metode yang dijalankan dalam KKN-Dikmas ini adalah metode observasi dan metode dokumentasi. Hasil yang diperoleh berupa bukti tugas-tugas yang berkaitan dengan kemampuan literasi menulis siswa yang berbeda-beda. Pentingnya kegiatan peningkatan literasi ini perlu untuk dikuasai agar siswa menjadi seseorang dengan kemam- puan bahasa yang baik. Kata Kunci: literasi menulis, media whatsapp selama masa pandemi Covid-19. Penu- laran yang sangat cepat melalui kontak an- tar individu menyebabkan perkumpulan dalam bentuk apapun dihindari. Hal ini 1. Pendahuluan Dewasa ini dunia pendidikan harus merubah sistem pendidikannya karena menyesuaikan dengan situasi dan kondisi Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 81 hampir terjadi di semua sektor kehidupan, baik sektor kesehatan, sektor bisnis dan perdagangan, sektor sosial, sektor politik, sektor budaya, sektor perkantoran terma- suk pada sektor Pendidikan (Sutopo et al., 2021). Maka dari itu, sekolah diadakan secara daring karena tidak bisa dipungkiri, dalam proses pembelajaran terjadi kontak antar individu dalam jumlah banyak. Pada masa pandemi corona ini, semua proses belajar mengajar dilakukan secara daring sesuai tema (Wisaksono et al., 2020). Pem- berlakuan praktik pendidikan dengan sis- tem daring tersebut pada dasarnya meru- pakan desain ideal untuk melaksanakan kegiatan pengajaran di tengah masa pan- demi saat ini (Lathifah & Utami, 2021). Peran teknologi sangat penting karena hubungan antara pembelajaran jarak jauh (PJJ) dengan pemanfaatan platform pem- belajaran sangat berdampak positif karena penggunaannya sangat praktis (Khikmawati et al., 2021). Pesatnya perkembangan teknologi zaman sekarang memberikan pengaruh yang begitu banyak bagi kemajuan dunia pendidikan khu- susnya dalam proses pembelajaran. Ada beberapa unsur pergeseran dalam proses pembelajaran, 1) dari pelatihan menjadi penampilan, 2) dari pembelajaran bersifat luring menjadi daring, 3) dari kertas men- jadi online atau saluran, 4) fasilitas berupa fisik menjadi fasilitas jaringan kerja, 5) serta dari waktu siklus menjadi waktu nyata (Suryadi, 2015). Selain itu kemajuan teknologi juga mempunyai peran penting dalam bidang pendidikan diantaranya: 1) hampir terjadi di semua sektor kehidupan, baik sektor kesehatan, sektor bisnis dan perdagangan, sektor sosial, sektor politik, sektor budaya, sektor perkantoran terma- suk pada sektor Pendidikan (Sutopo et al., 2021). Maka dari itu, sekolah diadakan secara daring karena tidak bisa dipungkiri, dalam proses pembelajaran terjadi kontak antar individu dalam jumlah banyak. Pada masa pandemi corona ini, semua proses belajar mengajar dilakukan secara daring sesuai tema (Wisaksono et al., 2020). Pem- berlakuan praktik pendidikan dengan sis- tem daring tersebut pada dasarnya meru- pakan desain ideal untuk melaksanakan kegiatan pengajaran di tengah masa pan- demi saat ini (Lathifah & Utami, 2021). Peran teknologi sangat penting karena hubungan antara pembelajaran jarak jauh (PJJ) dengan pemanfaatan platform pem- belajaran sangat berdampak positif karena penggunaannya sangat praktis (Khikmawati et al., 2021). Pesatnya perkembangan teknologi zaman sekarang memberikan pengaruh yang begitu banyak bagi kemajuan dunia pendidikan khu- susnya dalam proses pembelajaran. 1. Pendahuluan Ada beberapa unsur pergeseran dalam proses pembelajaran, 1) dari pelatihan menjadi penampilan, 2) dari pembelajaran bersifat luring menjadi daring, 3) dari kertas men- jadi online atau saluran, 4) fasilitas berupa fisik menjadi fasilitas jaringan kerja, 5) serta dari waktu siklus menjadi waktu nyata (Suryadi, 2015). Selain itu kemajuan teknologi juga mempunyai peran penting dalam bidang pendidikan diantaranya: 1) sebagai sumber ilmu dan pusat pendidi- kan, 2) munculnya metode pembelajaran baru yang dapat memudahkan tenaga pen- didik atau guru dalam proses pembelaja- ran, 3) system pembelajaran tidak harus ta- tap muka melainkan dengan daring atau secara online (Effendi, 2019). Kondisi pembelajaran dalam situasi daring seperti ini tentunya berdampak ter- hadap semangat belajar siswa. Tanpa disa- dari pelaksanaan pembelajaran dalam jaringan (daring) telah berjalan lebih dari satu tahun yang pengaruhnya amat besar bagi dunia pendidikan, khususnya peru- bahan perilaku anak yang dipaksa harus bersentuhan dengan teknologi tanpa bekal cukup mengontrol penggunaan gadget bila tanpa pendampingan dari orang tua secara maksimal (Nuroniyah, 2021). Selain itu juga ada beberapa hambatan dalam proses pembelajaran jarak jauh di masa pandemic. Selain keterbatasan sarana pen- dukung dan jaringan internet, faktor kesia- pan dari sumber daya manusia merupakan bagian penting dalam sebuah keberhasilan dalam pelaksanaan pembelajaran secara online (Latip, 2020). Namun, Pendidikan yang dil- aksanakan secara daring membuat peserta didik mengalami penurunan dalam minat dan keaktifan belajar (Ihwan et al., 2021). Pembelajaran secara daring dinilai kurang berjalan efektif. Hal ini dilihat dari respon siswa saat kegiatan pembelajaran daring yang cenderung tidak merespon ketika diskusi dan pengumpulan tugas (Choiru- din & Rahmasari, 2021). Banyak siswa yang kurang termotivasi dalam melaksanakan proses pembelajaran karena merasa dirinya tidak diawasi sehingga rasa tanggung jawab yang mereka miliki Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 82 menurun. Proses belajar mengajar yang dilakukan secara online selayaknya tidak dilaksanakan dalam durasi panjang dan memakan waktu lama, hal ini dikarenakan peserta didik akan mengalami kesulitan dalam mempertahankan konsentrasi lebih dari satu jam (Sumarsono et al., 2021). Ten- tunya permasalahan tersebut menjadikan Pendidikan tidak berjalan dengan seha- rusnya dan berimbas kepada proses dan hasil pembelajaran yang cacat (Sholihah et al., 2021). Namun, pendidikan harus tetap berjalan hingga tujuan pendidikan tercapai. Motivasi siswa terhadap kegiatan belajar harus ditingkatkan melalui pem- belajaran yang menyenangkan. 1. Pendahuluan Salah satunya adalah peningkatan kemampuan literasi siswa selama masa pandemi dengan didampingi oleh mahasiswa KKN- DIK Universitas Muhammadiyah Sura- karta. politik, dan juga meningkatkan kepekaan terhadap lingkungan. Upaya pengem- bangan literasi perlu dikembangkan secara terintegrasi, terutama melalui pendidikan. Ini karena, Pendidikan memiliki fungsi dan tujuan dalam memajukan peradaban bangsa (Agustina et al., 2020). Literasi tidak hanya sekadar kemampuan membaca dan menulis, tetapi literasi bisa berarti melek teknologi, politik, berpikir kritis, dan peka terhadapi lingkungan sekitar (Sunanda et al., 2020). Budaya literasi juga penting un- tuk bangsa karena dapat menggerakan ide- ide dan pengetahuan bangsa Indonesia, khususnya para anak muda di Indonesia (Ningrum et al., 2021). Mengajarkan lit- erasi pada intinya menjadikan manusia secara fungsional mampu berbaca-tulis, terdidik, cerdas dan menunjukkan apre- siasi terhadap sastra (Machromah et al., 2020). Literasi merupakan suatu gerakan yang bertujuan untuk mengatasi masalah buta huruf pada suatu lingkungan pen- didikan. Gerakan literasi sering dikaitkan dengan kegiatan membaca buku apa saja karena hal ini dapat meningkatkan moti- vasi siswa terhadap kegiatan membaca. Kemampuan literasi membaca merupakan aspek dasar yang harus dimiliki oleh siswa yang digunakan untuk menyerap berbagai sumber informasi yang diterima (Ma- hardhani et al., 2021). Menurut Hartati (2017) literasi merupakan kemampuan un- tuk memahami, mengelola, dan memakai informasi pada berbagai konteks. Literasi tidak hanya memberikan manfaat di bi- dang menulis dan membaca, namun juga memberikan manfaat di bidang teknologi, Menurut kamus besar bahasa Indone- sia (KBBI) yang dimaksud dengan literasi yaitu kemampuan menulis dan membaca; pengetahuan atau keterampilan pada bi- dang atau kegiatan tertentu; kemampuan individu untuk mengolah informasi serta pengetahuan untuk kecakapan hidup. Lit- erasi adalah istilah yang memiliki kaitan erat dengan kegiatan membaca dan menu- lis terutama dalam hal penggunaan bahasa dan fungsi bahasa tersebut. Menurut Kern (2000) dalam Firmansyah (2018: 12) literasi merupakan penggunaan praktik- praktik situasi sosial, dan historis, serta kultural dalam mewujudkan dan menafsirkan suatu makna melalui teks. Jadi dapat di- jelaskan bahwa literasi merupakan sebuah keahlian yang dimiliki oleh seseorang Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 83 untuk menuangkan dan menyajikan situasi-situasi dan berbagai fenomena atau kejadian melalui sebuah teks. Menurut Buckingham (2007) dalam Suhaimi (2017) literasi memiliki 4 elemen penting yakni representasi, bahasa, kreasi, dan khalayak. Bahasa termasuk salah satu komponen penting dalam literasi maka dari itu ke- mampuan berbahasa dapat ditingkatkan melalui sebuah literasi baik menulis atau membaca. lancar membaca. 1. Pendahuluan Hasil dari minggu kedua adalah cara membaca dan minat baca siswa bisa menjadi lebih lancar dan lebih baik dari sebelumnya. Kemudian hasil pada minggu ketiga peserta didik jadi lebih percaya diri untuk terampil berbicara di depan kelas dan kosa kata yang mereka gunakan dalam berbicara lebih banyak setelah pembiasaan budaya literasi dan public speaking yang diterapkan oleh peneliti. Dalam pelajaran Bahasa Indonesia, kegiatan literasi sangat banyak peran- annya karena literasi identik dengan mem- baca dan menulis. Walaupun dalam kon- disi daring seperti saat ini yaitu pembelaja- ran dilakukan secara online jarak jauh, ke- mampuan literasi siswa tetap perlu diasah dan ditingkatkan karena membaca dan menulis adalah sebuah kemampuan yang penting untuk dikuasai agar menjadi seseorang dengan kemampuan bahasa yang baik. Walaupun demikian, pen- guasaan literasi yang tinggi tentunya tidak boleh mengabaikan aspek sosiokultural, karena literasi merupakan bagian dari kul- tur atau budaya manusia (Kurniawan et al., 2019). Literasi yang dimiliki oleh siswa SMA Prawira Marta tergolong dalam kon- disi yang terbatas dikarenakan masih ban- yak siswa yang tidak peduli dan termoti- vasi untuk belajar menulis. Maka dari itu, pada kegiatan KKN-DIK yang dilakukan oleh mahasiswa Universitas Muhammadi- yah Surakarta di SMA Prawira Marta akan fokus kepada pendampingan guna meningkatkan kemampuan literasi menu- lis siswa SMA Prawira Marta. 2. Metode Metode yang dijalankan dalam KKN- Dikmas ini ialah metode observasi dan metode dokumentasi. Observasi merupa- kan salah satu kegiatan ilmiah empiris yang mendasarkan fakta-fakta lapangan maupun teks, melalui pengalaman panca indra tanpa menggunakan manipulasi apapun (Hasanah, 2017). Observasi dil- akukan dengan cara meninjau dan men- catat secara sistematiks semua hal yang ada di area SMA Prawira Marta. Pada metode ini, penulis terjun ke lokasi untuk meninjau secara langsung aktivitas-aktivi- tas dan kejadian-kejadian yang ditemui se- bagai pengaruh dari pelaksanaan KKN- Dalam kegiatan KKN yang dil- aksanakan oleh Saiful Jihad dan kelompok- nya pada tahun 2020 dengan judul kegiatan “Meningkatkan Budaya Literasi dan Public Speaking pada Kalangan Peserta Didik di MI Muhammadiyah Ngancar” menghasilkan beberapa hasil kegiatan tiap minggunya. Hasil minggu pertama, minat baca dari masing-masing dari tiap siswa menunjukkan bahwa siswa MI Muham- madiyah Ngancar banyak yang belum Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 84 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… Dikmas. Data yang dibutuhkan dalam metode ini yakni meninjau secaralangsung ke lokasi, proses pelaksanaan pembelaja- ran daring, dan aktivitas warga SMA Prawira Marta di situasi daring. Dikmas. Data yang dibutuhkan dalam metode ini yakni meninjau secaralangsung ke lokasi, proses pelaksanaan pembelaja- ran daring, dan aktivitas warga SMA Prawira Marta di situasi daring. Melaksanakan pendampingan belajar melalui media whatsapp metode: men- guraikan cara yang digunakan untuk me- mecahkan masalah. 3. Hasil dan Pembahasan Metode Dokumentasi adalah alat pengumpulan data yang digunakan untuk mencari, mengenal hal – hal atau variable yang berupa catatan, transkip buku, surat kabar, majalah, peraturan – peraturan, notulen rapat dan sebagainya (U. Hasanah, 2017). Metode dokumentasi dilakukan me- lalui cara menggabungkan sumber data berwujud catatan dokumen, foto-foto, dan video. Metode ini merupakan salah satu wujud pengumpulan data yang paling se- derhana, karena penulis hanya mengamati benda mati dan bilamana terdapat kesala- han mudah untuk memperbaikinya karena sumber datanya tetap dan tidak berubah. Hasil penelitian diuraikan berdasar- kan hasil pengamatan dalam proses meningkatkan literasi menulis siswa SMA Marta melalui pendampingan dalam pem- belajaran Bahasa Indonesia yang dil- akukan melalui aplikasi whatsapp chat dan grup. Hasil data ini berdasarkan data yang telah dikumpulkan melalui observasi dan catatan selama pembelajaran daring berlangsung. Dalam program meningkatkan lit- erasi menulis siswa ini, siswa diarahkan untuk menulis sebuah proposal kegiatan dan karya ilmiah. Penulisan proposal kegiatan dan karya ilmiah ini berdasarkan materi pelajaran yang sedang diajarkan pada pembelajaran Bahasa Indonesia. Selain dijadikan sebagai data program kerja yang sedang dilakukan dalam KKN- Dikmas, penulisan proposal kegiatan dan karya ilmiah juga dijadikan sebagai bentuk upaya meningkatkan literasi menulis siswa dijadikan sebagai nilai tugas pelaja- ran Bahasa Indonesia. Strategi pelaksanaan peningkatan rasa tanggung jawab siswa berkenaan tu- gas yang diberikan oleh guru ini dilakukan dalam bentuk pendampingan dan penyusunan bahan ajar melalui tahapan- tahapan sebagai berikut: a) Melakukan observasi pada ling- kungan pembelajaran dan mengamati proses pembelajaran dar- ing. b) Menyusun jadwal pertemuan dengan guru mata pelajaran BahasaIndone- sia. Saat pelaksanaan program tersebut siswa diarahkan untuk menulis sebuah proposal kegiatan dengan tema bebas dan siswa bebas mencarinya referensi di inter- net. Kemudian siswa menulis proposal kegiatan sesuai dengan sistematika yang telah ditentukan. Sistematika tersebut sesuai dengan yang telah disampaikan pada materi pelajaran Bahasa Indonesia. c) Mempersiapkan konsep materi yang akan disampaikan dalam kegiatan sesuai dengan kebutuhan siswa. d) Mengolah materi, pengetikan, dan latihan-latihan soal yang akan diberi- kan untuk siswa. Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 85 Guru menyampaikan materi pelajaran me- lalui whatsapp grup dengan mengirimkan modul sesuai materi yang diajarkan. (Lembar Kerja Siswa). Untuk mengem- bangkan baca siswa, perlu adanya pening- katan sumber rujukan yang me- nyenangkan dengan berbantuan modul pembelajaran digital yang dibuat lebih ringkas dan menarik untuk mengurangi kejenuhan siswa dan ketergantungan pada LKS. 3. Hasil dan Pembahasan Berikut merupakan laporan hasil pelaksanaan kegiatan yang dilaksanakan selama program KKN-Dikmas berlang- sung. Laporan hasil pelaksanaa KKN-Dik tersaji dalam bentuk rincian kegiatan yang dimulai pada tanggal 1 Februari 2021 sam- pai 6 Maret 2021. Pembelajaran daring mata pelajaran Bahasa Indonesia di SMA Prawira Marta dilaksanakan setiap hari Rabu pukul 07.30 WIB hingga pukul 12.00 WIB. Penyusunan materi pembelajaran yang berupa modul digital tersebut dirancang setiap hari Senin dan Selasa, dan diberikan kepada siswa SMA Prawira Marta di hari Rabu melalui media grup whatsapp. Berikut adalah be- berapa contoh modul pembelajaran yang diberikan ke siswa: c. Materi pelajaran dengan berbantuan modul digital tersampaikan kepada se- luruh siswa SMA Prawira Marta dengan lancar. Akan tetapi, masih ada sebagian siswa yang merespon dengan lambat bahkan tidak mau membaca modulnya sama sekali sehingga harus ditegur terus menerus. Untuk mengatasi masalah terse- but, pendidik selalu mengingatkan dan menghimbau siswa agar mereka mau bertekad meningkatkan kemampuan lit- erasi minat bacanya sehingga saat me- masuki tahap literasi menulis tidak akan mengalami kendala. Modul digital memiliki keunggulan dapat menampilkan beberapa materi dengan menggunakan media interaktif (Aprilliyah & Wahjudi, 2014; Sugianto, Ab- dullah, Elvyanti & Muladi, 2013; Sujanem Suswandi & Ganesha, 2012). Tujuan pem- buatan modul digital yakni untuk memu- dahkan siswa menguasai materi pelajaran yang diajarkan guru. Selain itu, modul digital dapat dipadukan dengan model pembelajaran inovatif yang dipandang mampu mening- katkan hasil belajar. Dalam modul digital, proyek bisa dijadikan sebagai salah satu teknik penilaian yang digunakan. Teknik ini mampu mendorong pembelajaran yang menantang dan meningkatkan kemam- puan siswa dalam memecahkan masalah (Winatha, Suharsono & Agustin, 2018). Oleh karena itu, pengembangan modul digital ini dalam bentuk proyek, dipan- dang penting untuk meningkatkan moti- vasi, pemahaman konsep, kemampuan siswa. a. Hasil Program berupa Modul Pem- belajaran Untuk mengembangkan kemampuan literasi menulis siswa dapat dimulai dari pengembangan minat baca siswa. Dalam tahap ini, pembelajaran Bahasa Indonesia di SMA Marta hanya mengacu pada variasi teks yang tersedia dalam format LKS Gambar 1. Tampilan Awal Modul Kelas XII Gambar 2. Tampilan Materi Modul Kelas XI Gambar 3. Tampilan Materi Modul Kelas X Gambar 1. Tampilan Awal Modul Kelas XII Gambar 2. Tampilan Materi Modul Kelas XI Gambar 1. Tampilan Awal Modul Kelas XII Gambar 3. Tampilan Materi Modul Kelas X Gambar 3. Tampilan Materi Modul Kelas X Gambar 1. Tampilan Awal Modul Kelas XII Gambar 2. Tampilan Materi Modul Kelas XI Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 86 b. Pembahasan Hasil Program Literasi Menulis (Modul Pembelajaran Dig- ital) dikembangkan memiliki dua manfaat yakni sebagai alat bantu belajar siswa di rumah dan bisa digunakan oleh guru se- bagai alat bantu mengajar di kelas. Jadi pembuatan modul digital tersebut dirancang dengan menarik serta materi yang dibuat lebih ringkas, supaya siswa se- mangat untuk membacanya dan akan mengembangkan kemampuan literasi lanjut. National Center For Vocational Educa- tion Research Ltd (Prastowo, 2014) mendefinisikan materi ajar sebagai segala bentuk materi yang dipakai untuk mem- bantu pendidik atau instruktur saat melaksanakan proses pembelajaran di ke- las. Salah satu bentuk materi ajar adalah modul (Tania & Susilo wibowo, 2017). Saat ini, modul terbagi menjadi dua kategori, yakni modul yang cetak dan modul digital. d. Hasil Program Literasi Menulis (Latihan Soal) Untuk mengembangkan kemampuan literasi menulis siswa di tahap berikutnya adalah dengan cara memberikan latihan soal. Semakin sering siswa berlatih mengerjakan latihan soal, maka kemam- puan literasi menulisnya pun akan se- makin meningkat. Dalam tahap ini, latihan soal pada pembelajaran Bahasa Indonesia di SMA Prawira Marta dituangkan dalam modul digital pada bagian akhir. Hasil Menguasai dan mengamalkan suatu draf tertentu (Zulhaini, 2016). Modul yang Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 87 jawabannya dituangkan kedalamselem- barkertasdandifotokanlalu dikirimkan ke nomor whatsapp pendidik sebagai bukti bahwa siswa telah selesai mengerjakan. kebutuhan dan kemampuan siswa. Jumlah soal-soal latihannya antara 3-6 butir soal saja, sehingga siswa tidak akan merasa terbebani apabila mendapatkan banyak butir soal. Berikut adalah beberapa contoh latihan soal yang diberikan ke siswa: Latihan soal dibuat dan dirancang dengan sederhana sesuai dengan Gambar 4. Hasil Latihan Soal Kelas X Gambar 5. Hasil Latihan Soal Kelas XI Gambar 6. Hasil Latihan Soal Kelas XI Gambar 6. Hasil Latihan Soal Kelas XI Gambar 5. Hasil Latihan Soal Kelas XI Gambar 4. Hasil Latihan Soal Kelas X Gambar 4. Hasil Latihan Soal Kelas X Gambar 5. Hasil Latihan Soal Kelas XI Gambar 5. Hasil Latihan Soal Kelas XI Gambar 4. Hasil Latihan Soal Kelas X Gambar 6. Hasil Latihan Soal Kelas XI Dengan menulis dapat melatih kita untuk berpikir kritis dan logis dalam memeca- hkan masalah (Ati and Widiyarto, 2020). e. Pembahasan Hasil Program Literasi g. Hasil Program Literasi Menulis (Pengayaan dan Penambahan Waktu) g. Hasil Program Literasi Menulis (Pengayaan dan Penambahan Waktu) Berdasarkan teori di atas, kemahiran pemecahan masalah adalah kemampuan untuk mengamalkan pemahaman yang di- miliki sebelumnya ke dalam kondisi baru yang mengaitkan proses berpikir tingkat tinggi. Kemahiran pemecahan masalah (latihan soal) siswa SMA Prawira Marta ditinjau dari motivasi belajarnya. Perihal tersebut bertujuan supaya pendidik dapat menunjang motivasi belajar, memperbaiki kesalahan dalam pemecahan masalah (lati- han soal), dan menangani kesulitan yang dialami oleh siswa. Jadi, semakin sering siswa berlatih mengerjakan latihan soal, maka semakin banyak kesempatan siswa melatih manajemen waktu pengerjaannya. Semakin rutin mengerjakan latihan soal, maka siswa akan semakin siap menghadapi ujian-ujian kedepannya. Untuk mengembangkan kemampuan literasi menulis siswa pada tahap terakhir adalah dengan cara memberikan penga- yaan dan penambahan waktu untuk siswa yang kemampuan literasi menulisnya ku- rang. Pemberian pengayaan dan penamba- han waktu tersebut sebagai bentuk kesem- patan kepada siswa yang mungkin belum sempat membaca modul dan mengerjakan latihan soal karena suatu halangan ter- tentu. Dalam tahap ini, pemberian penga- yaan dan penambahan waktu pada siswa SMA Prawira Marta yang kemampuan lit- erasi menulisnya kurang dibatasi dengan waktu yang telah ditentukan. Biasanya pemberian penambahan waktu pada siswa berkisar antara satu sampai tujuh hari, ter- gantung tingkat kesulitan tiap-tiap soal. Berikut adalah beberapa contoh bukti penambahanwaktu yang diberikan kesiswa yang kemampuan literasi menu- lisnya kurang: Si A e. Pembahasan Hasil Program Literasi e. Pembahasan Hasil Program Literasi Menulis (Latihan Soal) Kemahiran dalam pemecahan masalah (latihan soal) merupakan salah satu kemampuan pent- ing yang harus dikuasai siswa. Menurut Anderson (2009), pemecahan masalah ada- lah keahlian hidup yang melibatkan teknik menelaah, menguraikan, melogika, mem- prediksi, menilai, dan merefleksikan. Ren- dahnya kemampuan pemecahan masalah siswa salah satunya disebabkan oleh moti- vasi belajar. Selain itu, menulis juga meru- pakan suatu aktivitas komunikasi yang menggunakan bahasa sebagai medianya. Muzaki (2010) menemukan hasil bahwa kreativitas dan motivasi belajar siswa berdampak penting pada keahlian siswa dalam mengatasi persoalan. Menurut Sihombing (2021) siswa yang menguasai konsep dapat mengindetifikasi- kan dan mengerjakan soal baru yang lebih bervariasi. Selain itu, apabila anak me- mahami suatu konsep maka ia dapat menggeneralisasikan suatu objek dalam berbagai situasi lain yang tidak digunakan Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 88 kesempatan bagi siswa yang belum sempat mengerjakan latihan soal. dalam situasi belajar. Hal ini menyimpul- kan bahwa apabila siswa mempunyai kre- ativitas dan motivasi belajar yang tinggi maka kemahiran siswa dalam memeca- hkan persoalan juga akan baik. f. Evaluasi dan Tindak Lanjut Latihan-latihan soal yang dituangkan dalam modul digital pada bagian akhir ter- sampaikan kepada seluruh siswa SMA Prawira Marta dengan baik dan beberapa siswa mampu merespon serta mengerjakan dengan baik pula. Namun, ada pula siswa yang lambat bahkan ada yang tidak mengerjakannya sama sekali. Untuk mengatasi masalah tersebut, pen- didik selalu menghimbau dan memotivasi siswa supaya mau berusaha mengerjakan latihan soal semampunya dan memberikan Siswa B Siswa C Siswa C Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 89 Berdasarkan pemaparan di atas, ke- mampuan literasi menulis siswa SMA Prawira Marta berbeda-beda. Hal ini ter- bukti dari siswa yang mengumpulkan tu- gas ke guru tiap minggunya hanya sedikit. Bahkan, rata-rata siswanya hampir semua memiliki kemampuan literasi menulis yang kurang. Dengan demikian, pem- berian pengayaan dan penambahan waktu sangat penting dan dibutuhkan siswa un- tuk menyeimbangkan siswa satu dengan yang lain, serta meningkatkan ke- percayaan diri siswa-siswa yang lit- erasinya kurang supaya mereka yakin bahwa mereka bisa meningkatkan kemam- puannya seperti siswa lain yang kemam- puan literasinya baik. h. Pembahasan Hasil Program Literasi Menulis (Pengayaan dan Penamba- han Waktu) h. Pembahasan Hasil Program Literasi Menulis (Pengayaan dan Penamba- han Waktu) Kegiatan pengayaan yakni aktivitas yang diberikan kepada siswa agar mereka mampu mengembangkan potensinya secara optimal dengan memanfaatkan sisa waktu yang dimilikinya. Aktivitas tersebut bertujuan untuk memberikan kesempatan bagi siswa untuk memperdalam pema- hamannya tentang materi pelajaran yang bertautan dengan tugas yang sedang dik- erjakan, sehingga dapat mencapai tingkat perkembangan yang baik dan optimal. Jadi, dapat dikatakan bahwa pengayaan merupakan aktivitas yang diberikan pada siswa supaya mereka menjadi lebih kaya pengetahuan dan kemahirannya serta bisa mendalami materi pembelajaran yang se- dang mereka pelajari. i. Evaluasi dan Tindak Lanjut Pemberian pengayaan dan penamba- han waktu yang diberikankepada siswa SMA Prawira Marta yang memiliki ke- mampuan literasi menulisnya kurang dapat terlaksana dengan baik. Beberapa siswa mampu memanfaatkan kesempatan tersebut untuk mengerjakan tugas-tugas yang tertunda. Namun, masih ada pula siswa yang tidak memanfaatkan kesem- patan tersebut dengan baik. Untuk menga- tasi masalah tersebut, pendidik selalu memotivasi dan mendorong semangat siswa supaya mereka mampu memanfaat- kan kesempatan tambahan waktu tersebut dengan sebaik-baiknya untuk menutup nilai-nilai yang kosong dan dengan hara- pan mampu meningkatkan kemampuan literasi menulis mereka. Ketika guru merancang tugas yang akan diberikan kepada siswa untuk dik- erjakan di rumah, maka guru harus dapat memperhitungkan jangka waktu pengum- pulan tugas tersebut sesuai tingkat kesu- litan soal dan kemampuan masing- masing siswa. Jika tugas tersebut diselesaikan da- lam waktu 3 hari, maka guru dapat mem- berikan tambahan waktu sekitar 1-7 hari kepada siswa yang kemampuan literasi menulisnya kurang. Dengan begitu, siswa yang kemampuan literasi menulisnya ku- rang akan mampu mengimbangi siswa lain yang kemampuan literasi menulisnya baik. Bahkan, rata-rata siswanya hampir semua memiliki kemampuan literasi Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 90 Y., Agung, W., & Sholihah, I. (2020). Revitalisasi Perpustakaan untuk Meningkatkan Minat Literasi Siswa di SD Muhammadiyah Nurul Ilmi, Klaten. Buletin KKN Pendidikan, 1(2), 97–105. Y., Agung, W., & Sholihah, I. (2020). Revitalisasi Perpustakaan untuk Meningkatkan Minat Literasi Siswa di SD Muhammadiyah Nurul Ilmi, Klaten. Buletin KKN Pendidikan, 1(2), 97–105. menulis yang kurang. Dengan demikian, pemberian pengayaan dan penambahan waktu sangat penting dan dibutuhkan siswa untuk menyeimbangkan siswa satu dengan yang lain, serta meningkatkan ke- percayaan diri siswa-siswa yang lit- erasinya kurang supaya mereka yakin bahwa mereka bisa meningkatkan kemam- puannya seperti siswa lain yang kemam- puan literasinya baik. Ati, A.P. and Widiyarto, S. (2020) ‘Literasi Bahasa Dalam Meningkatkan Minat Baca Dan Menulis Pada Siswa Smp Kota Bekasi’, Basastra, 9(1), p. 105. Choirudin, F. M., & Rahmasari, S. N. (2021). Tingkat Cakap Tanggap Pe- serta Didik dalam Perangkat Google Classroom Selama Pembelajaran Dar- ing. Jurnal Ilmiah Kampus Mengajar, 62-69. 5. Daftar Pustaka Agustina, L., Arffianto, A., Khalishah, S. H., Indarwati, L., Putri, D. R., El- Majid, S. E., Rahayu, K. S., Nurleli, D. 4. Simpulan Kegiatan KKN-Dik di SMA Prawira Marta berjalan dengan lancar sesuai dengan apa yang direncanakan dalam pro- gram kerja. Adapun kesimpulan dari pelaksanaan program kerja KKN-Dik Te- matik ini yaitu: (1) Pelaksanaan kegiatan KKN-Dikmas di SMA Prawira Marta dim- ulai tanggal 1 Februari 2021 sampai dengan 6 Maret 2021; (2) Metode yang dijalankan dalam KKN- Dikmas ini dilakukan dengan cara mengamati dan mencatat secara siste- matik semua hal yang ada di area SMA Prawira Marta; (3) SMA Prawira Marta melaksanakan pembelajaran daring selama 5 hari dalam satu minggu dengan kurang lebih 20 tenaga pendidik dan 75siswa; (4) Proses pelaksanaan pembelaja- ran daring di SMA Prawira Marta menggunakan media whatsapp grub. Selama melaksanakan KKN Dik di SMA Prawira Marta, mahasiswa memperoleh banyak pengalaman berharga. Effendi, Darwin & Achmad Wahidy (2019). Pemanfaatan Teknologi Dalam Proses Pembelajaran Menuju Pem- belajaran Abad 21. Prosiding Seminar Nasional Pendidikan Program Pascasar- jana Universitas PGRI Palembang. Firmansyah, Bayu. 2017. Kompetensi Lit- erasi Mahasiswa dalam Pem- belajaran Mata Kuliah Seminar di Prodi Bahasa dan Sastra Indonesia. Jurnal Ilmiah Edukasi dan Sosial. Vol 8. No 1. Hasanah, H. (2017). Teknik-teknik ob- servasi (sebuah alternatif metode pengumpulan data kualitatif ilmu- ilmu sosial). At-Taqaddum, 8(1), 21– 46. Hasanah, U. (2017). Peningkatan Hasil Belajar Mata Pelajaran Fiqih Melalui Penerapan Metode PQRST (Preview, Question, Read, Summarize, Test) Pe- serta Didik Kelas V Di Mi Ismaria Al- Qur’aniyah Islamiyah Raja Basa Ban- dar Lampung Tahun Pelajaran Agustina, L., Arffianto, A., Khalishah, S. H., Indarwati, L., Putri, D. R., El- Majid, S. E., Rahayu, K. S., Nurleli, D. Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 91 Edukasi dan Teknologi Pembelajaran, Volume 1, No. 2 Edisi Juni. 2016/2017”. Al-Tadzkiyyah: Jurnal Pendidikan Islam, 8(1), 1–14. Ihwan, I., Lestari, N., Wulansari, F., Wulan- sari, W., & Khasanah, M. (2021). Pengembangan Diri Anak SD Ber- basis Seni pada Masa Pandemi di Kupang. Buletin KKN Pendidikan, 3(1), 33–44. Machromah, I. U., Mahendra, A., Dianing- sih, F. R., Indriani, N., Agustina, D. R., Fatimah, S., Arifah, N. Z. M., Al Khusna, M., Novianto, D., & Nugroho, F. C. (2020). Perpustakaan Dinding sebagai Program Gerakan Literasi Sekolah untuk Meningkatan Kemampuan Membaca Siswa MI Mu- hammadiyah Kuncen Cawas, Klaten. Buletin KKN Pendidikan, 2(2), 100–104. Irwandani, dkk. 2017. Modul Digital Inter- aktif Berbasis Articulate Stu- dio’13: Pengembangan Pada Materi Gerak Melingkar Kelas X. Jurnal Ilmiah Pendidikan Fisika. Vol. 6. No 2. Mahardhani, A. 4. Simpulan J., Prayitno, H. J., Huda, M., Fauziati, E., Aisah, N., & Praset- iyo, A. D. (2021). Pemberdayaan Siswa SD dalam Literasi Membaca melalui Media Bergambar di Magetan. Buletin KKN Pendidikan, 3(1), 11–22. Joyo, Aceng. 2018. Gerakan Literasi dalam Pembelajaran Bahasa Indonesia Berbasis Kearifan Lokal Menuju Siswa Berkarakter. Jurnal KIBASP (Kajian Bahasa, Sastra, dan Pengaja- rannya). Vol 1. No 2. Ningrum, F. W., Nurheni, A., Umami, S. A., Sufanti, M., & Rohmadi, R. (2021). Revitalisasi Budaya Literasi melalui Pemanfaatan Infografis di SMK Suka- wati Gemolong Kala Pandemi. Buletin KKN Pendidikan, 3(2), 161–168. Khikmawati, D. K., Alfian, R., Nugroho, A. A., Susilo, A., Rusnoto, R., & Choli- fah, N. (2021). Pemanfaatan E-book untuk Meningkatkan Minat Belajar Siswa Sekolah Dasar di Kudus. Buletin KKN Pendidikan, 3(1), 74–82. Suryadi, Sudi. (2015). Peranan Perkem- bangan Teknologi Informasi Dan Komunikasi Dalam Kegiatan Pem- belajaran Dan Perkembangan Dunia Pendidikan. Jurnal Ilmiah AMIK Labuhan Batu, Vol.3 No.3. Kurniawan, K. R. A., Latif, N., Suparno, R. R., Oktaviani, A., Zharifa, A. F., & Ar- yanti, S. Z. (2019). Revitalisasi Rumah Pintar Laskar Pelangi di Gantung me- lalui Budaya Literasi Humanitas. Buletin KKN Pendidikan, 1(1), 10–17. Nuroniyah, A. (2021). Home Teaching Era Pandemi Covid-19 pada Siswa SD se- bagai Upaya Mengatasi Problematika Pembelajaran Daring. Warta LPM, 24(3), 466–475. Lathifah, Z. K., & Utami, I. I. S. (2021). Bimbingan Teknis Aplikasi Portal Ru- mah Belajar pada Praktik Pembelaja- ran Daring di Masa Pandemi Covid- 19. Warta LPM, 24(2), 367–379. Puspitasari, Anggraini Diah. 2019. Penera- pan Media Pembelajaran Fisika Menggunakan Modul Cetak dan Modul Elektronik pada Siswa SMA. Latip, Abdul (2020). Peran Literasi Teknologi Informasi Dan Komu- nikasi Pada Pembelajaran Jarak Jauh Di Masa Pandemi Covid-19. Jurnal Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 92 Jurnal Pendidikan Fisika. Vol 7. No 1. H., Puspitasari, N. H., & Imaduddin, Z. (2020). Revitalisasi Perpustakaan Jurnal Pendidikan Fisika. Vol 7. No 1. H., Puspitasari, N. H., & Imaduddin, Z. (2020). 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Pem- anfaatan Aplikasi Evaluasi Belajar Berbasis Web Era Covid-19. Berdikari: Jurnal Inovasi Dan Penerapan Ipteks, 8(2), 104–114. Sunanda, A., Salma, I. A., Nugroho, Y. S., Aulia, K. M., Wilartono, R. Y., Farisa, D., Susilowati, E., Kusumaningrum, Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 e-ISSN 2828-6375
https://openalex.org/W4361814456
https://figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_A_Fully_Human_Insulin-Like_Growth_Factor-I_Receptor_Antibody_SCH_717454_Robatumumab_Has_Antitumor_Activity_as_a_Single_Agent_and_in_Combination_with_Cytotoxics_in_Pediatric_Tumor_Xenografts/22485270/1/files/39936825.pdf
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Supplementary Figure 3 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts
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https://openalex.org/W4205845434
https://www.cambridge.org/engage/api-gateway/coe/assets/orp/resource/item/61134722abc9e20d306ae1d0/original/robin-criterion-on-divisibility.pdf
English
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Robin Criterion on Divisibility
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Mathematics Subject Classification (2010) MSC 11M26 · MSC 11A41 · MSC 11A25 Mathematics Subject Classification (2010) MSC 11M26 · MSC 11A41 · MSC 11A25 Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) F. Vega CopSonic, 1471 Route de Saint-Nauphary 82000 Montauban, France ORCiD: 0000-0001-8210-4126 E-mail: vega.frank@gmail.com Robin Criterion on Divisibility Frank Vega the date of receipt and acceptance should be inserted later Abstract Robin criterion states that the Riemann Hypothesis is true if and only if the inequality σ(n) < eγ ×n×loglogn holds for all n > 5040, where σ(n) is the sum-of- divisors function and γ ≈0.57721 is the Euler-Mascheroni constant. We show that the Robin inequality is true for all n > 5040 which are not divisible by any prime number between 2 and 953. We prove that the Robin inequality holds when π2 6 ×loglogn′ ≤ loglogn for some n > 5040 where n′ is the square free kernel of the natural number n. The possible smallest counterexample n > 5040 of the Robin inequality complies that necessarily (logn)β < 1.2592 × log(Nm), where Nm = ∏m i=1 qi is the primorial number of order m and β = ∏m i=1 qai+1 i qai+1 i −1 when n is an Hardy-Ramanujan integer of the form ∏m i=1 qai i . the form ∏m i=1 qai i . Keywords Riemann hypothesis · Robin inequality · sum-of-divisors function · prime numbers Theorem 1.4 The smallest counterexample of the Robin inequality greater than 5040 must be a superabundant number [1]. On the other hand, suppose that n > 5040 might be the possible smallest coun- terexample of the Robin inequality, then we demonstrate that necessarily (logn)β < 1.2592 × log(Nm), where Nm = ∏m i=1 qi is the primorial number of order m and β = qai+1 ai ∏m i=1 q i i qai+1 i −1 when n is an Hardy-Ramanujan integer of the form ∏m i=1 qai i . ∏m i=1 qi qai+1 i −1 when n is an Hardy-Ramanujan integer of the form ∏m i=1 qai i . 1 Introduction In mathematics, the Riemann Hypothesis is a conjecture that the Riemann zeta func- tion has its zeros only at the negative even integers and complex numbers with real part 1 2 [6]. As usual σ(n) is the sum-of-divisors function of n [3]: ∑ d|n d 2 F. Vega F. Vega where d | n means the integer d divides to n and d ∤n means the integer d does not divide to n. Define f(n) to be σ(n) n . Say Robins(n) holds provided where d | n means the integer d divides to n and d ∤n means the integer d does not divide to n. Define f(n) to be σ(n) n . Say Robins(n) holds provided f(n) < eγ ×loglogn. f(n) < eγ ×loglogn. The constant γ ≈0.57721 is the Euler-Mascheroni constant, and log is the natural logarithm. The importance of this property is: Theorem 1.1 Robins(n) holds for all n > 5040 if and only if the Riemann Hypothesis is true [6]. Theorem 1.1 Robins(n) holds for all n > 5040 if and only if the Riemann Hypothesis is true [6]. It is known that Robins(n) holds for many classes of numbers n. Theorem 1.2 Robins(n) holds for all n > 5040 that are not divisible by 2 [3]. Theorem 1.2 Robins(n) holds for all n > 5040 that are not divisible by 2 [3]. On the one hand, we prove that Robins(n) holds for all n > 5040 that are not divisible by any prime number between 3 and 953. We recall that an integer n is said to be square free if for every prime divisor q of n we have q2 ∤n [3]. Robins(n) holds for all n > 5040 that are square free [3]. In addition, we show that Robins(n) holds for some n > 5040 when π2 6 ×loglogn′ ≤loglogn such that n′ is the square free kernel of the natural number n. Let q1 = 2,q2 = 3,...,qm denote the first m consecutive primes, then an integer of the form ∏m i=1 qai i with a1 ≥a2 ≥··· ≥am ≥0 is called an Hardy- Ramanujan integer [3]. A natural number n is called superabundant precisely when, for all m < n f(m) < f(n). Theorem 1.3 If n is superabundant, then n is an Hardy-Ramanujan integer [2]. Theorem 1.3 If n is superabundant, then n is an Hardy-Ramanujan integer [2]. Theorem 1.4 The smallest counterexample of the Robin inequality greater than 5040 must be a superabundant number [1]. 2 A Central Lemma These are known results: These are known results: These are known results: Lemma 2.1 [3]. For n > 1: Lemma 2.1 [3]. For n > 1: f(n) < ∏ q|n q q−1. (2.1) (2.1) Lemma 2.2 [4]. Lemma 2.2 [4]. ∞ ∏ k=1 1 1−1 q2 k = ζ(2) = π2 6 . (2.2) (2.2) 3 3 Robin Criterion on Divisibility The following is a key lemma. It gives an upper bound on f(n) that holds for all n. The bound is too weak to prove Robins(n) directly, but is critical because it holds for all n. Further the bound only uses the primes that divide n and not how many times they divide n. Lemma 2.3 Let n > 1 and let all its prime divisors be q1 < ··· < qm. Then, f(n) < π2 6 × m ∏ i=1 qi +1 qi . Proof We use that lemma 2.1: Proof We use that lemma 2.1: Proof We use that lemma 2.1: Now for q > 1, So So Then by lemma 2.2, Then by lemma 2.2, q Putting this together yields the proof: f(n) < m ∏ i=1 qi qi −1 ≤ m ∏ i=1 1 1−1 q2 i × qi +1 qi < π2 6 × m ∏ i=1 qi +1 qi . f(n) < m ∏ i=1 qi qi −1 ≤ m ∏ i=1 1 1−1 q2 i × qi +1 qi < π2 6 × m ∏ i=1 qi +1 qi . f(n) < m ∏ i=1 qi qi −1 ≤ m ∏ i=1 1 1−1 q2 i × qi +1 qi < π2 6 × m ∏ i=1 qi +1 qi . 3 About the p-adic order In basic number theory, for a given prime number p, the p-adic order of a natural number n is the highest exponent νp ≥1 such that pνp divides n. This is a known result: Lemma 3.1 In general, we know that Robins(n) holds for a natural number n > 5040 that satisfies either ν2(n) ≤19, ν3(n) ≤12 or ν7(n) ≤6, where νp(n) is the p-adic order of n [5]. F. Vega 4 We know the following lemmas: We know the following lemmas: Lemma 3.2 [5]. Let ∏m i=1 qai i be the representation of n as a product of primes q1 < ··· < qm with natural numbers as exponents a1,...,am. Then, f(n) = m ∏ i=1 qi qi −1 ! × m ∏ i=1 1− 1 qai+1 i ! . Lemma 3.3 [5]. Let n > ee23.762143 and let all its prime divisors be q1 < ··· < qm, then m ∏ i=1 qi qi −1 ! < 1771561 1771560 ×eγ ×loglogn. Lemma 3.4 Robins(n) holds for all 101010 ≥n > 5040 [5]. since 923520 923521 × 1771561 1771560 < 1. In light of lemma 3.4 and the fact that ee23.762143 < 101010, we then conclude that Robins(n) holds for n > 5040 when ν31(n) ≤3. Lemma 3.4 Robins(n) holds for all 101010 ≥n > 5040 [5]. Putting together all these results, then we obtain that Putting together all these results, then we obtain that Lemma 3.5 Robins(n) holds for n > 5040 when ν31(n) ≤3. Lemma 3.5 Robins(n) holds for n > 5040 when ν31(n) ≤3. Lemma 3.5 Robins(n) holds for n > 5040 when ν31(n) ≤3. Proof From lemma 3.2, we note that Proof From lemma 3.2, we note that f(n) = m ∏ i=1 qi qi −1 ! × m ∏ i=1 1− 1 qai+1 i ! ≤ m ∏ i=1 qi qi −1 ! ×  1− 1 31ν31(n)+1  when ν31(n) ≤3. We only need to look at the case where ν31(n) = 3 since the weaker cases follow because when ν31(n) ≤3. We only need to look at the case where ν31(n) = 3 since the weaker cases follow because  1− 1 311+1  <  1− 1 312+1  <  1− 1 313+1  . In this way, we obtain that In this way, we obtain that f(n) ≤ m ∏ i=1 qi qi −1 ! ×  1− 1 313+1  = 923520 923521 × m ∏ i=1 qi qi −1 ! when ν31(n) ≤3. With lemma 3.3, we have for n > ee23.762143 923520 923521 × m ∏ i=1 qi qi −1 ! < 923520 923521 × 1771561 1771560 ×eγ ×loglogn < eγ ×loglogn since 923520 923521 × 1771561 1771560 < 1. In light of lemma 3.4 and the fact that ee23.762143 < 101010, we then conclude that Robins(n) holds for n > 5040 when ν31(n) ≤3. since 923520 923521 × 1771561 1771560 < 1. In light of lemma 3.4 and the fact that ee23.762143 < 101010, we then conclude that Robins(n) holds for n > 5040 when ν31(n) ≤3. 5 Robin Criterion on Divisibility A Particular Case 4 A Particular Case 4 A Particular Case 4 A Particular Case We can easily prove that Robins(n) is true for certain kind of numbers: Lemma 4.1 Robins(n) holds for n > 5040 when q ≤7, where q is the largest prime divisor of n. Proof Let n > 5040 and let all its prime divisors be q1 < ··· < qm ≤5, then we need to prove Proof Let n > 5040 and let all its prime divisors be q1 < ··· < qm ≤5, then we need to prove f(n) < eγ ×loglogn f(n) < eγ ×loglogn that is true when that is true when m ∏ i=1 qi qi −1 ≤eγ ×loglogn according to the lemma 2.1. For q1 < ··· < qm ≤5, m ∏ i=1 qi qi −1 ≤eγ ×loglogn . For q1 < ··· < qm ≤5, m ∏ i=1 qi qi −1 ≤eγ ×loglogn according to the lemma 2.1. For q1 < ··· < qm ≤5, m ∏ i=1 qi qi −1 ≤2×3×5 1×2×4 = 3.75 < eγ ×loglog(5040) ≈3.81. However, we know for n > 5040 However, we know for n > 5040 eγ ×loglog(5040) < eγ ×loglogn and therefore, the proof is complete when q1 < ··· < qm ≤5. The remaining case is for n > 5040 when all its prime divisors are q1 < ··· < qm ≤7. Robins(n) holds for n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. Hence, it is enough to prove this for those natural numbers n > 5040 when 77 | n. For q1 < ··· < qm ≤7, m ∏ i=1 qi qi −1 ≤2×3×5×7 1×2×4×6 = 4.375 < eγ ×loglog(77) ≈4.65. However, for n > 5040 and 77 | n, we know that However, for n > 5040 and 77 | n, we know that eγ ×loglog(77) ≤eγ ×loglogn and as a consequence, the proof is complete when q1 < ··· < qm ≤7. and as a consequence, the proof is complete when q1 < ··· < qm ≤7. 5 A Better Bound This is a known result: This is a known result: Lemma 5.1 [7]. For x > 1: ∑ q≤x 1 q < loglogx+B+ 1 log2 x (5.1) where B = 0.2614972128··· denotes the (Meissel )Mertens constant [7] a 5.1 [7]. For x > 1: ∑ q≤x 1 q < loglogx+B+ 1 log2 x (5.1) ∑ q≤x 1 q < loglogx+B+ 1 log2 x (5.1) (5.1) where where B = 0.2614972128··· denotes the (Meissel-)Mertens constant [7]. denotes the (Meissel-)Mertens constant [7]. F. Vega 6 We show a better result: We show a better result: Lemma 5.2 For x ≥11, we have ∑ q≤x 1 q < loglogx+γ −0.12. ∑ q≤x 1 q < loglogx+γ −0.12. Proof Let’s define H = γ −B. The lemma 5.1 is the same as Proof Let’s define H = γ −B. The lemma 5.1 is the same as Proof Let s define H = γ −B. The lemma 5.1 is the same as ∑ q≤x 1 q < loglogx+γ −(H − 1 log2 x). For x ≥11, (H − 1 log2 x) > (0.31− 1 log2 11) > 0.12 and thus, ∑ q≤x 1 q < loglogx+γ −(H − 1 log2 x) < loglogx+γ −0.1 ∑ q≤x 1 q < loglogx+γ −(H − 1 log2 x). (H − 1 log2 x) > (0.31− 1 log2 11) > 0.12 ∑ q≤x 1 q < loglogx+γ −(H − 1 log2 x). For x ≥11, and thus, ∑ q≤x 1 q < loglogx+γ −(H − 1 log2 x) < loglogx+γ −0.12. 6 On a Square Free Number We know the following results: We know the following results: We know the following results: Lemma 6.1 [3]. For 0 < a < b: Lemma 6.1 [3]. For 0 < a < b: Lemma 6.1 [3]. For 0 < a < b: Lemma 6.1 [3]. For 0 < a < b: logb−loga b−a = 1 (b−a) Z b a dt t > 1 b. (6.1) (6.1) Lemma 6.2 [3]. For q > 0: log(q+1)−logq = Z q+1 q dt t < 1 q. (6.2) (6.2) We recall that an integer n is said to be square free if for every prime divisor q of n we have q2 ∤n [3]. Robins(n) holds for all n > 5040 that are square free [3]. Lemma 6.3 For a square free number Lemma 6.3 For a square free number n = q1 ×···×qm such that q1 < q2 < ··· < qm are odd prime numbers, qm ≥11 and 3 ∤n, then: π2 6 × 3 2 ×σ(n) ≤eγ ×n×loglog(219 ×n). 7 Robin Criterion on Divisibility Proof By induction with respect to ω(n), that is the number of distinct prime factors of n [3]. Put ω(n) = m [3]. We need to prove the assertion for those integers with m = 1. From a square free number n, we obtain σ(n) = (q1 +1)×(q2 +1)×···×(qm +1) (6.3) (6.3) when n = q1 × q2 × ··· × qm [3]. In this way, for every prime number qi ≥11, then we need to prove π2 6 × 3 2 ×(1+ 1 qi ) ≤eγ ×loglog(219 ×qi). (6.4) (6.4) For qi = 11, we have π2 6 × 3 2 ×(1+ 1 11) ≤eγ ×loglog(219 ×11) is actually true. For another prime number qi > 11, we have is actually true. For another prime number qi > 11, we have (1+ 1 qi ) < (1+ 1 11) and loglog(219 ×11) < loglog(219 ×qi) which clearly implies that the inequality (6.4) is true for every prime number qi ≥11. Now, suppose it is true for m−1, with m ≥2 and let us consider the assertion for those square free n with ω(n) = m [3]. So let n = q1 ×···×qm be a square free number and assume that q1 < ··· < qm for qm ≥11. 6 On a Square Free Number q1 qm qm ≥ Case 1: qm ≥log(219 ×q1 ×···×qm−1 ×qm) = lo q1 qm qm Case 1: qm ≥log(219 ×q1 ×···×qm−1 ×qm) = log(219 ×n). q1 qm qm Case 1: qm ≥log(219 ×q1 ×···×qm−1 ×qm) = log(219 ×n). ( By the induction hypothesis we have π2 6 × 3 2 ×(q1+1)×···×(qm−1+1) ≤eγ ×q1×···×qm−1×loglog(219×q1×···×qm−1) and hence and hence π2 6 × 3 2 ×(q1 +1)×···×(qm−1 +1)×(qm +1) ≤ eγ ×q1 ×···×qm−1 ×(qm +1)×loglog(219 ×q1 ×···×qm−1) when we multiply the both sides of the inequality by (qm +1). We want to show when we multiply the both sides of the inequality by (qm +1). We want to show eγ ×q1 ×···×qm−1 ×(qm +1)×loglog(219 ×q1 ×···×qm−1) ≤ eγ ×q1×···×qm−1×qm×loglog(219×q1×···×qm−1×qm) = eγ ×n×loglog(219×n). Indeed the previous inequality is equivalent with qm ×loglog(219 ×q1 ×···×qm−1 ×qm) ≥(qm +1)×loglog(219 ×q1 ×···×qm−1) or alternatively eγ ×q1 ×···×qm−1 ×(qm +1)×loglog(219 ×q1 ×···×qm−1) ≤ eγ ×q1×···×qm−1×qm×loglog(219×q1×···×qm−1×qm) = eγ ×n×loglog(219×n). I d d h i i li i i l i h Indeed the previous inequality is equivalent with qm ×loglog(219 ×q1 ×···×qm−1 ×qm) ≥(qm +1)×loglog(219 ×q1 ×···×qm−1) or alternatively qm ×(loglog(219 ×q1 ×···×qm−1 ×qm)−loglog(219 ×q1 ×···×qm−1)) logqm ≥ logqm F. Vega 8 We can apply the inequality in lemma 6.1 just using b = log(219 ×q1 ×···×qm−1 × qm) and a = log(219 ×q1 ×···×qm−1). Certainly, we have We can apply the inequality in lemma 6.1 just using b = log(219 ×q1 ×···×qm−1 × qm) and a = log(219 ×q1 ×···×qm−1). Certainly, we have We can apply the inequality in lemma 6.1 just using b = log(21 qm) and a = log(219 ×q1 ×···×qm−1). Certainly, we have log(219 ×q1 ×···×qm−1 ×qm)−log(219 ×q1 ×···×qm−1) = log 219 ×q1 ×···×qm−1 ×qm 219 ×q1 ×···×qm−1 = logqm. In this way, we obtain In this way, we obtain qm ×(loglog(219 ×q1 ×···×qm−1 ×qm)−loglog(219 ×q1 ×···×qm−1)) logqm > qm log(219 ×q1 ×···×qm). qm ×(loglog(219 ×q1 ×···×qm−1 ×qm)−loglog(219 ×q1 ×···×qm−1)) logqm > Using this result we infer that the original inequality is certainly satisfied if the next inequality is satisfied qm log(219 ×q1 ×···×qm) ≥loglog(219 ×q1 ×···×qm−1) logqm which is trivially true for qm ≥log(219 ×q1 ×···×qm−1 ×qm) [3]. Case 2: qm < log(219 ×q1 ×···×qm−1 ×qm) = log(219 ×n). We need to prove π2 6 × 3 2 × σ(n) n ≤eγ ×loglog(219 ×n). We know 3 2 < 1.503 < 4 2.66. 6 On a Square Free Number Nevertheless, we could have We know 3 2 < 1.503 < 4 2.66. Nevertheless, we could have 3 2 × σ(n) n × π2 6 < 4×σ(n) 3×n × π2 2×2.66 and therefore, we only need to prove and therefore, we only need to prove σ(3×n) 3×n × π2 5.32 ≤eγ ×loglog(219 ×n) where this is possible because of 3 ∤n. If we apply the logarithm to the both sides of the inequality, then we obtain log( π2 5.32)+(log(3+1)−log3)+ m ∑ i=1 (log(qi +1)−logqi) ≤γ +logloglog(219 ×n). In addition, note that log( π2 5.32) < 1 2 +0.12. However, we know 2) < 1 2 +0.12. However, we know In addition, note that log( π2 5.32) < 1 2 +0.12. However, we know In addition, note that log( π2 5.32) < 1 2 +0.12. However, we know In addition, note that log( π2 5.32) < 1 2 +0.12. However, we know γ +loglogqm < γ +logloglog(219 ×n) In addition, note that log( π2 5.32) < 1 2 +0.12. However, we know γ +loglogqm < γ +logloglog(219 ×n) γ +loglogqm < γ +logloglog(219 ×n) 9 Robin Criterion on Divisibility since qm < log(219 ×n). We use that lemma 6.2 for each term log(q+1)−logq and thus, since qm < log(219 ×n). We use that lemma 6.2 for each term log(q+1)−logq and thus 0.12+ 1 2 + 1 3 + 1 q1 +···+ 1 qm ≤0.12+ ∑ q≤qm 1 q ≤γ +loglogqm where qm ≥11. Hence, it is enough to prove where qm ≥11. Hence, it is enough to prove ∑ q≤qm 1 q ≤γ +loglogqm −0.12 ∑ q≤qm 1 q ≤γ +loglogqm −0.12 but this is true according to the lemma 5.2 for qm ≥11. In this way, we finally show the lemma is indeed satisfied. but this is true according to the lemma 5.2 for qm ≥11. In this way, we finally show the lemma is indeed satisfied. 7 Robin on Divisibility Robins(n) holds for every n > 5040 that is not divisible by 2 [3]. We extend this property to other prime numbers: Lemma 7.1 Robins(n) holds for all n > 5040 when 3 ∤n. More precisely: every pos- sible counterexample n > 5040 of the Robin inequality must comply with (220 ×313) | n. Proof We will check the Robin inequality is true for every natural number n = qa1 1 × qa2 2 × ··· × qam m > 5040 such that q1,q2,··· ,qm are distinct prime numbers, a1,a2,··· ,am are natural numbers and 3 ∤n. We know this is true when the great- est prime divisor of n > 5040 is lesser than or equal to 7 according to the lemma 4.1. Therefore, the remaining case is when the greatest prime divisor of n > 5040 is greater than or equal to 11. We need to prove f(n) < eγ ×loglogn that is true when that is true when π2 6 × m ∏ i=1 qi +1 qi ≤eγ ×loglogn according to the lemma 2.3. Using the formula (6.3) for the square free numbers, then we obtain that is equivalent to π2 6 × σ(n′) n′ ≤eγ ×loglogn where n′ = q1 × ··· × qm is the square free kernel of the natural number n [3]. The Robin inequality has been proved for all integers n not divisible by 2 (which are bigger than 10) [3]. Hence, we only need to prove the Robin inequality is true when 2 | n′. In addition, we know that Robins(n) holds for every n > 5040 when ν2(n) ≤19 according to the lemma 3.1 [5]. Consequently, we only need to prove that Robins(n) holds for n > 5040 when 220 | n and thus, eγ ×n′ ×loglog(219 × n′ 2 ) ≤eγ ×n′ ×loglogn F. Vega 10 because of 219 × n′ 2 ≤n where 220 | n and 2 | n′. So, because of 219 × n′ 2 ≤n where 220 | n and 2 | n′. So, because of 219 × n′ 2 ≤n where 220 | n and 2 | n′. So, π2 6 ×σ(n′) ≤eγ ×n′ ×loglog(219 × n′ 2 ). 7 Robin on Divisibility According to the formula (6.3) for the square free numbers and 2 | n′, then, According to the formula (6.3) for the square free numbers and 2 | n′, then, π2 6 ×3×σ(n′ 2 ) ≤eγ ×2× n′ 2 ×loglog(219 × n′ 2 ) which is the same as which is the same as π2 6 × 3 2 ×σ(n′ 2 ) ≤eγ × n′ 2 ×loglog(219 × n′ 2 ) where this is true according to the lemma 6.3 when 3 ∤n′ 2 . In addition, we know that Robins(n) holds for every n > 5040 when ν3(n) ≤12 according to the lemma 3.1 [5]. Hence, we only need to prove that Robins(n) holds for every n > 5040 when 220 | n and 313 | n. To sum up, the proof is complete. Lemma 7.2 Robins(n) holds for all n > 5040 when 5 ∤n or 7 ∤n. Lemma 7.2 Robins(n) holds for all n > 5040 when 5 ∤n or 7 ∤n. Proof We need to prove Proof We need to prove f(n) < eγ ×loglogn when (220 ×313) | n. Suppose that n = 2a ×3b ×m, where a ≥20, b ≥13, 2 ∤m, 3 ∤m and 5 ∤m or 7 ∤m. Therefore, we need to prove f(2a ×3b ×m) < eγ ×loglog(2a ×3b ×m). We know Lemma 7.3 Robins(n) holds for all n > 5040 when a prime number 11 ≤q ≤4 complies with q ∤n. Proof We know that Robins(n) holds for every n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. We need to prove Proof We know that Robins(n) holds for every n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. We need to prove f(n) < eγ ×loglogn when (220 ×313 ×77) | n. Suppose that n = 2a ×3b ×7c ×m, where a ≥20, b ≥13, c ≥7, 2 ∤m, 3 ∤m, 7 ∤m, q ∤m and 11 ≤q ≤47. Therefore, we need to prove when (220 ×313 ×77) | n. Suppose that n = 2a ×3b ×7c ×m, where a ≥20, b ≥13, c ≥7, 2 ∤m, 3 ∤m, 7 ∤m, q ∤m and 11 ≤q ≤47. Therefore, we need to prove f(2a ×3b ×7c ×m) < eγ ×loglog(2a ×3b ×7c ×m). f(2a ×3b ×7c ×m) < eγ ×loglog(2a ×3b ×7c ×m). We know We know f(2a ×3b ×m) = f(3b)× f(2a ×m) since f is multiplicative [3]. In addition, we know f(3b) < 3 2 for every natural number b [3]. In this way, we have f(3b)× f(2a ×m) < 3 2 × f(2a ×m). However, that would be equivalent to However, that would be equivalent to 3 2 × f(2a ×m) = 9 8 × f(3)× f(2a ×m) = 9 8 × f(2a ×3×m) where f(3) = 4 3 since f is multiplicative [3]. Nevertheless, we have where f(3) = 4 3 since f is multiplicative [3]. Nevertheless, we have 9 8 × f(2a ×3×m) < f(5)× f(2a ×3×m) = f(2a ×3×5×m) 9 8 × f(2a ×3×m) < f(5)× f(2a ×3×m) = f(2a ×3×5×m) and 9 8 × f(2a ×3×m) < f(7)× f(2a ×3×m) = f(2a ×3×7×m) 11 Robin Criterion on Divisibility where 5 ∤m or 7 ∤m, f(5) = 6 5 and f(7) = 8 7. We know the Robin inequality is true for 2a ×3×5×m and 2a ×3×7×m when a ≥20, since this is true for every natural number n > 5040 when ν3(n) ≤12 according to the lemma 3.1 [5]. Hence, we would h f(2a ×3×7×m) < eγ ×loglog(2a ×3×7×m) < eγ ×loglog(2a ×3b ×m) when b ≥13. Lemma 7.3 Robins(n) holds for all n > 5040 when a prime number 11 ≤q ≤47 complies with q ∤n. f(n) < eγ ×loglogn f(n) < eγ ×loglogn when (220 ×313 ×314) | n. Suppose that n = 2a ×3b ×31c ×m, where a ≥20, b ≥13, c ≥4, 2 ∤m, 3 ∤m, 31 ∤m, q ∤m and 53 ≤q ≤953. Therefore, we need to prove that f(2a ×3b ×31c ×m) < eγ ×loglog(2a ×3b ×31c ×m). f(2a ×3b ×31c ×m) < eγ ×loglog(2a ×3b ×31c ×m). We know that We know f(2a ×3b ×7c ×m) = f(7c)× f(2a ×3b ×m) f(2a ×3b ×7c ×m) = f(7c)× f(2a ×3b ×m) since f is multiplicative [3]. In addition, we know f(7c) < 7 6 for every natural number c [3]. In this way, we have f(7c)× f(2a ×3b ×m) < 7 6 × f(2a ×3b ×m). However, that would be equivalent to However, that would be equivalent to 7 6 × f(2a ×3b ×m) = 49 48 × f(7)× f(2a ×3b ×m) = 49 48 × f(2a ×3b ×7×m) where f(7) = 8 7 since f is multiplicative [3] In addition we know where f(7) = 8 7 since f is multiplicative [3]. In addition, we know 49 48 × f(2a ×3b ×7×m) < f(q)× f(2a ×3b ×7×m) = f(2a ×3b ×7×q×m) 49 48 × f(2a ×3b ×7×m) < f(q)× f(2a ×3b ×7×m) = f(2a ×3b ×7×q×m) where q ∤m, f(q) = q+1 q and 11 ≤q ≤47. Nevertheless, we know the Robin inequality is true for 2a × 3b × 7 × q × m when a ≥20 and b ≥13, since this is true for every natural number n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. Hence, we would have where q ∤m, f(q) = q+1 q and 11 ≤q ≤47. Nevertheless, we know the Robin inequality is true for 2a × 3b × 7 × q × m when a ≥20 and b ≥13, since this is true for every natural number n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. Hence, we would have f(2a ×3b ×7×q×m) < eγ ×loglog(2a ×3b ×7×q×m) < eγ ×loglog(2a ×3b ×7c ×m) when c ≥7 and 11 ≤q ≤47. when c ≥7 and 11 ≤q ≤47. 12 F. Vega F. Vega Lemma 7.4 Robins(n) holds for all n > 5040 when a prime number 53 ≤q ≤953 complies with q ∤n. Lemma 7.4 Robins(n) holds for all n > 5040 when a prime number 53 ≤q ≤953 complies with q ∤n. Lemma 7.4 Robins(n) holds for all n > 5040 when a prime number 53 ≤q ≤953 complies with q ∤n. Proof We know that Robins(n) holds for every n > 5040 when ν31(n) ≤3 according to the lemma 3.5. We need to prove that We know that f(2a ×3b ×31c ×m) = f(31c)× f(2a ×3b ×m) since f is multiplicative [3]. In addition, we know that f(31c) < 31 30 for every natural number c [3]. In this way, we have that f(31c)× f(2a ×3b ×m) < 31 30 × f(2a ×3b ×m). However, that would be equivalent to 31 30 × f(2a ×3b ×m) = 961 960 × f(31)× f(2a ×3b ×m) = 961 960 × f(2a ×3b ×31×m) where f(31) = 32 31 since f is multiplicative [3]. In addition, we know that 961 960 × f(2a ×3b ×31×m) < f(q)× f(2a ×3b ×31×m) = f(2a ×3b ×31×q×m) where q ∤m, f(q) = q+1 q and 53 ≤q ≤953. Nevertheless, we know the Robin in- equality is true for 2a ×3b ×31×q×m when a ≥20 and b ≥13, since this is true for every natural number n > 5040 when ν31(n) ≤3 according to the lemma 3.5. Hence, we would have that f(2a ×3b ×31×q×m) < eγ ×loglog(2a ×3b ×31×q×m) < eγ ×loglog(2a ×3b ×31c ×m) when c ≥4 and 53 ≤q ≤953. 13 Robin Criterion on Divisibility 13 8 Helpful Lemmas For every prime number pn > 2, we define sequence Yn = e 1 2×log(pn) (1− 1 log(pn) ). Lemma 8.1 For every prime number pn > 2, the sequence Yn is strictly decreasing. Proof For every real value x ≥3, we state the function Proof For every real value x ≥3, we state the function f(x) = e 1 2×log(x) (1− 1 log(x)) which is equivalent to f(x) = g(x)×h(u) where g(x) = e 1 2×log(x) and h(u) = u u−1 for u = log(x). We know that g(x) decreases as x ≥3 increases, Moreover, we note that h(u) decreases as u > 1 increases where u = log(x) > 1 for x ≥3. In conclusion, we can see that the function f(x) is monotonically decreasing for every real value x ≥3 and therefore, the sequence Yn is monotonically decreasing as well. In addition, Yn is essentially a strictly decreasing sequence, since there is not any natural number n > 1 such that Yn = Yn+1. where g(x) = e 1 2×log(x) and h(u) = u u−1 for u = log(x). We know that g(x) decreases as x ≥3 increases, Moreover, we note that h(u) decreases as u > 1 increases where u = log(x) > 1 for x ≥3. In conclusion, we can see that the function f(x) is monotonically decreasing for every real value x ≥3 and therefore, the sequence Yn is monotonically decreasing as well. In addition, Yn is essentially a strictly decreasing sequence, since there is not any natural number n > 1 such that Yn = Yn+1. In mathematics, the Chebyshev function θ(x) is given by Due to the theorem 8.3, we prove that Due to the theorem 8.3, we prove that m ∏ i=1 qi qi −1 < eγ ×(logqm + 1 2×log(qm)) < eγ ×log(Ym ×θ(qm)) m ∏ i=1 qi qi −1 < eγ ×(logqm + 1 2×log(qm)) < eγ ×log(Ym ×θ(qm)) when qm > 286. when qm > 286. In mathematics, the Chebyshev function θ(x) is given by In mathematics, the Chebyshev function θ(x) is given by θ(x) = ∑ p≤x log p where p ≤x means all the prime numbers p that are less than or equal to x. Lemma 8.2 [7]. For x ≥41: Lemma 8.2 [7]. For x ≥41: θ(x) > (1− 1 log(x))×x. Besides, we know that Besides, we know that Lemma 8.3 [7]. For x ≥286: Lemma 8.3 [7]. For x ≥286: ∏ q≤x q q−1 < eγ ×(logx+ 1 2×log(x)). ∏ q≤x q q−1 < eγ ×(logx+ 1 2×log(x)). We will prove another important inequality: We will prove another important inequality: Theorem 8.4 Let q1,q2,...,qm denote the first m consecutive primes such that q1 < q2 < ··· < qm and qm > 286. Then Theorem 8.4 Let q1,q2,...,qm denote the first m consecutive primes such that q1 < q2 < ··· < qm and qm > 286. Then m ∏ i=1 qi qi −1 < eγ ×log(Ym ×θ(qm)). F. Vega 14 Proof From the theorem 8.2, we know that Proof From the theorem 8.2, we know that Proof From the theorem 8.2, we know that θ(qm) > (1− 1 log(qm))×qm. In this way, we can show that log(Ym ×θ(qm)) > log  Ym ×(1− 1 log(qm))×qm  = logqm +log  Ym ×(1− 1 log(qm))  . We know that log  Ym ×(1− 1 log(qm))  = log   e 1 2×log(qm) (1− 1 log(qm)) ×(1− 1 log(qm))   = log  e 1 2×log(qm)  = 1 2×log(qm). Consequently, we obtain that logqm +log  Ym ×(1− 1 log(qm))  ≥(logqm + 1 2×log(qm)). Due to the theorem 8.3, we prove that m ∏ i=1 qi qi −1 < eγ ×(logqm + 1 2×log(qm)) < eγ ×log(Ym ×θ(qm)) when qm > 286. θ(qm) > (1− 1 log(qm))×qm. In this way, we can show that log(Ym ×θ(qm)) > log  Ym ×(1− 1 log(qm))×qm  = logqm +log  Ym ×(1− 1 log(qm))  . We know that log  Ym ×(1− 1 log(qm))  = log   e 1 2×log(qm) (1− 1 log(qm)) ×(1− 1 log(qm))   = log  e 1 2×log(qm)  = 1 2×log(qm). Consequently, we obtain that Consequently, we obtain that logqm +log  Ym ×(1− 1 log(qm))  ≥(logqm + 1 2×log(qm)). 9 Proof of Main Theorems Theorem 9.1 Robins(n) holds for all n > 5040 when a prime number q ≤953 com- plies with q ∤n. Proof This is a compendium of the results from the theorem 1.2 and the lemmas 7.1, 7.2, 7.3 and 7.4. Proof This is a compendium of the results from the theorem 1.2 and the lemmas 7.1, 7.2, 7.3 and 7.4. Theorem 9.2 Let π2 6 × loglogn′ ≤loglogn for some n > 5040 such that n′ is the square free kernel of the natural number n. Then Robins(n) holds. 15 Robin Criterion on Divisibility Proof Let n′ be the square free kernel of the natural number n. Let n′ be the product of the distinct primes q1,...,qm. By assumption we have that π2 6 ×loglogn′ ≤loglogn. π2 6 ×loglogn′ ≤loglogn. π2 6 ×loglogn′ ≤loglogn. For all square free n′ ≤5040, Robins(n′) holds if and only if n′ /∈{2,3,5,6,10,30} [3]. However, Robins(n) holds for all n > 5040 when n′ ∈{2,3,5,6,10,15,30} due to the lemma 4.1. When n′ > 5040, we know that Robins(n′) holds and so f(n′) < eγ ×loglogn′. By the previous lemma 2.3: f(n) < π2 6 × m ∏ i=1 qi +1 qi . f(n) < π2 6 × m ∏ i=1 qi +1 qi . Suppose by way of contradiction that Robins(n) fails. Then Suppose by way of contradiction that Robins(n) fails. Then f(n) ≥eγ ×loglogn. f(n) ≥eγ ×loglogn. f(n) ≥eγ ×loglogn. We claim that We claim that π2 6 × m ∏ i=1 qi +1 qi > eγ ×loglogn. Since otherwise we would have a contradiction. This shows that π2 6 × m ∏ i=1 qi +1 qi > π2 6 ×eγ ×loglogn′. Thus Thus m ∏ i=1 qi +1 qi > eγ ×loglogn′, m ∏ i=1 qi +1 qi > eγ ×loglogn′, and and m ∏ i=1 qi +1 qi > f(n′), m ∏ i=1 qi +1 qi > f(n′), This is a contradiction since f(n′) is equal to This is a contradiction since f(n′) is equal to (q1 +1)×···×(qm +1) q1 ×···×qm according to the formula (6.3) for the square free numbers. according to the formula (6.3) for the square free numbers. Theorem 9.3 Let ∏m i=1 qai i be the representation of n as a product of the first m consecutive primes q1 < ··· < qm with natural numbers as exponents a1,...,am. 9 Proof of Main Theorems If n > 5040 is the smallest integer such that Robins(n) does not hold, then (logn)β < Ym × log(Nm), where Nm = ∏m i=1 qi is the primorial number of order m and β = ∏m i=1 qai+1 i qai+1 1. Theorem 9.3 Let ∏m i=1 qai i be the representation of n as a product of the first m consecutive primes q1 < ··· < qm with natural numbers as exponents a1,...,am. If n > 5040 is the smallest integer such that Robins(n) does not hold, then (logn)β < Ym × log(Nm), where Nm = ∏m i=1 qi is the primorial number of order m and β = ∏m i 1 qai+1 i F. Vega F. Vega 16 Proof According to the theorems 1.3 and 1.4, the primes q1 < ··· < qm must be the first m consecutive primes since n > 5040 should be an Hardy-Ramanujan integer. From the theorem 9.1, we know that necessarily qm ≥953. From the lemma 3.2, we note that f(n) = m ∏ i=1 qi qi −1 ! × m ∏ i=1 1− 1 qai+1 i ! . However, we know that m ∏ i=1 qi qi −1 < eγ ×log(Ym ×log(Nm)) because of the lemma 8.4 when qm ≥953. If we multiply by ∏m i=1  1− 1 qai+1 i  the both sides of the previous inequality, then we obtain that f(n) < eγ ×log(Ym ×log(Nm))× m ∏ i=1 1− 1 qai+1 i ! . If n is the smallest integer exceeding 5040 that does not satisfy the Robin inequality, then eγ ×loglogn < eγ ×log(Ym ×log(Nm))× m ∏ i=1 1− 1 qai+1 i ! because of eγ ×loglogn ≤f(n). eγ ×loglogn ≤f(n). That is the same as m ∏ i=1 qai+1 i qai+1 i −1 ×loglogn < log(Ym ×log(Nm)) which is equivalent to which is equivalent to (logn)β < Ym ×log(Nm) where β = ∏m i=1 qai+1 i qai+1 i −1. Therefore, the proof is done. where β = ∏m i=1 qai+1 i qai+1 i −1. Therefore, the proof is done. Theorem 9.4 Let ∏m i=1 qai i be the representation of n as a product of the first m consecutive primes q1 < ··· < qm with natural numbers as exponents a1,...,am. I thank Richard J. Lipton for helpful comments. I thank Richard J. Lipton for helpful comments. 9 Proof of Main Theorems If n > 5040 is the smallest integer such that Robins(n) does not hold, then (logn)β < 1.2592 × log(Nm), where Nm = ∏m i=1 qi is the primorial number of order m and β = ∏m i=1 qai+1 i ai+1 1. β = ∏m i=1 qai+1 i qai+1 i −1. Proof From the theorem 9.1, we know that necessarily qm ≥953. Using the theorem 9.3, we obtain that β Proof From the theorem 9.1, we know that necessarily qm ≥953. Using the theorem 9.3, we obtain that β (logn)β < 1.2592×log(Nm) because of the lemma 8.1 since we have that Ym < 1.2592 when qm ≥953. because of the lemma 8.1 since we have that Ym < 1.2592 when qm ≥953. 17 Robin Criterion on Divisibility Acknowledgments I thank Richard J. Lipton for helpful comments. References 1. Akbary, A., Friggstad, Z.: Superabundant numbers and the Riemann hypothesis. The American Math- ematical Monthly 116(3), 273–275 (2009). DOI doi:10.4169/193009709X470128 2. Alaoglu, L., Erd˝os, P.: On highly composite and similar numbers. Transactions of the American Math- ematical Society 56(3), 448–469 (1944). DOI doi:10.2307/1990319 3. Choie, Y., Lichiardopol, N., Moree, P., Sol´e, P.: On Robin’s criterion for the Riemann hypothesis. Journal de Th´eorie des Nombres de Bordeaux 19(2), 357–372 (2007). DOI doi:10.5802/jtnb.591 4. Edwards, H.M.: Riemann’s Zeta Function. Dover Publications (2001) 5. Hertlein, A.: Robin’s Inequality for New Families of Integers. Integers 18 (2018) 6. Robin, G.: Grandes valeurs de la fonction somme des diviseurs et hypoth`ese de Riemann. J. Math. pures appl 63(2), 187–213 (1984) 7. Rosser, J.B., Schoenfeld, L.: Approximate Formulas for Some Functions of Prime Numbers. Illinois Journal of Mathematics 6(1), 64–94 (1962). DOI doi:10.1215/ijm/1255631807 1. Akbary, A., Friggstad, Z.: Superabundant numbers and the Riemann hypothesis. The American Math- ematical Monthly 116(3), 273–275 (2009). DOI doi:10.4169/193009709X470128 1. Akbary, A., Friggstad, Z.: Superabundant numbers and the Riemann hypothesis. The American Math- ematical Monthly 116(3), 273–275 (2009). DOI doi:10.4169/193009709X470128 2. Alaoglu, L., Erd˝os, P.: On highly composite and similar numbers. Transactions of the American Math- ematical Society 56(3), 448–469 (1944). DOI doi:10.2307/1990319 y 3. Choie, Y., Lichiardopol, N., Moree, P., Sol´e, P.: On Robin’s criterion for the Riemann hypothesis. Journal de Th´eorie des Nombres de Bordeaux 19(2), 357–372 (2007). DOI doi:10.5802/jtnb.591 4. Edwards, H.M.: Riemann’s Zeta Function. Dover Publications (2001) 5. Hertlein, A.: Robin’s Inequality for New Families of Integers. Integers 18 (2018) 5. Hertlein, A.: Robin’s Inequality for New Families of Integers. Integers 18 (2018) 6. Robin, G.: Grandes valeurs de la fonction somme des diviseurs et hypoth`ese de Riemann. J. Math. pures appl 63(2), 187–213 (1984) p pp 7. Rosser, J.B., Schoenfeld, L.: Approximate Formulas for Some Functions of Prime Numbers. Illinois Journal of Mathematics 6(1), 64–94 (1962). DOI doi:10.1215/ijm/1255631807
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Nursing care time and quality indicators for adult intensive care: correlation analysis
Revista latino-americana de enfermagem
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Corresponding Author: Paulo Carlos Garcia Universidade de São Paulo. Hospital Universitário Av. Professor Lineu Prestes, 2565 Bairro: Butantã CEP: 05508-000, São Paulo, SP, Brasil E-mail: paulogarcia@usp.br 1 Paper extracted from Master’s Thesis “Tempo de assistência de enfermagem em UTI e indicadores de qualidade assistencial: análise correlacional” presented to Escola de Enfermagem, Universidade de São Paulo, Brazil. 2 RN, MSc, Hospital Universitário, Universidade de São Paulo, Brazil. 3 PhD, Associate Professor, Escola de Enfermagem, Universidade de São Paulo, Brazil. Original Article Original Article Original Article Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8 www.eerp.usp.br/rlae Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8 www.eerp.usp.br/rlae Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8 www.eerp.usp.br/rlae Tempo de assistência de enfermagem em unidade de terapia intensiva adulto e indicadores de qualidade assistencial: análise correlacional Trata-se de studo de abordagem quantitativa, correlacional e descritivo, elaborado com o objetivo de analisar o tempo utilizado pela equipe de Enfermagem para assistir aos pacientes internados em unidade de terapia intensiva adulto, bem como verificar sua correlação com os indicadores de qualidade assistencial. A identificação do tempo médio de assistência despendido e dos indicadores de qualidade assistencial foi efetivada por meio de consulta aos instrumentos de gestão, utilizados pela chefia de Enfermagem da unidade. O tempo médio de assistência despendido com os pacientes manteve-se equilibrado, porém, inferior aos indicados pelos órgãos oficiais brasileiros. A correlacão entre o tempo de assistência de Enfermagem despendido por enfermeiros e o indicador incidência de extubação acidental indicou que a incidência de extubação acidental diminui, à medida que aumenta o tempo de assistência de Enfermagem despendido por enfermeiros. Os resultados desta investigação demonstram a influência do tempo de assistência de Enfermagem, provido por enfermeiros, no resultado do cuidado ministrado. Descritores: Enfermagem; Administração de Recursos Humanos em Enfermagem; Carga de Trabalho; Indicadores de Qualidade em Assistência à Saúde. Nursing care time and quality indicators for adult intensive care: correlation analysis1 Paulo Carlos Garcia2 Fernanda Maria Togeiro Fugulin3 The objective of this quantitative, correlational and descriptive study was to analyze the time the nursing staff spends to assist patients in Adult Intensive Care Units, as well as to verify its correlation with quality care indicators. The average length of time spent on care and the quality care indicators were identified by consulting management instruments the nursing head of the Unit employs. The average hours of nursing care delivered to patients remained stable, but lower than official Brazilian agencies’ indications. The correlation between time of nursing care and the incidence of accidental extubation indicator indicated that it decreases with increasing nursing care delivered by nurses. The results of this investigation showed the influence of nursing care time, provided by nurses, in the outcome of care delivery. Descriptors: Nursing; Nursing Administration of Human Resources; Workload; Quality Indicators in Health Care. 652 Tiempo de atención de enfermería en la unidad de cuidados intensivos de adultos y los indicadores de atención de calidad: análisis de correlación. Un estudio de enfoque cuantitativo, de correlación y descriptiva, elaborado con objetivo de analizar el tiempo empleado por equipo de enfermería para asistir pacientes hospitalizados en Unidad de Cuidados Intensivos de Adultos y verificar su correlación con indicadores de calidad de la atención. La identificación del promedio de tiempo de cuidados y de indicadores de calidad asistencial fue realizada por medio de consulta a las herramientas de gestión utilizadas por el jefe de enfermería de la Unidad. El promedio de tiempo de cuidado ministrado a los pacientes se mantuvo equilibrado, pero inferior a los indicados por organismos oficiales en Brasil. La correlación entre tiempo de cuidados de enfermería e indicador de incidencia extubación accidental indicó que esta disminuye con aumento de tiempo de atención de enfermería utilizado por enfermeras. Los resultados de esta investigación demuestran la influencia del tiempo de atención de enfermería por las enfermeras, en los resultados de la atención prestada. Descriptores: Enfermería; Gestión de Recursos Humanos en la Carga de Trabajo de Enfermería; Los Indicadores de Calidad en la Atención de la Salud. www.eerp.usp.br/rlae Method A systematic literature review(4) appoints that concerns with patient safety and care quality are determining the accomplishment of research on clinical practice and the cost-effectiveness relation of health interventions are care, including the distribution of human resources. This is particularly important in intensive care, where a large part of hospitals’ financial resources are consumed and the nursing staff represents the main cost item. A quantitative and descriptive correlation study was developed at the AICU of the HU-USP. The AICU offers 12 beds. Four of these are structured and preferably reserved for the hospitalization of patients who need isolation. It should be highlighted that the Unit elaborates monthly reports, as a management instrument, which are forwarded to the Nursing Department (ND), informing on the number of care hours delivered to hospitalized patients during the period, with a view to supporting human resource management decisions, so as to maintain quality care. Another literature review(5) indicates that research done in recent decades shows an association between the nursing staff and patient care outcomes. Most of these studies were developed at intensive care units and picture the direct relation between nursing staff and increased infection rates, postoperative complications, mortality and accidental extubation, also described as unplanned extubation. The nursing time spent is calculated with the help of an electronic worksheet called “Worksheet to calculate the mean nursing care time spent”(8). Another characteristic of the Unit where the research was accomplished is the use of care quality indicators. The selected indicators, according to the Institution’s Group of Quality Indicators, are: Incidence of nasogastric tube loss for nutrition; Incidence of central venous catheter loss; Incidence of accidental extubation; Incidence of pressure ulcer; Incidence of fall; Incidence of medication error. The author of that research(5) concluded that the analysis of nursing staff needs at intensive care units is important, mainly for the managers of these units, to the extent that they have the power to make decisions and distribute resources. Thus, the challenge for intensive care nurses is to develop and quantify evidence to demonstrate that a larger proportion of nurses positively influences the outcomes of care delivery to patients and their families, through studies that relate staff number with quality care indicators(6). These indicators represent care practice at the Institution and are validated and recommended in Brazilian(9) and international literature(10). Method One exception is the central venous catheter loss indicator, which was developed according to the need evidenced at HU-USP. In addition, these indicators are frequently used in the Brazilian hospital network, permitting intra and extra- institutional comparability. In this perspective, the use of quality care indicators represents a powerful management instrument for nurses, as it permits showing the relevance of quantitative and qualitative staff dimensioning with a view to the promotion of excellent care delivery to health service users. Introduction Alliance, with a view to promoting and developing patient safety health practices and policies at global level(2). In the global context, the challenge of improving the safety and quality of patient care delivery in health services is not new. A document the International Council of Nurses published also considers that the improvement of nursing practice can guarantee patient safety and constitutes a global priority goal(3). According to the campaign launched by the World Health Organization(1), we are in the “Age of Safety” and, since 2002, the organization has addressed this theme as a priority issue. The program was strengthened in 2004, through the creation of the Global Patient Safety The same document(3) highlights that organizational culture and management directly influence the results www.eerp.usp.br/rlae 653 Garcia PC, Fugulin FMT. rigor, they permit comparative analyses among health services, with a view to planning quality improvements in care delivery to patients/service users(7). reached in health institutions. Quality in the nursing work environment, in turn, interferes in the quality and safety of patient care and is influenced by the size of the nursing staff, the number of professionals according to professional category and the nursing/patient rate. The analysis of Brazilian literature, however, shows a lack of studies that demonstrate the relation between nursing staff conformity and care indicators. In that sense, the aim of this study is to analyze the time the nursing team uses to see to patients/clients hospitalized at the Adult Intensive Care Unit (AICU) of the University of São Paulo Teaching Hospital (HU-USP), as well as to check its correlation quality care indicators. In that context, it is verified that issues related to the nursing staff dimensioning process gain relevance and are under investigation to produce technical and scientific evidence to enhance awareness of the meaning of health staff that sees not only to patients and health institutions’ needs, but also to patients and nursing team professionals’ safety. www.eerp.usp.br/rlae Data collection procedures Data were collected monthly from the management instruments the Unit nursing head uses: “Worksheet to calculate the mean nursing care time spent”, and “Worksheet to obtain nursing quality indicators, AICU The use of clinical or quality indicators to assess the care offered represents an important approach to document attendance. In addition, when the indicators are validated and tested with transparency and scientific Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8. 654 pk = mean productivity of professional category k (85%); tk = work journey of professional category k (six hours); n = mean daily number of patients attended. Unit”, with the approval of the HU-USP Research Ethics Committee (CEP) (registration CEP-HU/USP: 982/10 – Registration SISNEP – CAAE: 0009.0.198.196-10). Unit”, with the approval of the HU-USP Research Ethics Committee (CEP) (registration CEP-HU/USP: 982/10 – Registration SISNEP – CAAE: 0009.0.198.196-10). Unit”, with the approval of the HU-USP Research Ethics Committee (CEP) (registration CEP-HU/USP: 982/10 – Registration SISNEP – CAAE: 0009.0.198.196-10). No. of central venous catheter losses No. of patients with central venous catheter/day x 100 Spearman’s correlation coefficient, while Pearson’s correlation coefficient was used for the other quality indicators. Data treatment and analysis The results were presented through tables with central trend and variability measures. To correlate the mean care time spent on AICU patients with the quality care indicators, initially, the Kolmogorov-Smirnov test was applied to assess the normality distribution. It was ascertained that the central venous catheter loss indicator showed no normal distribution. Thus, for this variable, correlation analysis was developed using Survey of quality care indicators at the AICU of the HU-USP The “Worksheets to calculate the mean nursing care time spent”, which are part the Unit head nurse’s monthly reports, were consulted to identify the mean monthly care time spent on patients hospitalized at the Adult ICU of the HU-USP, between 01/01/2008 and 12/31/2009. The choice of the quality indicators used in this research was based on the criteria recommended in literature(7) and on data accessibility at the Unit. Among the indicators assessed at the HU-USP, the decision was made to use those inherent in the intensive care context: incidence of nasogastric tube loss for nutrition; incidence of central venous catheter loss, incidence of accidental extubation and incidence of pressure ulcer. The mean nursing care time was calculated electronically, using the worksheet developed in Microsoft Excel®, with the help of equation (1)(11): qk.pk.tk n hk= (1) qk.pk.tk n hk= (1) Thus, the quality care indicators were collected monthly from the “Worksheet to obtain nursing quality indicator data, AICU Unit”, which the Unit nursing head uses to make calculations according to the equations indicated in the Manual of Nursing Indicators(9). One exception is the central venous loss indicator, whose equation the institution developed by itself. Where: hk = mean nursing care time per patient, spend by workers from professional category k; k = professional category; k = professional category; qk= mean number of nursing staff members in professional category k; Incidence of central venous catheter loss = No. of central venous catheter losses No. of patients with central venous catheter/day Results C.(%) 3.49 8.13 3.79 5.22 5.44 5.50 As for the distribution of nursing care time between professional categories, the mean care hours both nurses and nursing technicians spent remained the same in 2008 and 2009 (4.3 and 4.4 hours, respectively, for nurses and 9.6 for technicians). On average, the proportion of nurses’ care time corresponded to 31% and that of nursing technicians to 69%. Slight variations were observed in the time spent during the study period (minimum 13.3 hours and maximum 14.6 hours in 2008 and minimum 13.1 hours and maximum 15.4 hours in 2009). The mean number of nursing care hours spent on the patients during the two years studied is practically equivalent (13.9 h/patient/ day in 2008 and 14.1 h/patient/day in 2009), with a difference of only 0.2 hours, i.e. 12 minutes. Table 2 – Mean scores of Nursing quality care indicators at AICU of HU-USP between Jan/2008 and Dec/2009. São Paulo, SP, Brazil, 2011 Variables 2008 2009 Incidence of Accidental Extubation Incidence of NGT loss Incidence of CVC loss Incidence of PU Incidence of Accidental Extubation Incidence of NGT loss Incidence of CVC loss Incidence of PU January 1.14 0.00 0.00 18.60 0.00 4.69 0.00 3.85 February 1.26 1.51 1.08 10.20 0.00 4.61 1.65 5.77 March 0.62 4.05 0.00 5.26 0.43 1.79 0.00 14.58 April 0.74 1.88 1.37 6.25 0.53 3.13 0.00 6.67 May 0.48 2.78 1.20 12.82 1.18 1.06 0.00 2.38 June 0.72 1.44 0.00 7.27 0.00 1.72 0.00 7.50 July 1.88 3.32 0.00 16.67 0.00 1.03 0.00 6.56 August 1.09 0.87 1.17 10.71 0.00 2.97 0.00 0.00 September 0.00 4.48 0.79 12.50 1.75 6.19 0.00 5.56 October 0.00 0.87 0.39 3.85 0.97 0.83 0.37 7.94 November 0.00 4.52 0.00 0.00 0.60 1.06 0.43 2.22 December 0.83 2.25 0.50 12.50 0.00 1.32 0.00 17.65 Mean 0.73 2.33 0.54 9.72 0.46 2.53 0.20 6.72 sd 0.57 1.51 0.55 5.40 0.58 1.79 0.48 5.03 VAR. C.(%) 0.33 2.27 0.31 29.12 0.34 3.19 0.23 25.28 ores of Nursing quality care indicators at AICU of HU-USP between Jan/2008 and Dec/2009. São Table 2 – Mean scores of Nursing quality care indicators at AICU of HU-USP between Jan/2008 and Dec/2009. São Paulo, SP, Brazil, 2011 Table 2 shows the performance of nursing quality care indicators used at the AICU of the HU-USP during the study period. Results Table 1, presented next, demonstrates the mean care time spent on patients hospitalized at the Adult ICU of the HU-USP between 01/01/2008 and 12/31/2009. Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo, SP Brazil 2011 Variables Mean Time (2008) Mean Time (2009) Total Nurses Nursing Technician Total Nurses Nursing Technician (h) (%) (h) (%) (h) (%) (h) (%) (h) (%) (h) (%) January 14.5 100 4.2 28.9 10.4 71.1 13.5 100 4.2 31.1 9.3 68.9 February 13.8 100 3.9 28.2 9.8 71.8 15.1 100 4.7 31.1 10.4 68.9 March 13.5 100 3.9 28.8 9.6 71.2 14.7 100 4.6 31.3 10.1 68.7 April 13.3 100 4.1 30.8 9.2 69.2 13.1 100 4.1 31.3 9.1 68.7 May 13.3 100 3.7 27.8 9.5 72.2 13.4 100 4.2 31.3 9.3 68.7 Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo, SP, Brazil, 2011 (continue...) ursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo, Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/ SP, Brazil, 2011 Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo, SP, Brazil, 2011 655 Garcia PC, Fugulin FMT. Table 1 - (continuation) Variables Mean Time (2008) Mean Time (2009) Total Nurses Nursing Technician Total Nurses Nursing Technician (h) (%) (h) (%) (h) (%) (h) (%) (h) (%) (h) (%) June 14.0 100 4.4 31.4 9.6 68.6 13.3 100 4.3 32.3 9.0 67.7 July 13.3 100 4.1 30.8 9.2 69.2 13.9 100 4.5 32.4 9.4 67.6 August 14.5 100 4.6 31.7 9.9 68.3 15.4 100 4.8 31.1 10.6 68.9 September 13.8 100 4.4 31.8 9.3 68.2 14.0 100 4.3 30.7 9.7 69.3 October 13.6 100 4.6 33.8 9.0 66.2 14.0 100 4.3 30.7 9.7 69.3 November 14.6 100 4.9 33.5 9.6 66.5 14.7 100 4.7 32.0 10.0 68.0 December 14.1 100 4.4 31.2 9.7 68.8 13.7 100 4.6 33.5 9.2 66.5 Mean 13.9 100 4.3 30.9 9.6 69.1 14.1 100 4.4 31.2 9.6 68.9 sd 0.48 0.35 0.36 0.73 0.24 0.53 VAR. www.eerp.usp.br/rlae Discussion The mean care time spent on patients hospitalized at the AICU of the HU-USP between 01/01/2008 and 12/31/2009 corresponded to approximately 14 hours. Based on the comparison between the present research results and those of a study(12) that identified the mean nursing care hours at the AICU of the HU-USP between 2001 and 2005, it was ascertained that less nursing care hours were spent on AICU patients in 2008 and 2009 than in the years the author analyzed (mean 15.4 hours), although the percentage distribution the same researcher found did correspond (30% for nurses and 70% for nursing technicians)(12). The percentage of nursing care time AICU nurses spent, however, is higher than that identified in RDC(14) No. 7, proposed by the Joint Board of ANVISA(14) (20%). The percentage of nursing care time AICU nurses spent, however, is higher than that identified in RDC(14) No. 7, proposed by the Joint Board of ANVISA(14) (20%). In that sense, studies available in international literature demonstrate an inverse relation between the number of nurses and the occurrence of adverse events in patients, correlating the number of care hours these professionals spend with the quality of care delivery, concluding that a larger number of care hours delivered by nurses is associated with a drop in mortality and adverse event rates (urinary tract infection, pressure ulcer, hospital pneumonia, wound infections, central venous access complications, shock, thrombosis, medication errors, postoperative complications), as well as with a decrease in mortality rates deriving from these events(18-20). Concerning the times established by the Federal Nursing Council(13) (COFEN) (17.9 h) and the National Health Surveillance Agency(14) (ANVISA) (15 h), the mean nursing care hours spent on patients hospitalized at the AICU of HU-USP, except in February and August 2009, remained below these entities’ indications. This result suggests the possibility that the nursing team is overburdened and evidences the need to revise the Unit’s existing staff. Nevertheless, the workload patients demand should also be assessed through the application of specific instruments(15-16), as the care time patients demand can differ from official entities’ indications and their use in putting in practice dimensioning methods could cause an over or under- dimensioning of nursing staff. This scenario evidences that improving the percentage distribution of care hours attributed to nurses represents not only a perspective for the AICU at HU-USP, but also a challenge for Brazilian nursing. Results The CVC loss incidence indicator showed the smallest variation coefficient (0.31% in 2008 and 0.23% in 2009), while the PU incidence indicator showed the highest coefficient (29.12% in 2008 and 25.28% in 2009). Data in Table 3 demonstrate that the nursing care time nurses spent and the quality indicator incidence of accidental extubation showed a Pearson’s correlation coefficient of (r =- 0.454), with p=0.026. For the other variables, no statistically significant correlations were found. 656 Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8. Table 3 - Correlation between mean care time spent, according to professional category, and quality care indicators at the Adult ICU of HU-USP, between January 1st 2008 and December 31st 2009. HU-USP, São Paulo, Brazil, 2011 Table 3 - Correlation between mean care time spent, according to professional category, and quality care indicators at the Adult ICU of HU-USP, between January 1st 2008 and December 31st 2009. HU-USP, São Paulo, Brazil, 2011 Variables Incidence of NGT loss Incidence of CVC loss* Incidence of accidental extubation Incidence of PU Pearson’s correlation p Spearman’s Correlation p Pearson’s correlation p Pearson’s correlation p Time spent -0.015 0.944 0.033 0.878 -0.174 0.416 -0.162 0.450 Time spent, nurses -0.003 0.987 -0.013 0.953 -0.454 0.026† -0.306 0.146 Time spent, tech./aux. -0.019 0.931 0.208 0.330 0.067 0.756 -0.017 0.939 * The CVC loss incidence variable shows no normal distribution, so that Spearman’s non-parametric correlation was used. † Significance level for p < 0.05. * The CVC loss incidence variable shows no normal distribution, so that Spearman’s non-parametric correlation was used. † Significance level for p < 0.05. an official reference for nursing staff dimensioning at hospital institutions, found that, at the six ICUs studies, the proportion of hours attributed to the nurses was smaller than that attributed to nursing technicians and auxiliaries, remaining far below COFEN recommendations(13). www.eerp.usp.br/rlae Discussion The analysis of AICU indicators suggests improved care quality at the Unit in 2009, when compared with 2008, as the mean incidence levels of accidental extubation, CVC loss and PU dropped. Due to the absence of Brazilian research and the lack of international studies, however, which used the same indicators and method as adopted in the present research, AICU/HU-USP data could not be confronted with those of other services. As for the percentage distribution of care time between the professional categories who are part of the nursing team, it is observed that the proportion of time AICU nurses spent remains below the minimum percentage recommended in COFEN Resolution(13) 293/2004 (52%). Concerning the analysis of the correlation between nursing care time spent by nurses and the incidence of accidental extubation quality indicator, it can be inferred that the incidence of accidental extubation decreases as the nursing care time spent by nurses increases. No An exploratory and descriptive study(17) developed in fourteen hospitalization units of seven hospitals in São Paulo City, aimed at assessing the parameters recommended in COFEN Resolution(13) 293/04, as www.eerp.usp.br/rlae 657 Garcia PC, Fugulin FMT. 31st 2009 remained below COFEN and ANVISA recommendations. studies were found with similar methods to the present study. Some studies describe the influence of the nursing staff variable on the incidence of unplanned extubation and support the results found. The nursing care time spent by nurses and the incidence of accidental extubation quality indicator showed a negative Pearson’s correlation coefficient, indicating that the incidence of accidental extubation drops as the number of nursing care hours spent by nurses increases. Concerning the other quality indicators assessed in this study, i.e. the incidence of nasogastric tube loss for feeding, incidence of central venous catheter loss and incidence of pressure ulcer, no statistically significant correlations were found. A study(21) that assessed the association between the nursing workload and the probability of unplanned extubations at a Pediatric Intensive Care Unit appointed that the logistic regression model revealed positive associations between unplanned extubations and the patient/nurse ratio. In the same study, it was concluded that the number of unplanned extubations may increase in situations when the patient/nurse ratio is higher. References 1. World Health Organization (WHO). World Alliance for Patient Safety. Forward program 2006- 2007. [acesso 13 jan 2011]. Disponível em: www.who.int/patientesafety 2. Pedreira MLG, MJCS Harada. Enfermagem dia a dia: segurança do paciente. São Caetano do Sul: Yendis Editora; 2009. Thus, the present research results demonstrate the influence of nursing care time, provided by nurses, on the outcomes of care delivered to patients attended at AICU. 3. Braumann A. Positive practice environments: quality workforce, quality patient care. Geneva (SZ): International Council of Nurses; 2007. 65 p. 4. West E, Mays N , Rafferty AM , Rowan K , Sanderson C. Nursing resources and patient outcomes in intensive care: A systematic review of the literature. Int J Nurs Stud. 2009;46:993-1011. Discussion A research(22) aimed at exploring the effects of nursing care on the occurrence and consequences of unplanned tracheal extubation at adult Intensive Care Units reported that self-extubations were more frequent at night and under routine care of nurses with less professional experience. Those authors concluded that an adequate patient/nurse ratio can generate better work and safety conditions during nursing procedures. The limitations of the present research, i.e. the fact that it was accomplished at a single unit of a single institution, put constraints on its generalization. It does offer support and equips nurses though, through the presentation of a systematic method and the indicator time series, with a view to reproducing the study in different contexts, contributing to the validation of this finding and to comparability among health services, as no studies have been published that correlate these variables in the Brazilian context. A study developed to identify high-risk patients for unplanned extubation and determine professionals’ beliefs on perceived unplanned extubation risks revealed that 60% of the interviewed professionals believe that the nursing staff or the patient/nurse ratio are closely related with accidental extubation cases(23). The accomplishment of this study also contributes to understand the importance and impact of the nursing staff on care outcomes and patient safety, supporting nurses’ administrative and political decisions, as well as staff negotiations with hospital managers, with a view to the excellence of care offered to these services’ users. A recent publication(24) aimed at assessing risk factors and outcomes after unplanned extubation at ICU indicated that non-accidental extubation is a frequent event during mechanical ventilation in critically ill patients and can be associated with increased morbidity and mortality. In the same study, the incidence of extubation was considered relatively low (2.1% for patients under mechanical ventilation and 0.4% per ventilation day), due to the high nurse/patient ratio in the units where the field research was accomplished. www.eerp.usp.br/rlae Conclusion As a result of this study, the mean nursing care time spent on patients hospitalized at the AICU of the HU-USP could be identified and analyzed, between 01/01/2008 and 12/31/2009, and its correlation with quality care indicators used at the Unit could be ascertained. 5. Penoyer DA. Nurse staffing and patient outcomes in critical care: A concise review. Crit Care Med. 2010;38(7):1521-8. 5. Penoyer DA. Nurse staffing and patient outcomes in critical care: A concise review. Crit Care Med. 2010;38(7):1521-8. 5. Penoyer DA. Nurse staffing and patient outcomes in critical care: A concise review. Crit Care Med. 2010;38(7):1521-8. 6. Bray K, Wren I, Baldwin A, Ledger US, Gibson V, Goodman S, Walsh D. Standards for nurse staffing in critical care units determined by: The British Association of 6. Bray K, Wren I, Baldwin A, Ledger US, Gibson V, Goodman S, Walsh D. Standards for nurse staffing in critical care units determined by: The British Association of The nursing care hours spent on AICU patients at the HU-USP between January 1st 2008 and December Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8. 658 18. Aiken LH, Clarke SP, Cheung RB, Sloane DM, Silber JH. Educational Levels of Hospital Nurses and Surgical Patient Mortality. JAMA. 2003;290(12):1617-23. 19. Needleman J, Buerhaus P, Matte S, Stewart M, Zelevinsky K. Nurse-staffing levels and the quality of care in hospitals. N Engl J Med. 2002;346(22):1715-22. 20. Needleman J, Buerhaus P, Pankratz SV, Leibson CL, Stevens SR, Harris M. Nurse Staffing and Inpatient Hospital Mortality. N Engl J Med. 2011;364(11):1037-45. 21. Silva PSL, Carvalho BW. Unplanned extubation in pediatric critically ill patients: 18. Aiken LH, Clarke SP, Cheung RB, Sloane DM, Silber JH. Educational Levels of Hospital Nurses and Surgical Patient Mortality. JAMA. 2003;290(12):1617-23. 19. Needleman J, Buerhaus P, Matte S, Stewart M, Zelevinsky K. Nurse-staffing levels and the quality of care in hospitals. N Engl J Med. 2002;346(22):1715-22. 20. Needleman J, Buerhaus P, Pankratz SV, Leibson CL, Stevens SR, Harris M. Nurse Staffing and Inpatient Hospital Mortality. N Engl J Med. 2011;364(11):1037-45. 21. Silva PSL, Carvalho BW. Unplanned extubation in pediatric critically ill patients: Critical Care Nurses, The Critical Care Networks National Critical Care Nurses, The Critical Care Networks National Nurse Leads. Nurs Crit Care.2010;15(3):109-11. Critical Care Nurses, The Critical Care Networks National Nurse Leads. Nurs Crit Care.2010;15(3):109-11. Nurse Leads. Nurs Crit Care.2010;15(3):109-11. 7. Mainz J. Developing evidence-based clinical indicators: a state of the art methods primer. Received: May 5th 2011 Accepted: Apr. 3th 2012 Conclusion Int J Qual in Heal Care. 2003;15 Suppl 1:i5-i11. 8. Gaidzinski RR, Fugulin FMT, Castilho V. O uso de ferramentas tecnológicas no processo de dimensionamento de pessoal. In: Prado C, Peres HHC, 8. Gaidzinski RR, Fugulin FMT, Castilho V. O uso de ferramentas tecnológicas no processo de dimensionamento de pessoal. In: Prado C, Peres HHC, Leite MMJ. Tecnologia da informação e comunicação na enfermagem. São Paulo: Atheneu; 2011. p. 35-43. Leite MMJ. Tecnologia da informação e comunicação na enfermagem. São Paulo: Atheneu; 2011. p. 35-43. 9. CQH. Programa de Qualidade Hospitalar: manual de indicadores de enfermagem NAGEH. São Paulo: APM/ CREMESP; 2006. A systematic review and best practice recommendations. Pediatr Crit Care Med. 2010;11(2):287-94. A systematic review and best practice recommendations. Pediatr Crit Care Med. 2010;11(2):287-94. Pediatr Crit Care Med. 2010;11(2):287-94. 22. Yeh SH, Lee LN, Ho TH, Chiang MC, Lin LW. Implications of nursing care in the occurrence and consequences of unplanned extubation in adult intensive care units. Int J Nurs Stud. 2004;41:255-62. 10. American Nurses Association. National Database of Nursing Quality Indicators (NDQI 2003-2011) .[acesso 13 jan 2012]. Disponível em: https://www. nursingquality.org/ 22. Yeh SH, Lee LN, Ho TH, Chiang MC, Lin LW. Implications of nursing care in the occurrence and consequences of unplanned extubation in adult intensive care units. Int J Nurs Stud. 2004;41:255-62. 10. American Nurses Association. National Database of Nursing Quality Indicators (NDQI 2003-2011) .[acesso 13 jan 2012]. Disponível em: https://www. nursingquality.org/ 23. Tanios MA, Epstein SK, Livelo J, Teres D. Can We Identify Patients at High Risk for Unplanned Extubation? A Large-Scale Multidisciplinary Survey. Respir Care. 2010;55(5):561-8. 11. Gaidzinski RR, Fugulin FMT. Dimensionamento de pessoal de enfermagem em unidade de terapia intensiva. In: Associação Brasileira de Enfermagem. Programa de atualização em Enfermagem: Saúde do adulto (PROENF) – Ciclo 3 – Módulo 3. Porto Alegre: Artmed/Panamericana; 2008. p. 65-96. 12. Rogenski KE, Fugulin FMT, Gaidzinski RR, Rogenski NMB. Tempo de assistência de enfermagem em instituição hospitalar de ensino. Rev Esc Enferm USP. 2011;45(1):223-9. 23. Tanios MA, Epstein SK, Livelo J, Teres D. Can We Identify Patients at High Risk for Unplanned Extubation? A Large-Scale Multidisciplinary Survey. Respir Care. 2010;55(5):561-8. 11. Gaidzinski RR, Fugulin FMT. Dimensionamento de pessoal de enfermagem em unidade de terapia intensiva. In: Associação Brasileira de Enfermagem. Programa de atualização em Enfermagem: Saúde do adulto (PROENF) – Ciclo 3 – Módulo 3. Porto Alegre: Artmed/Panamericana; 2008. p. 65-96. 11. Gaidzinski RR, Fugulin FMT. Conclusion Dimensionamento de pessoal de enfermagem em unidade de terapia intensiva. In: Associação Brasileira de Enfermagem. Programa de atualização em Enfermagem: Saúde do adulto (PROENF) – Ciclo 3 – Módulo 3. Porto Alegre: Artmed/Panamericana; 2008. p. 65-96. 24. Groot RI, Dekkers OM, Herold IHF, Jonge E, Arbous MS. Risk factors and outcomes after unplanned extubations on the ICU: a case-control study. Crit Care. 2011;15(19):1-9. 24. Groot RI, Dekkers OM, Herold IHF, Jonge E, Arbous MS. Risk factors and outcomes after unplanned extubations on the ICU: a case-control study. Crit Care. 2011;15(19):1-9. 12. Rogenski KE, Fugulin FMT, Gaidzinski RR, Rogenski NMB. Tempo de assistência de enfermagem em instituição hospitalar de ensino. Rev Esc Enferm USP. 2011;45(1):223-9. 12. Rogenski KE, Fugulin FMT, Gaidzinski RR, Rogenski NMB. Tempo de assistência de enfermagem em instituição hospitalar de ensino. Rev Esc Enferm USP. 2011;45(1):223-9. 13. Conselho Federal de Enfermagem (BR). Resolução n. 293, de 21 de setembro de 2004. Fixa e Estabelece parâmetros para Dimensionamento do Quadro de Profissionais de enfermagem nas Unidades Assistenciais das Instituições de Saúde e Assemelhados [Internet]. [acesso 13 jan 2012]; Rio de Janeiro; 2004. Disponível em: http://corensp.org.br/072005/ 14. Agência Nacional de Vigilância Sanitária (BR). RDC nº 7 de 24 de fevereiro de 2010. [acesso 13 jan 2012]; Disponível em: http://www.brasilsus.com.br/ legislacoes/rdc/102985-7.html 14. Agência Nacional de Vigilância Sanitária (BR). RDC nº 7 de 24 de fevereiro de 2010. [acesso 13 jan 2012]; Disponível em: http://www.brasilsus.com.br/ legislacoes/rdc/102985-7.html 15. Miranda DR, Rijk AD, Schaufeli W, Iapichino G. Nursing activities score. Crit Care Med. 2003;31(2):374-82. 16. Panunto MR, Guirardello EB. Nursing workload at a gastroenterology unit. Rev. Latino-Am. Enfermagem. 2009;17(6):1009-14. 17. Fugulin FMT. Parâmetros oficiais para o dimensionamento de profissionais de enfermagem em instituições hospitalares: análise da resolução COFEN nº 293/04. [tese Livre-docência]. São Paulo : Escola de Enfermagem da Universidade de São Paulo; 2010. Received: May 5th 2011 Accepted: Apr. 3th 2012 www.eerp.usp.br/rlae
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The feather pattern <i>autosomal barring</i> in chicken is strongly associated with segregation at the <i>MC1R</i> locus
Pigment cell & melanoma research
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j INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH PIGMENT CELL & MELANOMA Research The feather pattern autosomal barring in chicken is strongly associated with segregation at the MC1R locus Doreen Schwochow | Susanne Bornelöv | Tingxing Jiang | Jingyi Li | David Gourichon | Bertrand Bed’Hom | Ben J. Dorshorst | Cheng-Ming Chuong | Michèle Tixier-Boichard| Leif Andersson If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here K E Y W O R D S chicken, feather patterning, genetics, MC1R, pigmentation, SOX10 O R I G I N A L A R T I C L E Doreen Schwochow1,2  | Susanne Bornelöv3 | Tingxing Jiang4  | Jingyi Li5,6  | David Gourichon7 | Bertrand Bed’Hom2  | Ben J. Dorshorst5 | Cheng-­Ming Chuong4  | Michèle Tixier-­Boichard2  | Leif Andersson1,3,6 Doreen Schwochow1,2  | Susanne Bornelöv3 | Tingxing Jiang4  | Jingyi Li5,6  | David Gourichon7 | Bertrand Bed’Hom2  | Ben J. Dorshorst5 | Cheng-­Ming Chuong4  | Michèle Tixier-­Boichard2  | Leif Andersson1,3,6 Doreen Schwochow1,2  | Susanne Bornelöv3 | Tingxing Jiang4  | Jingyi Li5,6  | David Gourichon7 | Bertrand Bed’Hom2  | Ben J. Dorshorst5 | Cheng-­Ming Chuong4  | Michèle Tixier-­Boichard2  | Leif Andersson1,3,6 1Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden 2Université Paris-­Saclay, INRAE, AgroParisTech, GABI, Jouy-­en-­Josas, France 3Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden 4Department of Pathology, University of Southern California, Los Angeles, CS, USA 5Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA 6Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA 7INRAE, PEAT, Nouzilly, France Abstract bst act Color patterns within individual feathers are common in birds but little is known about the genetic mechanisms causing such patterns. Here, we investigate the genetic basis for autosomal barring in chicken, a horizontal striping pattern on individual feath- ers. Using an informative backcross, we demonstrate that the MC1R locus is strongly associated with this phenotype. A deletion at SOX10, underlying the dark brown phenotype on its own, affects the manifestation of the barring pattern. The coding variant L133Q in MC1R is the most likely causal mutation for autosomal barring in this pedigree. Furthermore, a genetic screen across six different breeds showing dif- ferent patterning phenotypes revealed that the most striking shared characteristics among these breeds were that they all carried the MC1R alleles Birchen or brown. Our data suggest that the presence of activating MC1R mutations enhancing pigment synthesis is an important mechanism underlying pigmentation patterns on individual feathers in chicken. We propose that MC1R and its antagonist ASIP play a critical role for determining within-­feather pigmentation patterns in birds by acting as activator and inhibitor possibly in a Turing reaction–­diffusion model. Correspondence Leif Andersson, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. Email: leif.andersson@imbim.uu.se EMAIL ALERTS Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Received: 18 February 2021  |  Accepted: 26 March 2021 DOI: 10.1111/pcmr.12975 Significance Within-­feather patterns are an important part of the stunning diversity of plumage color in birds. Barring is a common feather pigmentation pattern present, for instance, in hawks and owls. This study dissects the genetic basis for autosomal barring in the domestic chicken using pedigree analysis and molecular characterization. We show that the autosomal barring pheno- type co-­segregates with a missense mutation L133Q in the melanocortin 1 receptor (MC1R) gene. A general observation across breeds was that various feather pigmentation patterns in chicken are associated with missense mutations in the MC1R gene. Pigment Cell Melanoma Res. 2021;34:1015–1028. This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2021 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd. 1016  |     1 | INTRODUCTION 1016 SCHWOCHOW et al. located on chromosome 1 in close linkage with Db and Melanotic (Ml) (Carefoot, 1999; Moore & Smyth, 1972). Pigmentation variation among birds is astonishing and is a conse- quence of the ability to create complex patterns through varied pig- ment deposition across the body as well as on individual feathers. Due to the usual homogeneous plumage appearance within species, it is challenging to pinpoint the underlying genetic cause for the ob- served phenotype diversity among species. The domestic chicken is a widely used model for genetic studies of the bewildering pig- ment variation in birds and has been used successfully so in the past (Andersson et al., 2020). Chickens exhibit a variety of within-­ feather pigmentation patterns of which five have been described to be the consequence of the interaction between a proposed major locus called Patterning (Pg) and other loci (Figure 1; Figure S1) (Smyth Jr, 1990). Two of these interacting loci, Extension (E) and Dark brown (Db), have been identified at the molecular level. E corresponds to the melanocortin 1 receptor gene (MC1R), one of the major pigmentation genes in vertebrates (see below). Db is caused by a 8.3 kb deletion 14 kb upstream of exon 1 in SOX10 on chromosome 1 (Gunnarsson et al., 2011). Pg is assumed to affect the distribution of dark pigment on individual feathers and to underlie penciling, which is character- ized by elliptic bands of eumelanin on the feather (Figure  1b). Pg has been described as an incompletely dominant, autosomal gene There are two distinct types of barring patterns in chicken, au- tosomal barring, and sex-­linked barring. While sex-­linked barring is adding a white bar on a pigmented background, autosomal barring is adding a black bar on a gold or silver background on individual feathers (Smyth Jr, 1990). Sex-­linked barring is caused by the com- bined effect of regulatory mutation(s) and missense mutations in the tumor suppressor gene CDKN2A (Hellström et al., 2010; Schwochow Thalmann et al., 2017). Autosomal barring on the other hand has been suggested to be caused by the combined effect of the three autoso- mal loci Db, E, and Pg (Andersson et al., 2020; Carefoot, 1984,1999; Moore & Smyth, 1972) and is the trademark of some chicken breeds such as Fayoumi, Campine, and Westfälische Totleger. Funding information Pigment Cell Melanoma Res. 2021;34:1015–1028.   |  1015 wileyonlinelibrary.com/journal/pcmr This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2021 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2021 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd. |  1015 wileyonlinelibrary.com/journal/pcmr |  101 wileyonlinelibrary.com/journal/pcmr Pigment Cell Melanoma Res. 2021;34:1015–1028. (eumelanin), whereas low or no activity leads to the default pro- duction of red pigment (pheomelanin) (Garcia-­Borron et al., 2005). In birds, associations of MC1R variants with pigmentation traits have been found both in domesticated species (Davila et al., 2014; Kerje et al., 2003; Nadeau et al., 2006; Ran et al., 2016; Takeuchi et al., 1996) as well as in wild birds (Baiao & Parker, 2012; Johnson et al., 2012; Lamichhaney et al., 2016; Mundy et al., 2004; San-­Jose et al., 2015; Theron et al., 2001). There are at least six phenotypically distinct Extension/MC1R alleles described in chicken (Andersson et al., 2020), which are here listed according to dominance and de- creasing amount of eumelanin: Extended black (E*E), Birchen (E*R), wild-­type (E*N), brown (E*B), buttercup (E*BC), and wheaten (E*WH or E*Y depending on dominance). These alleles determine the dis- tribution of black eumelanin across the body and MC1R is therefore considered to be involved in primary pattern formation (Andersson et al., 2020; Smyth Jr, 1990). Birds carrying the dominant E and R alleles usually exhibit very dark to black adult plumage while the re- maining alleles create patterns of brown, salmon and wheat color in different body regions. In adult males, only two alleles (E and R) can be distinguished from wild-­type, while the other variant alleles only cause distinct phenotypic effects in female plumage (Andersson et al., 2020; Smyth Jr, 1990). with complete genotype and phenotype data, except that sex re- cordings were missing for two individuals. The chicks were phe- notyped at hatch and at 12 weeks of age as well as genotyped for Db/SOX10 and the E*R(Fay) allele. This protocol received the permit 02410.02 delivered by the French authority for animal experiments, after advice from the INRA Val de Loire ethical committee for animal experimentation. Feather samples for gene expression were collected after sacri- ficing the animals, using electronarcosis followed by bleeding. The feather shafts were frozen in liquid nitrogen and stored at −80°C until further processing. DNA samples for genotyping were obtained from blood samples. Blood samples were taken from the wing vein and submerged with EDTA as an anti-­clotting agent. Blood samples from other breeds showing various patterning phenotypes were ob- tained from fancy breeders. 2.2 | Whole-­genome resequencing of the Fayoumi backcross For the Fayoumi backcross, between 71 and 102 DNA samples per phenotype category were pooled in equimolar quantities and sequenced to 45× coverage on Illumina HiSeq2500 with 2  ×  125 paired-­end reads. In the present study, we generated an informative backcross involving the Fayoumi breed showing autosomal barring (expected genotype Pg/Pg, Db/Db, and R/R at the E/MC1R locus) and an in- bred line of Light Brown Leghorn (LBL) showing no feather pattern and assumed to be wild-­type at all the major loci known to influence plumage color. We used pooled sequencing to map the loci affecting feather pigmentation patterns. Our data show that the major locus controlling the segregation of autosomal barring in this cross is lo- cated at the distal end of chromosome 11 where the E/MC1R locus is located. We also show that Db is not required for autosomal barring but that it affects the manifestation of this phenotype, and we did not find any evidence for the segregation at the proposed Pg locus. Sequencing adapters and low-­quality bases were removed using Trim Galore! (with “-­-­stringency 6 -­q 15”). The trimmed reads were aligned to the chicken reference genome (GRCg6a/Galgal6) using bwa mem with the “-­M” option. PCR duplicates were identified using Picard MarkDuplicates, and variant calling was done using the GATK toolkit (McKenna et  al.,  2010) v4.1.1.0. First, base quality scores were recalibrated using known variants in dbSNP150. Next, the HaplotypeCaller was used to call variants in gVCF format. All gVCF files were combined and the raw variants were scored and filtered by the VariantRecalibrator module using dbSNP150 as a training set (-­-­truth-­sensitivity-­filter-­level 90.0), followed by a hard filter which excluded SNPs with QD <2.0, FS >60.0, MQ <40.0, MQRankSum < −12.5, or ReadPosRankSum < −8.0 and indels with QD <2.0, FS >200.0 or ReadPosRankSum < −20.0. 2.1 | Animals Variants used in the linkage analysis were selected based on an earlier version of the analysis, which was performed as described above with the following differences: alignments were done against Galgal4 using bwa aln followed by bwa sampe, the gatk toolkit v3.2.2 was used, an IndelRealigner step was included, all recalibration steps were done using dbSNP140, and “-­-­ts_filter_level 95.0” was used for the VariantRecalibrator. All genomic coordinates from this analysis have been updated to Galgal6 using the liftOver tool. The Fayoumi population used in this study has been kept at an ex- perimental farm over several decades and has shown none or little deviation from the characteristic pigmentation phenotype described for this breed. Feather samples and phenotype data were obtained from a Fayoumi backcross which was produced at the PEAT Poultry Experimental Facility (INRAE, Nouzilly; https://doi.org/10.15454/​ 1.55723​26250​88729​2E12). Five silver autosomal barred Fayoumi dams (presumed genotype: Pg/Pg, Db/Db, S/W) were crossed with two Light Brown Leghorn males (wild-­type genotype for all feather color loci to the best of our knowledge). From the F1 generation, twelve gold autosomal barred females (presumed genotype Pg/pg+, Db/db+, s+/W) were selected and crossed with another Light Brown Leghorn male to generate 365 backcross progenies in three batches 1016  |     1 | INTRODUCTION MC1R is a key locus in pigmentation biology, and MC1R muta- tions are underlying pigmentation polymorphisms in many ver- tebrates. It codes for a G-­protein-­coupled receptor located in the plasma membrane of melanocytes. Upon activation by its agonist α-­melanocyte-­stimulating hormone (αMSH), MC1R goes through a conformation change triggering a signaling cascade, which even- tually leads to an increase of intracellular cAMP levels, which in turn activates transcription of genes involved in pigment produc- tion. MC1R signaling promotes production of black/brown pigment FI G U R E 1 Phenotypic characterization. (a) Pigmentation phenotypes of the breeds used to generate the intercross, Light Brown Leghorn (left) showing wild-­type color and Fayoumi (right) showing autosomal barring. Photograph credit: Laurence Verrier for the LBL male and David Gourichon for the Fayoumi female. (b) Within-­feather pigmentation pattern in chickens. The proposed major Patterning (Pg) locus interacts with several other loci causing complex variation in melanin distribution across individual feathers. Wild-­type alleles are indicated by “+.” E/ MC1R—­Extension/Melanocortin-­1 receptor, Ml—­Melanotic, Co—­Columbian, Db—­Dark Brown/SOX10. Modified after Andersson et al (2020) FI G U R E 1 Phenotypic characterization. (a) Pigmentation phenotypes of the breeds used to generate the intercross, Light Brown Leghorn (left) showing wild-­type color and Fayoumi (right) showing autosomal barring. Photograph credit: Laurence Verrier for the LBL male and David Gourichon for the Fayoumi female. (b) Within-­feather pigmentation pattern in chickens. The proposed major Patterning (Pg) locus interacts with several other loci causing complex variation in melanin distribution across individual feathers. Wild-­type alleles are indicated by “+.” E/ MC1R—­Extension/Melanocortin-­1 receptor, Ml—­Melanotic, Co—­Columbian, Db—­Dark Brown/SOX10. Modified after Andersson et al. (2020) FI G U R E 1 Phenotypic characterization. (a) Pigmentation phenotypes of the breeds used to generate the intercross, Light Brown Leghorn (left) showing wild-­type color and Fayoumi (right) showing autosomal barring. Photograph credit: Laurence Verrier for the LBL male and David Gourichon for the Fayoumi female. (b) Within-­feather pigmentation pattern in chickens. The proposed major Patterning (Pg) locus interacts with several other loci causing complex variation in melanin distribution across individual feathers. Wild-­type alleles are indicated by “+.” E/ MC1R—­Extension/Melanocortin-­1 receptor, Ml—­Melanotic, Co—­Columbian, Db—­Dark Brown/SOX10. Modified after Andersson et al. (2020) SCHWOCHOW et al. 1017 2.5 | Linkage analysis Linkage analysis was performed using the cri-­map 2.504 software and 76 SNPs obtained through the custom-­made genotyping assay as well as genotype data at MC1R and Db/SOX10 (Tables  S2 and S3). The initial analyses were carried out using the entire pedigree. Genetic studies have predicted Pg to be located on chromosome 1 (Carefoot, 1999; Moore & Smyth, 1972), but evaluation of phe- notype and genotype at Db in our cross suggested that Db is not required for autosomal barring. We therefore eliminated backcross individuals with wild-­type plumage and only used those that showed pattern (autosomal barring or unclear) and carried the Fayoumi allele at MC1R as those could reveal a potential-­associated locus on chro- mosome 1 if there was any. We also noticed a strong sex bias within the phenotype group “unclear” (Table 2), which was dominated by 2.4 | Genotyping and Sanger sequencing of MC1R and Db/SOX10 RNA extraction from growing feather follicles, primer design, primer testing, gene expression assays as well as allelic imbalance testing was performed as described (Schwochow Thalmann et  al.,  2017). All primers used for expression analysis are provided in Table S10. Relative gene expression levels of target genes MC1R, NQO1, CDH1, and WWP2 were calculated using the ΔΔCt method and normal- ized with two housekeeping genes: eukaryotic translation elonga- tion factor (EEF2) and β-­actin (Schwochow Thalmann et al., 2017). The following sequence variants were used for the allelic imbalance assays: MC1R (located at chr11:18,841,043), NQO1 (T to C change; chr11:19,037,574), and CDH1 (A to T change; chr11:18,874,575). The entire Fayoumi pedigree was genotyped for Db, an 8.4 kb dele- tion upstream of SOX10, by using primers and PCR conditions de- scribed elsewhere (Gunnarsson et  al.,  2011). The MC1R genotype was first evaluated by using an allele discrimination assay on a 7900 HT Fast Real-­Time PCR System machine (LifeTechnologies). In brief, 10 ng of DNA was amplified in a reaction using 1× TaqMan Universal PCR mastermix (LifeTechnologies) with 1× Assay mix containing probe and primers and filled up with water to a total reaction vol- ume of 5 μl/sample. The reaction mix was subjected to 40 cycles as follows: 15 s at 92°C and 1 min at 60°C, preceded by 1 × 10 min at 95°C. The data were analyzed using the software sds 2.3. Custom-­ made probes for the L133Q mutation were obtained from Thermo Fisher. females. We therefore performed part of the analysis by only using E*R(Fay)/E*N backcross females. excluding regions with a very low number of variants. The scores for each region were transformed to a Z score, and regions with a score corresponding to p < .05 after Bonferroni correction were reported. 2.7 | Immunohistochemistry and in situ hybridization In order to select genetic markers for fine mapping, we used the resequencing data and extracted SNPs located between position 40,155,407 and 59,843,680 bp on chromosome 1, which occurred at a frequency of 0.4 to 0.6 in the “autosomal barring” pool and <0.2 frequency in the “wild-­type” pool. Each SNP had to be covered by at least 20 reads. The same procedure was used for retrieving SNPs from chromosome 11 located between 15,539,481 and 19,932,279. Some high FST SNPs (FST  >  0.35) between 6,258,804  bp and 71,048,324 on chromosome 2 were also included. The final com- position of SNPs was chosen randomly by keeping the distance be- tween neighboring SNPs in the range 189–­322 kb on chromosomes 1 and 11. For chromosome 2, the SNPs were spread out across the chromosome, as there was no distinct haplotype. The genotyping service was provided by Neogen Europe Ltd, Geneseek. The haplo- type composition was evaluated using Excel. For section immunostaining and in situ hybridization, fixed skin tissue was embedded in paraffin and sectioned at 6–­7  µm. After de-­paraffination, sections were processed for immunohistochem- istry or in situ hybridization. The MITF antibody was from Abcam (ab12039, 1:200 dilution). The peroxidase staining was used after primary antibody treatment as described (Jiang & Chuong,  1992). Non-­radioactive in situ hybridization was performed as described (Chuong et al., 1996). Briefly, the sections were treated with pro- teinase K (10 µg/ml in PBS) for 20 min, re-­fixed with 0.2% glutar- aldehyde/4% paraformaldehyde, and rinsed with PBT. The sections were then prehybridized in hybridization buffer (containing 50% formamide, 5× sodium citrate/sodium chloride buffer, 1% sodium dodecyl sulfate, 50 µg/ml heparin, 50 µg/ml tRNA) at 65°C for 1 hr. After prehybridization, sections were placed in new prehybridiza- tion buffer containing 1–­3  µg/ml digoxigenin-­labeled riboprobes and hybridized overnight at 65°C. Finally, sections were incubated with alkaline phosphatase-­conjugated anti-­digoxigenin Fab (Roche, Indianapolis, IN) overnight. Positive signals were detected by incu- bating the specimens with NBT (nitro-­blue tetrazolium)/BCIP (5-­b romo-­4-­chloro-­3'-­indolyphosphate) substrates (Promega, Madison). 2.3 | Calculation of the fixation index (FST) FST was estimated for each called variant as FST  =  (πbetween –­ πwithin) / πbetween. Variant-­level FST values were then averaged across 30  kb sliding windows and visualized as a Manhattan plot after 1018 SCHWOCHOW et al. Variants were called with HaplotypeCaller from the gatk toolkit (v3.8) followed by filtering using the VariantRecalibrator and hard filters as described above. Variants were called with HaplotypeCaller from the gatk toolkit (v3.8) followed by filtering using the VariantRecalibrator and hard filters as described above. 21 males initially phenotyped as AB at hatch and 7, which exhib- ited an irregular pattern, the pattern had completely disappeared at 12 weeks of age (Table S1, Figure S2), and instead, they developed a reddish taint in the belly region and were termed “red belly” (RB). This group also includes four males, which were scored as wild-­type at hatch but developed the red belly phenotype by 12 weeks. These 28 + 4 male progenies classified as red belly were excluded from most analyses. Pooled heterozygosity was calculated as described (Rubin et  al.,  2010). For each observed SNP, the read count of the major allele, nMAJ, and the minor allele, nMIN, was calculated across all breeds. A pooled heterozygosity, Hp, was calculated as Hp = 2 * ∑(nMAJ) * ∑(nMIN ) / (∑(nMAJ) + ∑(nMIN ))2 across all variants within a 30 kb window and then transformed to a Z score according to ZHp = (Hp − μ) / σ. Negative Z-­scores correspond to regions with less than expected heterozygosity, indicating that they may be iden- tical by descent (IDB). Windows with Z-­scores less than −4.70 were considered significant, which correspond to a p-­value less than .05 after Bonferroni correction. The resulting Z score was visualized in Manhattan plot-­style, omitting windows with too few variants. According to the assumed inheritance pattern of autosomal bar- ring, we expected that 25%–­50% of the backcross progeny should be heterozygous carriers of Pg and Db and show this phenotype, the reason for this range in expected frequency is because Pg has been reported to be linked to Db but no precise estimate of the recom- bination rate is available. We observed 102 AB versus 201 WT, 28 birds with unclear phenotype classification, and 32 with the red belly phenotype (Table  1). By genotyping, we confirmed homozygosity for the causal mutation at Db/SOX10 (Gunnarsson et al., 2011) in all Fayoumi founder females (Db/Db) and all F1 females were heterozy- gous Db/N as expected. 3.1 | Segregation of plumage color in a Fayoumi backcross population We crossed five Fayoumi females (presumed genotype Pg/Pg, Db/ Db) displaying the typical black and white autosomal barring pattern with two Light Brown Leghorn males showing no patterning (wild-­ type at the Pg and Db loci) (Figure 1a). Twelve F1 females (heterozy- gous carriers of Pg and Db) showing the typical autosomal barring pattern of the Fayoumi breed were backcrossed with another Light Brown Leghorn male generating a total of 365 backcross progeny with complete phenotype information that were used for the ge- netic analysis. Of these 365, 102 (43 males and 59 females) chick- ens exhibited the typical autosomal barring pattern (AB), whereas 203 (99 males, 102 females, and 2 unrecorded sex) were classified as wild type (WT) for patterning (Table 1, Figure S2). A total of 60 birds were more difficult to categorize, 28 (9 males and 19 females) were neither plain nor did they have the typical autosomal barring pattern and were classified as “unclear” (Figure 2a). Furthermore, for Surprisingly, not all of the autosomal barred backcross progeny were genotyped as carriers of Db based on the presence of the deletion upstream of SOX10 (66 heterozygous car- riers of Db and 36 non-­carriers) whereas the “unclear” group was almost entirely (27 out of 32 chickens) wild-­type for the SOX10 dele- tion (Table 2). The WT group contained an almost equal distribution with 100 heterozygous carriers of Db and 103 non-­carriers. Thus, the segregation and genotyping data question the critical role of the SOX10 deletion for autosomal barring as well as the linkage between Pg and Db. For the visualization of all haplotypes near MC1R (Figure 5), we extracted all SNPs (including low-­confidence ones) ±5kb (n = 105) or ±50kb (n = 1,424) of the MC1R gene. aSex recordings missing. 2.8 | Whole-­genome resequencing of chicken pools representing different within-­feather patterning phenotypes Six samples with pooled DNA from five individuals each of the Brahma, Buttercup, Fayoumi, Hamburg, Plymouth Rock, and Sebright breeds were sequenced to about 10× coverage on Illumina HiSeq 2,500 with 2  ×  100 bp paired-­end reads. Genome align- ment and variant calling were done against Galgal6 using bwa mem. SCHWOCHOW et al. 1019 Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular pattern; WT, wild-­type. TA B LE 2 Phenotype—­genotype associations among backcross progenies of the Fayoumi/Light Brown Leghorn cross TA B LE 2 Phenotype—­genotype associations among backcross progenies of the Fayoumi/Light Brown Leghorn cross MC1R haplotype (E*R(Fay)) inherited from the Fayoumi founder fe- males. Linkage analysis revealed highly significant LOD scores for loci located at the distal end of chromosome 11 (LOD scores = 28.4–­ 69.1), whereas only weak associations between autosomal barring and some markers on chromosome 1 were observed, irrespective of which subset of backcross progeny was used (Tables S4–­S6). Genotype Phenotype AB WT unclear RB Total SOX10 Db*N/N 36 103 25 27 191 Db*Db/N 66 100 3 5 174 MC1R E*N/N 0 203 0 3 206 E*R(Fay)/N 102 0 28 29 159 Total 102 203 28 32 365 Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular pattern; WT, wild-­type. The 1 Mb interval on chromosome 11 showing a complete asso- ciation with patterning contains 29 genes including MC1R (Table S7). We used our resequencing data to extract SNPs that differed be- tween autosomal barred and wild-­type backcross progeny within the non-­recombining region. The L133Q mutation was not extracted, most likely because it did not meet the coverage requirements of at least 20 reads/SNP. The obtained variant SNPs were analyzed using the online tool UCSC Variant Annotation Integrator (VAI), which re- sulted in a total of 6,627 variants. As expected, the great majority of those variants were located in non-­coding regions such as inter- genic regions (2,342), introns (2,298) as well as down-­ or upstream of genes (967 and 852, respectively; Figure  S4), 19 variants were detected as potential splice variants and 149 as exonic variants. Of the 149 exonic variants, 107 were synonymous while 41 were non-­ synonymous (Figure S4). Ten of the genes in the interval showing no recombination harbored non-­synonymous changes (Table  S8). To further predict the possible effect of the missense mutations on protein function, we used PROVEAN (http://prove​an.jcvi.org/ index.php), an online tool, which generates a score indicating how likely it is that a missense mutation affects protein function (Choi & Chan, 2015; Choi et al., 2012). Except for the known L133Q mutation in the MC1R E*R(Fay) allele, none of the other missense mutations as- sociated with the Fayoumi haplotype from this region was predicted as having a deleterious effect on protein function (Table S8). Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular pattern; WT, wild-­type. TA B LE 2 Phenotype—­genotype associations among backcross progenies of the Fayoumi/Light Brown Leghorn cross covered the region 18.399–­19.527 Mb (near the chromosome end) with the highest signal around 18.702–­19.185 Mb, which included MC1R located at 18,840,646–­18,841,590 bp. The signal on chromo- some 11 was detected both when the “autosomal barring” pool and the “unclear” pool were compared with either one or both of the wild-­type pools (Figure 2b). 3.2 | A major locus underlying autosomal barring maps to a region on chromosome 11 Based on the phenotype data and the genotype data at the Db/SOX10 locus described above, we set up four pools for whole-­ genome resequencing using Illumina HiSeq technology in an at- tempt to map the Pg locus. We used two different wild-­type pools but with different genotypes at Db/SOX10 (heterozygous carrier of Db, n = 99 or wild-­type, n = 102) whereas the two other pools either contained clearly autosomal barred chickens (heterozygous carrier of Db, n = 71) or chickens with a less clear barring phenotype includ- ing all “unclear” progenies as well as some with a less pronounced autosomal barring pattern (wild-­type at Db, n = 72). The pools were constructed taking into account the genotype at the Db/SOX10 locus in order to maximize the chance to detect other loci affecting the patterning phenotype. All four pools were compared to each other, and regions of high differentiation between pools were determined using the Fixation index (FST) in 30 kb sliding windows (Figure 2b; Figure S3). Depending on which pools were compared, two regions with high FST, one on chromosome 1 and one on chromosome 11, were detected. The signal on chromosome 1 encompassing the Db/ SOX10 locus was expected since we constructed the pools on the basis of the genotypes at this locus. The signal on chromosome 11 TA B LE 1 Segregation of four different phenotypes in the Fayoumi/Light Brown Leghorn backcross Sex Phenotype at 12 weeks of age Total AB WT unclear RB Males 43 99 9 32 183 Females 59 102 19 0 180 Unknowna  0 2 0 0 2 Total 102 203 28 32 365 Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular pattern; WT, wild-­type. aS di i i SCHWOCHOW et al. 1020 1021 SCHWOCHOW et al. FI G U R E 2 A 1 Mb region on chromosome 11 is associated with autosomal barring. (a) Segregation of pigmentation phenotypes among backcross progeny. Photograph credit: David Gourichon, INRA. (b) Genome-­wide screen for genetic differentiation between three different pools (“autosomal barred,” “unclear” and “wild-­type”) of Fayoumi/Light Brown Leghorn backcross offspring using FST values in 30 kb sliding windows. (c) SNP scoring within 20 Mb and 3.5 Mb regions on chromosomes 1 and 11, respectively, in parents (P) and offspring progeny (BC) showing autosomal barring (AB), “unclear” patterning or wild-­type plumage. The positions of MC1R and SOX10 are indicated. Please note that a higher density of SNPs was used toward the end of chromosome 11 visually suggesting that the 1 Mb interval is taking up half of the 3.5 Mb region. The borders of the 1 Mb non-­recombining interval are based on a single recombinant. Yellow color indicates homozygosity for an allele inherited from the wild-­type Light Brown Leghorn parental (WT), whereas red indicates homozygosity for a Fayoumi-­derived allele. Orange color indicates heterozygosity. The panel for chromosome 11 represents 18 SNPs with the first situated at 15.8 Mb and the last one at 19.7 Mb. Forty-­six SNPs were placed within the 20 Mb region on chromosome 1 with the first SNP located at 40 Mb and the last at 59.8 Mb. White fields indicate missing genotypes. 3.4 | MC1R, NQO1, and CDH1 are over-­expressed in barred feathers but do not show allelic imbalance 3.4 | MC1R, NQO1, and CDH1 are over-­expressed in barred feathers but do not show allelic imbalance at the MC1R locus among the backcross progeny deviated signifi- cantly from the expected 1:1 ratio (χ2 = 6.05, df = 1, p < .05). This is not specific for MC1R but applies to all markers in the 1 Mb region that did not recombine in this pedigree data. We decided to explore possible regulatory changes in gene expres- sion for genes located in the 1 Mb interval on chromosome 11 asso- ciated with patterning and with a putative role in melanocyte biology (Figure 3a; Table S7). In addition to the obvious positional candidate MC1R, we considered the NAD(P)H Quinone Dehydrogenase 1 (NQO1) gene that codes for a cytoplasmatic 2-­electron reductase, which is reducing quinones to hydroquinones and has been shown to affect the regulation of tyrosinase, thereby enhancing melanogenesis (Choi et al., 2010). We also examined the expression of Cadherin 1 (CDH1), a calcium-­dependent cell-­cell adhesion protein, which among others, To summarize, our genotyping data revealed a perfect associa- tion between a patterning phenotype and the variant E*R(Fay) allele at MC1R. It further revealed an incomplete association between the Db allele at SOX10 and autosomal barring, which suggests that Db is not required to form the pattern but makes it more pronounced and easier to phenotype. There may be a third locus segregating in our cross, which is responsible for the red belly phenotype only present in males. Evaluating the genetic basis for this phenotype will be a subject for future studies. g g p yp subject for future studies. , ) p ( a calcium-­dependent cell-­cell adhesion protein, which among othe FI G U R E 3 Expression analysis of candidate genes on chromosome 11. (a) Genes in the interval lacking recombination in the Fayoumi/ Light Brown Leghorn cross. Positional candidate genes are highlighted with red text. (b) Relative gene expression levels of candidate genes on chromosome 11 in autosomal barred and non-­barred feathers. EEF2 and βACTIN were used as housekeeping genes. Significant differences are indicated by stars (Student's t test; *p < .05, **p < .01, ***p < .001). 3.3 | Fine mapping revealed a region of low recombination on chromosome 11 We used our resequencing data to identify a total of 100 highly in- formative SNPs. The SNPs were selected from the candidate regions on chromosome 1 (n = 63) and chromosome 11 (n = 21), and in ad- dition a few relatively high FST SNPs from chromosome 2 (n = 16) (Table  S2). The entire pedigree was genotyped for these markers to validate the observed associations and improve the map resolu- tion on chromosomes 1 and 11 (Table S3). A total of 46 SNPs on chromosome 1 were informative and revealed no haplotype that was shared among all individuals of either phenotype category (Figure 2c). However, a large proportion of the backcross progeny showing autosomal barring carried the Db haplotype whereas only three that were classified as unclear carried this haplotype reveal- ing a clear association to a patterning phenotype. For chromosome 11, 20 SNPs were highly informative and revealed a region of 1 Mb without any recombination starting from about 18.8 Mb to the end of chromosome 11 (Figure  2c, Table  S3). All birds phenotypically classified as autosomal barred or unclear were heterozygous for the Thus, the screen for missense mutations within the 1  Mb re- gion on chromosome 11 revealed only one missense mutation, the one in MC1R, that was predicted to affect protein function. Since MC1R is an obvious candidate gene for a pigmentation phenotype, we genotyped the entire pedigree for this missense (L133Q) muta- tion (Table 2). All chicken displaying autosomal barring (AB) or some kind of pattern (“unclear”) carried the variant allele at this position, whereas all plain (WT) chicken had the wild-­type allele at MC1R. Even the 28 males that initially were phenotyped as AB but did not show any patterning at 12 weeks of age and were grouped into the red belly group carried the variant allele (Table S1). The segregation data 1022 SCHWOCHOW et al. 3.5 | The expression of MITF, ASIP, and KIT in Fayoumi feather follicles Pigment bars can form due to the presence/absence of melanocytes or by differences in melanin production (eumelanin, pheomela- nin, or no melanin) of the melanocytes that are present (Andersson et al., 2020; Lin et al., 2013). Autosomal barring is composed of alter- nating black and non-­black bars (Figure 1). The non-­black bars may be yellowish due to the presence of pheomelanin (Figure 1b) or more or less non-­pigmented, like in Fayoumi chicken (Figure 1a) due to the presence of the Silver allele at SLC25A2 that inhibits expression of pheomelanin (Gunnarsson et al., 2007). To characterize the mecha- nism underlying autosomal barring in Fayoumi chicken, we performed immunostaining of MITF (microphthalmia-­associated transcription factor), a marker of melanocyte progenitor cells. MITF-­positive cells are present in the proximal follicle where melanocyte stem cells are present, as shown by positive staining for KIT (Figure 4a,c). Toward the distal feather, barb branches start to form and pigment bars emerge. In both pigmented and non-­pigmented regions, we ob- serve the presence of MITF-­positive melanocyte progenitors. This is particularly clear in the border region (Figure 4a,b). We explored what may repress the activity of these melanocyte progenitors in We next reasoned that if the elevated expression levels are the result of cis-­regulatory changes, we expect to observe allelic imbalance in gene expression at one or more of the three genes (Figure 3c). We used SNPs in the transcripts to assess the relative expression of alleles and genomic DNA as control in which we ex- pect a perfect 50:50 ratio. We used the T to A polymorphism caus- ing the MC1R/E*R(Fay) allele (L133Q; chr11: 18,841,043 bp), and SNPs in NQO1 (T to C change; chr11: 19,037,574 bp) and CDH1 (A to T change; chr11:18,874,575 bp) to assess allelic expression in 15 heterozygous chickens utilizing cDNA from growing feathers and the pyrosequencing technology. The expression of the Fayoumi al- lele associated with each investigated gene was 43.1 ± 0.75% for MC1R, 47.7 ± 0.43% for NQO1, and 59.4 ± 1.11% for CDH1. anchors melanocytes to surrounding keratinocytes (Vasioukhin et  al.,  2000) and is implicated in diseases such as vitiligo (Tarle et al., 2015). The last gene we evaluated is coding for an E3 ubiquitin-­ protein ligase (ww domain-­containing protein 1, WWP1) involved in the tanning response following UV exposure in melanocytes (Cao et al., 2013). We measured the expression of these genes in growing chicken feathers from 10 to 12 non-­barred and 10 autosomal barred birds. Three out of the four genes showed a statistically significant up-­regulation of expression in barred feathers compared with non-­ barred feathers (Figure 3b): MC1R (fold change 2.3 ± 0.2, p = .0007, Student's t test), NQO1 (fold change 3.2 ± 0.7, p = .004) and CDH1 (fold change 1.7 ± 0.2, p = .008). WWP1 showed no statistically sig- nificant differential expression (p = .75, Student's t test). To summarize, MC1R, NQO1, and CDH1 located within the non-­ recombining region show up-­regulated expression in growing auto- somal barred feathers but this up-­regulation does not appear to be mediated by a cis-­regulatory effect since none of the three genes shows clear allelic imbalance in favor of the Fayoumi allele. A possi- ble explanation for this up-­regulated expression is that the Fayoumi allele underlying autosomal barring results in a higher proliferation of melanocytes. 3.4 | MC1R, NQO1, and CDH1 are over-­expressed in barred feathers but do not show allelic imbalance (c) Relative proportion of the Fayoumi (F) allele at MC1R NQO1, and CDH1 in either cDNA samples from F/-­ feathers (light gray) or genomic DNA from different genotypes (dark gray; F/-­, -­/-­ and F/F FI G U R E 3 Expression analysis of candidate genes on chromosome 11. (a) Genes in the interval lacking recombination in the Fayoumi/ Light Brown Leghorn cross. Positional candidate genes are highlighted with red text. (b) Relative gene expression levels of candidate genes on chromosome 11 in autosomal barred and non-­barred feathers. EEF2 and βACTIN were used as housekeeping genes. Significant differences are indicated by stars (Student's t test; *p < .05, **p < .01, ***p < .001). (c) Relative proportion of the Fayoumi (F) allele at MC1R, NQO1, and CDH1 in either cDNA samples from F/-­ feathers (light gray) or genomic DNA from different genotypes (dark gray; F/-­, -­/-­ and F/F) SCHWOCHOW et al. 1023 3.6 | Patterning phenotypes are strongly associated with certain MC1R (E) alleles A number of chicken breeds are assumed to be fixed for the Pg allele based on the interpretation of the genetic basis for various plum- age color variants (Figure  1b; Figure  S1). We therefore performed whole-­genome pooled sequencing of birds from six chicken breeds (n = 5 each), exhibiting a variety of pigmentation patterns: Partridge Plymouth Rock—­penciling, Buttercup—­autosomal barring, Silver Sebright—­single lacing, Brahma—­penciling, Hamburg—­spangling, and Fayoumi—­autosomal barring (Figure 1; Figure S1). This was carried out with two purposes (i) to explore whether all breeds predicted to be fixed for the Pg allele share any region of the genome that is identical FI G U R E 5 Region near MC1R reveals no shared haplotype associated with patterning. Predicted variants within ±5kb (top) or ±50kb (bottom) from the MC1R locus in Fayoumi and five other breeds (Brahma, Buttercup, Hamburg, Plymouth Rock, and Sebright) showing patterning phenotypes. The heatmaps show individual variants with vertical black lines indicating their position at the MC1R-­flanking region on chromosome 11. Each breed was sequenced as a pool of five individuals and heterozygous positions thus represent a ~50/50 split within the pool. The MC1R coding sequence is indicated using red lines. The listed MC1R alleles use the following definitions: E*R(Fay) = 133Q, E*B = 71T-­92K-­215P, and E*R = 92K. The question mark for the Hamburg pool indicates that it was predicted to be variable for E92K, V126I, and L133P (note the P instead of Q) and thus is not fixed for E*R. Based on the data presented for Hamburg in Table S9, we deduce that the bird sequenced here carried the two following alleles: 92K-­126I-­133L (E*R) and 92E-­126V-­133P (E*?) FI G U R E 5 Region near MC1R reveals no shared haplotype associated with patterning. Predicted variants within ±5kb (top) or ±50kb (bottom) from the MC1R locus in Fayoumi and five other breeds (Brahma, Buttercup, Hamburg, Plymouth Rock, and Sebright) showing patterning phenotypes. The heatmaps show individual variants with vertical black lines indicating their position at the MC1R-­flanking region on chromosome 11. Each breed was sequenced as a pool of five individuals and heterozygous positions thus represent a ~50/50 split within the pool. The MC1R coding sequence is indicated using red lines. The listed MC1R alleles use the following definitions: E*R(Fay) = 133Q, E*B = 71T-­92K-­215P, and E*R = 92K. by descent (IBD) and (ii) if these breeds share an IBD region within the 1  Mb region on chromosome 11 defined in our Fayoumi backcross population. Thus, the reason for using only five individuals per pool was that our aim was not to estimate allele frequencies, but to identify sequence variants that are fixed in these breeds since all individuals within the breeds show a patterning phenotype. The analysis of the pooled data revealed no striking IBD region shared by all populations neither within the chromosome 11 interval nor in the entire genome (Figure S5). However, the most striking finding was that all populations carried only two alleles Birchen (E*R or E*R(Fay)) or brown (E*B) at MC1R, while not sharing any common haplotype around MC1R (Figure 5). the non-­black region. In Silver Laced Wyandotte chicken, ASIP was found to be present in the peripheral pulp facing the white region and suppress eumelanogenesis of MITF-­positive progenitor cells (Inaba & Chuong, 2020; Lin et al., 2013). Using in situ hybridization, we de- tected expression of ASIP in the non-­black regions. This is particularly clear in a cross section (Figure 4b). Thus, the non-­black bars in autoso- mal barring of Fayoumi chicken are not caused by the absence of mel- anocytes, as seen in sex-­linked barring (Lin et al., 2013; Schwochow Thalmann et al., 2017). Our results suggest that eumelanogenesis is suppressed due to the expression of ASIP in the non-­black regions. A striking finding when comparing the 10 kb region harboring MC1R is the lack of linkage disequilibrium between sequence vari- ants in this region (Figure 5), a total contrast to the lack of recombi- nation over a 1 Mb region reported in this study based on pedigree analysis. This analysis also demonstrates that the Birchen (E*R) allele previously defined based on its phenotypic effect on plumage color is genetically heterogenous both as regards the MC1R coding se- quence as well as the flanking sequences that may harbor regulatory variants affecting MC1R expression. For instance, the Brahma breed is considered fixed for the E*R allele but the Brahma birds included in this study are apparently segregating for at least two alleles both carrying the E92K mutation but differing as regards other missense mutations in MC1R as well as the haplotype upstream of the coding sequence (Figure 5). 3.5 | The expression of MITF, ASIP, and KIT in Fayoumi feather follicles These cDNA data and the genomic DNA control differed significantly from each other (MC1R: Student's t test p = .0001; NQO1: Student's t test p = .008; CDH1: Student's t test p = 1 × 10−6), but these minor differ- ences do not support a typical allelic imbalance expected in the pres- ence of cis-­regulatory effects, and we believe that they are unlikely to reflect actual expression differences with biological significance. FI G U R E 4 The expression of MITF, ASIP, and KIT in the Fayoumi feather follicle. (a) MITF immunostaining. Longitudinal feather sections with enlargement shown in the right column. a–­c, black and white barred region. d, proximal follicle in collar bulge region. MITF-­positive cells are seen in the basal layer of the feather filament epidermis in the stem cells region, in both black and white barb ridges. MITF-­ positive cells (red) are highlighted by box a, b, c, and d. (b) Longitudinal feather sections with ASIP in situ hybridization. ASIP is expressed in feather peripheral pulp facing non-­pigmented bar regions (a’ and b’; b’ is a cross section). (c) Longitudinal feather sections with KIT in situ hybridization showing KIT-­positive melanocyte stem cells at the follicle base near collar bulge region box a’’ and b’’ FI G U R E 4 The expression of MITF, ASIP, and KIT in the Fayoumi feather follicle. (a) MITF immunostaining. Longitudinal feather sections with enlargement shown in the right column. a–­c, black and white barred region. d, proximal follicle in collar bulge region. MITF-­positive cells are seen in the basal layer of the feather filament epidermis in the stem cells region, in both black and white barb ridges. MITF-­ positive cells (red) are highlighted by box a, b, c, and d. (b) Longitudinal feather sections with ASIP in situ hybridization. ASIP is expressed in feather peripheral pulp facing non-­pigmented bar regions (a’ and b’; b’ is a cross section). (c) Longitudinal feather sections with KIT in situ hybridization showing KIT-­positive melanocyte stem cells at the follicle base near collar bulge region box a’’ and b’’ SCHWOCHOW et al. 1024 3.6 | Patterning phenotypes are strongly associated with certain MC1R (E) alleles The question mark for the Hamburg pool indicates that it was predicted to be variable for E92K, V126I, and L133P (note the P instead of Q) and thus is not fixed for E*R. Based on the data presented for Hamburg in Table S9, we deduce that the bird sequenced here carried the two following alleles: 92K-­126I-­133L (E*R) and 92E-­126V-­133P (E*?) SCHWOCHOW et al. 1025 Since there was no shared haplotype detected for breeds with a patterning phenotype at MC1R or elsewhere in the genome (Figure 5 and S5), but they all carry an MC1R allele with an activating mutation, we were considering the hypothesis that different MC1R alleles with similar functional effects are required for patterning phenotypes. Genotyping and subsequent sequencing of 60 Fayoumi chicken from different generations from the flock initially used for linkage mapping revealed that they are not fixed for E*R(Fay) (L133Q) but also segre- gate for the E*R allele (Table S9), which involves a missense mutation (E92K) resulting in constitutive activation (Benned-­Jensen et al., 2011; Ling et al., 2003; Robbins et al., 1993) and known to be associated with melanism in other species (Baiao & Parker, 2012; Kerje et al., 2003; Mundy et al., 2004; Nadeau et al., 2006). Despite the segregation of two different MC1R/E alleles, there has been no obvious heterogene- ity in the phenotypic appearance of autosomal barring in this Fayoumi line suggesting that the two alleles have a similar effect on patterning in this breed. We further extended our MC1R sequencing efforts to 16 additional individuals each from three of the Pg breeds (Partridge Plymouth Rock, Hamburg Silver Spangled and Sebright Silver) and found that all carried the E92K mutation (Table S9). that regulatory mutations affecting MC1R expression also contribute to the complex inheritance of within-­feather patterns in chicken as previously suggested (Ling et al., 2003). One third of the autosomal barred backcross offspring were wild-­type at the SOX10 locus, demonstrating that the Db allele is not required for the autosomal barring phenotype. This is in con- trast to previous reports in which Db was claimed to be required to exhibit autosomal barring in Fayoumi chicken (Carefoot, 1984,1999; Moore & Smyth, 1972). 4 | DISCUSSION The Patterning locus has been considered the major locus control- ling within-­feather pigmentation patterns in chickens (Figure  1b). Previous studies assigned this locus to chicken linkage group 3 (Carefoot,  1987; Moore & Smyth,  1972), now known to reside on chromosome 1. According to this model, we expected that the seg- regation of autosomal barring in our Fayoumi × Light Brown Leghorn backcross should be controlled by a locus on chromosome 1 in combi- nation with the Dark brown/SOX10 locus located on the same chromo- some. However, the present study demonstrates that a locus located at the distal end of chromosome 11 is underlying autosomal barring in this pedigree. Our linkage data did not pinpoint a single gene associ- ated with this phenotype due to the lack of recombination in a 1 Mb region. However, other data strongly suggest that autosomal barring in this pedigree is caused by the E*R(Fay) allele, characterized by the missense mutation L133Q, at the MC1R locus located in this interval. Pooled genome resequencing of six different breeds all exhibiting var- ious types of patterning (previously assumed to be controlled by the Patterning locus) did not reveal any IBD region, which was expected if they were sharing the same causal mutation. However, the most striking feature was that all six breeds carried either Birchen (E*R) or brown (E*B) alleles at the MC1R locus. We observe that MC1R exhibits a high level of genetic diversity in its flanking sequences in addition to diversity within the coding sequence. We propose that MC1R is the major patterning locus in chickens; that is, different mutations causing altered regulation of MC1R signaling promote the develop- ment of feather patterns in interaction with other loci. One such in- teracting locus is definitely Dark brown/SOX10, others are melanotic (Ml) and Columbian restriction (Co) for which no underlying causal gene has been reported yet (Figure 1b). Furthermore, it is possible The presence of an activating MC1R/E mutation may be permis- sive but not sufficient to cause feather patterning because there are breeds carrying the activating E92K mutation but do not show pat- terning. In fact, none of the breeds examined in this study carried the top dominant allele Extended Black (E), which also has the E92K mu- tation and is associated with solid black colored feathers (Andersson et al., 2020). 3.6 | Patterning phenotypes are strongly associated with certain MC1R (E) alleles However, as almost all individuals carrying the causal MC1R allele but with an “unclear” feather pattern were wild-­type at Db, we propose that the Db mutation is contributing to the regularity and clearness of autosomal barring, although not re- quired for its formation. It is therefore likely that breeders specif- ically selected Db carriers for a more appealing phenotype, which may have led to the assumption that Db is required for autosomal barring. Thus, the genetic basis for patterning in chicken needs to be reconsidered since it is clear that there is not a universal pattering gene such as Pg, but that the MC1R/E locus plays a major role for patterning. However, the present study does not completely rule out the possibility of the existence of a Patterning locus in addition to MC1R/E, because it is possible, but unlikely, that the Fayoumi and Light Brown Leghorn founders used in the present study are all ho- mozygous Pg/Pg despite the fact the latter do not show any pattern- ing phenotype (Figure 1a). Nevertheless, our study provides strong evidence against the previous assumption that a Patterning locus on chromosome 1 has a predominant role for the presence/absence of within-­feather patterns in chicken (Smyth Jr, 1990). Ling et al. (2003) investigated the functional effects of differ- ent chicken MC1R alleles using transfection studies. They found that the E92K mutation present in the E*E, E*R, and E*B alleles is causing constitutive activation of the receptor. In contrast, they did not observe a similar effect for the L133Q mutation underlying the E*R(Fay) allele, which has been phenotypically assigned to the E*R group of MC1R alleles in chicken. However, these pharmacological studies carried out in mammalian cells do not perfectly replicate the endogenous conditions for chicken MC1R; for instance, im- portant cofactors may be absent during the assay. Furthermore, these missense mutations may occur in strong linkage disequilib- rium with regulatory mutations that affect MC1R expression. Thus, an important functional effect may not be manifested under these experimental conditions. In fact, the segregation of both the E92K and L133Q alleles in the line of Fayoumi chickens used in the pres- ent study without an obvious heterogeneity in phenotype shows that the two alleles must have similar effects on autosomal barring. expression of MC1R, also the closely linked NQO1 and CDH1 genes show higher expression in autosomal barred feathers; both genes have an established role in pigment cell biology (Choi et al., 2010; Vasioukhin et al., 2000). Thus, the presence of the E*R(Fay) allele re- sults in more active melanocytes and/or an expansion of the number of melanocytes that may affect the interaction of different cell types that together determine within-­feather pigmentation patterns. This model can be tested by gene editing and/or in vivo transfection ex- periment manipulating MC1R and ASIP expression. The present study is also relevant for understanding the basis for camouflage color in birds. Stippling (Figure 1b) is a wild-­type camou- flage pattern typical for female birds nesting on the ground, and some juvenile birds present on the ground before they can fly, like gulls, terns and certain shorebirds. Our results support a role for ASIP-­ MC1R interactions in the formation of within-­feather patterns, at least in some birds, and that increased MC1R signaling not only leads to a darker plumage but can also adjust the appearance of camouflage patterns. The importance of ASIP-­MC1R interactions for camouflage patterns is also evident in mammals (Andersson,  2020). Dominant MC1R mutations causing constitutive activation as well as recessive mutations inactivating MC1R function disrupt the characteristic cam- ouflage pattern in piglets (Kijas et al., 2001), which is also observed in chickens since the dorsal stripes of a wild-­type day-­old chick are not observed in fully black breeds, such as the Black Castellana, as well as in the fully red breeds, such as the Rhode Island Red. In the case of the Fayoumi, the day-­old chicks show incomplete stripes on the back. Furthermore, ASIP-­MC1R interactions are underlying winter white camouflage color in snow-­shoe hares (Jones et al., 2018). In birds, distinct pigment patterns can form across the body or within a feather (Inaba & Chuong,  2020). Horizontal barring in feathers is a striking pattern. Genetic studies of sex-­linked barring established that this phenotype is caused by the combined effect of regulatory mutations and changes in the coding sequence of CDKN2A (Hellström et al., 2010; Schwochow Thalmann et al., 2017). Interestingly, MITF-­positive cells are absent in the white barred re- gions, and there is no ASIP expression in the associated peripheral pulp of sex-­linked barring feathers (Lin et  al.,  2013). Feathers are made from the distal to the proximal end (Chen et al., 2015). Careful analyses revealed that the depletion of melanocytes is considered to be based on CDKN2A-­dependent precocious differentiation of me- lanocytes. When melanocyte stem cells sense the absence of mela- nocytes, another wave of melanocyte progenitor cells is produced to make the next black bar (Lin et al., 2013). In contrast, in the autosomal barring phenotype, we observed MITF-­positive cells to be present in both the black and non-­black barred regions during their forma- tion. The pattern appears to be controlled by ASIP in the peripheral pulp region. How ASIP expression in the dermal cells is controlled is not known. In developing Japanese quail embryos, longitudinal pigmented stripes form on the dorsal trunk, and Japanese quail me- lanocytes transplanted to embryonic day 6 White Leghorn chicken embryos can induce ASIP in adjacent dermis (Inaba et  al.,  2019). Thus, we can speculate that melanocytes within the feather follicle at certain functional states may also induce ASIP expression. The present finding that genetic variation at MC1R is affecting pigmentation patterns in chicken is interesting in relation to reports that the MC1R antagonist ASIP has a role in feather pigmentation (Lin et al., 2013; Yoshihara et al., 2011; Zhang et al., 2013) and that ASIP is strongly associated with differences in throat pigmentation in golden-­winged and blue-­winged warblers (Toews et al., 2016) and a plumage trait in white wagtails (Semenov et al., 2021). These data together suggest that ASIP-­MC1R interaction may play a pivotal role for pigmentation patterns in birds in general and that this interaction may have a greater impact in the growing feather that will develop into a two-­dimension structure, as compared to the single dimension of the hair developing from mammalian hair follicles. 1026  | 1026 SCHWOCHOW et al. 4 | DISCUSSION Thus, further pedigree experiments are required before we fully understand the genetic basis for the rich phenotypic diver- sity in within-­feather patterning in this species (Figure 1 and S1). We observed a surprisingly low rate of recombination in the 1 Mb interval at the distal end of chromosome 11 given the fact that the average recombination rate in chicken is about 4 cM/Mb. A possible explanation for a low rate of recombination is the presence of an inversion. However, it is unlikely that this is the case for this region because a similarly low rate of recombination has beem reported in previous studies (Groenen et al., 2009; Pengelly et al., 2016). The expression data in feather follicles are consistent with our interpretation that autosomal barring is caused by an activating coding mutation at the MC1R locus. We documented up-­regulated expression of MC1R, CDH1, and NQO1 in autosomal barred feath- ers compared with wild-­type feathers consistent with increased pigment production or an expansion of the number of pigment cells (Figure 4b). However, none of the three loci showed a clear allelic imbalance as expected if the causal mutation was a cis-­acting reg- ulatory mutation affecting the expression of one or more of these genes. 1026  | REFERENCES Andersson, L. (2020). Mutations in domestic animals disrupting or creat- ing pigmentation patterns. Frontiers in Ecology and Evolution, 8, 116. https://doi.org/10.3389/fevo.2020.00116 Gunnarsson, U., Hellström, A. R., Tixier-­Boichard, M., Minvielle, F., Bed'hom, B., Ito, S., Jensen, P., Rattink, A., & Vereijken, A., & Andersson, L. (2007). Mutations in SLC45A2 cause plumage color variation in chicken and Japanese quail. Genetics, 175, 867–­877. Andersson, L., Bed’hom, B., Chuong, C.-­M., Inaba, M., Okimoto, R., & Tixier-­Boichard, M. (2020). The genetic basis for pigmentation phe- notypes in poultry. In S. E. Aggrey, H. Zhou, M. Tixier-­Boichard, & D. D. Rhoads (Eds.), Advances in poultry genetics and genomics. Burleigh Dodds Science Publishing. Gunnarsson, U., Kerje, S., Bed’hom, B., Sahlqvist, A.-­S., Ekwall, O., Tixier-­ Boichard, M., Kämpe, O., & Andersson, L. (2011). The Dark brown plumage color in chickens is caused by an 8.3-­kb deletion upstream of SOX10. Pigment Cell and Melanoma Research, 24, 268–­274. https:// doi.org/10.1111/j.1755-­148X.2011.00825.x Baiao, P. C., & Parker, P. G. (2012). Evolution of the melanocortin-­1 re- ceptor (MC1R) in boobies and gannets (Aves, Suliformes). Journal of Heredity, 103, 322–­329. https://doi.org/10.1093/jhere​d/esr151 Hellström, A. R., Sundström, E., Gunnarsson, U., Bed’Hom, B., Tixier-­ Boichard, M., Honaker, C. F., Sahlqvist, A.-­S., Jensen, P., Kämpe, O., Siegel, P. B., & Kerje, S., & Andersson, L. (2010). Sex-­linked barring in chickens is controlled by the CDKN2A/B tumour suppressor locus. Pigment Cell and Melanoma Research, 23, 521–­530. Benned-­Jensen, T., Mokrosinski, J., & Rosenkilde, M. M. (2011). The E92K melanocortin 1 receptor mutant induces cAMP production and arrestin recruitment but not ERK activity indicating biased constitutive signaling. PLoS One, 6, e24644. https://doi.org/10.1371/journ​al.pone.0024644 Cao, J., Wan, L., Hacker, E., Dai, X., Lenna, S., Jimenez-­Cervantes, C., Wang, Y., Leslie, N. R., Xu, G. X., Widlund, H., Ryu, B., Alani, R. M., Dutton-­Regester, K., Goding, C., Hayward, N. K., Wei, W., & Cui, R. (2013). MC1R is a potent regulator of PTEN after UV exposure in melanocytes. Molecular Cell, 51, 409–­422. https://doi.org/10.1016/j. molcel.2013.08.010 Inaba, M., & Chuong, C. M. (2020). Avian pigment pattern formation: Developmental control of macro-­ (across the body) and micro-­ (within a feather) level of pigment patterns. Frontiers in Cell and Developmental Biology, 8, 620. https://doi.org/10.3389/fcell.2020.00620 Inaba, M., Jiang, T.-­X., Liang, Y.-­C., Tsai, S., Lai, Y.-­C., Widelitz, R. B., & Chuong, C. M. (2019). Instructive role of melanocytes during pig- ment pattern formation of the avian skin. Proceedings of the National Academy of Sciences USA, 116, 6884–­6890. https://doi.org/10.1073/ pnas.18161​07116 Carefoot, W. ACKNOWLEDGEMENTS The help of the animal caretakers of the INRA PEAT experimental unit is gratefully acknowledged as well as Mr. Frédéric Theme and Mr. Jacques Berge who provided blood samples from various breeds showing feather pigmentation patterns. We are also thankful to the wet laboratory support by Erika Manlig and thank Marie Allen and Magdalena Bus for instructions and use of their pyrosequenc- ing instrument. LA is supported by the Knut and Alice Wallenberg Foundation and Vetenskapsrådet. CMC and TXJ are supported by NIH NIAMS R37 AR060306. DS benefited from an Erasmus Mundus fellowship within the framework of the European School of Animal Breeding and Genetics. The National Genomics Infrastructure Why is the presence of some MC1R mutations enhancing MC1R signaling and melanogenesis associated with within-­feather pig- mentation patterns as shown here, rather than just make feathers darker as predicted from the effects of MC1R mutations on hair pig- mentation in mammals? Prum and Williamson (2002) proposed that within-­feather pigmentation patterns might be caused by a Turing reaction–­diffusion model. MC1R signaling stimulates melanogenesis, and it is possible that this, by an unknown mechanism, also induces ASIP expression in neighboring cells, and that ASIP in turn suppresses MC1R signaling as indicated in this study (Figure 4). This model gains some support from our finding that in addition to up-­regulated SCHWOCHOW et al. 1027 Chen, C.-­F., Foley, J., Tang, P.-­C., Li, A., Jiang, T. X., Wu, P., Widelitz, R. B., & Chuong, C. M. (2015). Development, regeneration, and evolution of feathers. Annual Review of Animal Biosciences, 3, 169–­195. https:// doi.org/10.1146/annur​ev-­anima​l-­02251​3-­114127 (NGI)/Uppsala Genome Center and UPPMAX provided service in massive parallel sequencing and computational infrastructure. Work performed at NGI/Uppsala Genome Center has been funded by RFI/ VR and Science for Life Laboratory, Sweden. Choi, T.-­Y., Sohn, K.-­C., Kim, J.-­H., Kim, S.-­M., Kim, C.-­H., Hwang, J.-­ S., Lee, J.-­H., Kim, C. D., & Yoon, T.-­J. (2010). Impact of NAD(P) H:Quinone oxidoreductase-­1 on pigmentation. Journal of Investigative Dermatology, 130, 784–­792. https://doi.org/10.1038/jid.2009.280 Authors declare no competing interests. Choi, Y., & Chan, A. P. (2015). PROVEAN web server: A tool to pre- dict the functional effect of amino acid substitutions and indels. Bioinformatics, 31, 2745–­2747. https://doi.org/10.1093/bioin​forma​ tics/btv195 ORCID Doreen Schwochow  https://orcid.org/0000-0002-0090-4355 Tingxing Jiang  https://orcid.org/0000-0003-1972-8985 Jingyi Li  https://orcid.org/0000-0001-6775-5190 Bertrand Bed’Hom  https://orcid.org/0000-0002-0825-0886 Cheng-­Ming Chuong  https://orcid.org/0000-0001-9673-3994 Michèle Tixier-­Boichard  https://orcid.org/0000-0001-5944-7884 Leif Andersson  https://orcid.org/0000-0002-4085-6968 Garcia-­Borron, J. C., Sanchez-­Laorden, B. L., & Jimenez-­Cervantes, C. (2005). Melanocortin-­1 receptor structure and functional regulation. Pigment Cell Research, 18, 393–­410. https://doi. org/10.1111/j.1600-­0749.2005.00278.x Groenen, M. A., Wahlberg, P., Foglio, M., Cheng, H. H., Megens, H.-­J., Crooijmans, R. P., Besnier, F., Lathrop, M., Muir, W. M., Wong, G.-­ K.-­ S., Gut, I., & Andersson, L. (2009). A high-­density SNP-­based linkage map of the chicken genome reveals sequence features correlated with recombination rate. Genome Research, 19, 510–­519. https://doi. org/10.1101/gr.086538.108 DATA AVAILABILITY STATEMENT All sequencing data from this study are available through NCBI Sequence Read Archive BioProject PRJNA694957 (SAMN17582112, Autosomal barred pool; SAMN17582113, Dark brown (Db) pool; SAMN17582114, Wild-­type pool; SAMN17582115, unclear pool) and PRJNA679787 (SAMN16846355, Sebright; SAMN16846356, Brahma; SAMN16846357, Hamburg; SAMN16846358, Plymouth_ Rock; SAMN16846359, Buttercup; SAMN16846360, Fayoumi). Choi, Y., Sims, G. E., Murphy, S., Miller, J. R., & Chan, A. P. (2012). Predicting the functional effect of amino acid substitutions and indels. PLoS One, 7, e46688. https://doi.org/10.1371/journ​al.pone.​ 0046688 Chuong, C.-­M., Widelitz, R. B., Ting-­Berreth, S., & Jiang, T.-­X. (1996). Early events during avian skin appendage regeneration: Dependence on epithelial-­mesenchymal interaction and order of molecular reap- pearance. Journal of Investigative Dermatology, 107, 639–­646. https:// doi.org/10.1111/1523-­1747.ep125​84254 Davila, S. G., Gil, M. G., Resino-­Talavan, P., & Campo, J. L. (2014). Association between polymorphism in the melanocortin 1 recep- tor gene and E locus plumage color phenotype. Poultry Science, 93, 1089–­1096. https://doi.org/10.3382/ps.2013-­03611 CONFLICT OF INTEREST Authors declare no competing interests. melanocortin-­1 receptor gene. 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Melanocortin 1-­receptor (MC1R) mutations are associated with plumage colour in chicken. Animal Genetics, 34, 241–­248. Schwochow Thalmann, D., Ring, H., Sundström, E., Cao, X., Larsson, M., Kerje, S., Höglund, A., Fogelholm, J., Wright, D., Jemth, P., Hallböök, F., Bed'Hom, B., Dorshorst, B., & Tixier-­Boichard, M., & Andersson, L. (2017). The evo- lution of Sex-­linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A. PLoS Genetics, 13, e1006665. Kijas, J. M. H., Moller, M., Plastow, G., & Andersson, L. (2001). A frame- shift mutation in MC1R and a high frequency of somatic reversions cause black spotting in pigs. Genetics, 158, 779–­785. Semenov, G. A., Linck, E., Enbody, E. D., Harris, R. B., Khaydarov, D. R., Alström, P., Andersson, L., & Taylor, S. A. (2021). Asymmetric introgression reveals the genetic architecture of a plumage trait. 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Series B: Biological Sciences, 269, 781–­792. https:// doi.org/10.1098/rspb.2001.1896 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. Ran, J. S., You, X. Y., Jin, J., Zhou, Y. G., Wang, Y., Lan, D., Ren, P., & Liu, Y. P. (2016). The relationship between MC1R mutation and plum- age color variation in pigeons. BioMed Research International, 2016, 3059756. https://doi.org/10.1155/2016/3059756 How to cite this article: Schwochow D, Bornelöv S, Jiang T, et al. The feather pattern autosomal barring in chicken is strongly associated with segregation at the MC1R locus. Pigment Cell Melanoma Res. 2021;34:1015–­1028. https://doi. org/10.1111/pcmr.12975 How to cite this article: Schwochow D, Bornelöv S, Jiang T, et al. The feather pattern autosomal barring in chicken is strongly associated with segregation at the MC1R locus. Pigment Cell Melanoma Res. 2021;34:1015–­1028. https://doi. org/10.1111/pcmr.12975 Robbins, L. S., Nadeau, J. H., Johnson, K. R., Kelly, M. A., Roselli-­Rehfuss, L., Baack, E., Mountjoy, K. G., & Cone, R. D. (1993). Pigmentation phenotypes of variant extension locus alleles result from point mu- tations that alter MSH receptor function. Cell, 72, 827–­834. https:// doi.org/10.1016/0092-­8674(93)90572​-­8 Rubin, C.-­J., Zody, M. C., Eriksson, J., Meadows, J. R. S., Sherwood, E., Webster, M. T., Jiang, L., Ingman, M., Sharpe, T., Ka, S., Hallböök, F.,
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Contribution of Information and Communication Technology (ICT) in Country'S H-Index
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1MARYAM FARHADI1*, HADI SALEHI2,3, MOHAMED AMIN EMBI2, MASOOD FOOLADI1, HADI FARHADI4, AREZOO AGHAEI CHADEGANI1, and NADER ALE EBRAHIM5 *Corresponding author: Maryam Farhadi, Email: farhadim58@gmail.com *Corresponding author: Maryam Farhadi, Email: farhadim58@gmail.com Journal of Theoretical and Applied Information Technology 10th November 2013. Vol. 57 No.1 © 2005 - 2013 JATIT & LLS. All rights reserved. Journal of Theoretical and Applied Information Technology 10th November 2013. Vol. 57 No.1 © 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org ABSTRACT The aim of this study is to examine the effect of Information and Communication Technology (ICT) development on country’s scientific ranking as measured by H-index. Moreover, this study applies ICT development sub-indices including ICT Use, ICT Access and ICT skill to find the distinct effect of these sub-indices on country’s H-index. To this purpose, required data for the panel of 14 Middle East countries over the period 1995 to 2009 is collected. Findings of the current study show that ICT development increases the H-index of the sample countries. The results also indicate that ICT Use and ICT Skill sub- indices positively contribute to higher H-index but the effect of ICT access on country’s H-index is not clear Keywords: Information and Communication Technology (ICT) development, H-index, Middle East CONTRIBUTION OF INFORMATION AND COMMUNICATION TECHNOLOGY (ICT) IN COUNTRY’S H- INDEX 1MARYAM FARHADI1*, HADI SALEHI2,3, MOHAMED AMIN EMBI2, MASOOD FOOLADI1, HADI FARHADI4, AREZOO AGHAEI CHADEGANI1, and NADER ALE EBRAHIM5 1Department of Accounting, Mobarakeh Branch, Islamic Azad University, Mobarakeh, Isfahan, Iran 2Faculty of Education, Universiti Kebangsaan Malaysia (UKM), Bangi, 43600, Malaysia 3Faculty of Literature and Humanities, Najafabad Branch, Islamic Azad University, Najafabad, Isfahan, Iran 4School of Psychology and Human Development, Faculty of Social Sciences and Humanities, Universiti Kebangsaan Malaysia (UKM), Malaysia 5Department of Engineering Design and Manufacture, Faculty of Engineering, University of Malaya, Kuala Lumpur, Malaysia *Corresponding author: Maryam Farhadi, Email: farhadim58@gmail.com www.jatit.org it 0 1 it 2 it 3 it it H Access Use Skill µ µ µ µ δ = + + + + (2) (2) where 's µ are parameters to be estimated, H shows H-index and Access, Use and Skill stand for ICT development sub-indices. Moreover, subscripts i and t indicate the ith country in tth year, respectively, and δ is the error term which is assumed to be independent of Access, Use and Skill variables. where 's µ are parameters to be estimated, H shows H-index and Access, Use and Skill stand for ICT development sub-indices. Moreover, subscripts i and t indicate the ith country in tth year, respectively, and δ is the error term which is assumed to be independent of Access, Use and Skill variables. H-index can measure the quantity and quality of papers simultaneously since the total number of published articles and the number of citations received by that articles are considered in the calculation of H-index. In addition, there are many studies, which reveal the benefits of H- index as an appropriate and fair tool for evaluating and ranking scientists, laboratories, scientific journals, universities, institutions and countries [17, 18, 19, 20, 21]. 2.2.1. ICT development The IDI, as an indicator of ICT development is computed based on Principal Component Analysis (PCA), which combines various indicators in order to make single value. For the first time, IDI is introduced in 2009 by Measuring the Information Society [23] and considers the degree of progress in ICTs in more than 150 countries. Composition of this indicator includes ICT Access showing infrastructure and access, ICT Skill referring to ICT skill or capability and ICT Use displaying the extent of using ICT. Required ICT data to compute the IDI is extracted from United Nations Educational Scientific and Cultural Organization (UNESCO) and ITU. Nowadays, ICT is considered as an important factor in development of countries, which are moving towards knowledge or Hirsch [15] proposes that H-index can be used to appraise not only the scientists but also countries and institutions. Hence, following Jacso [22], current study also uses H-index as a proxy to measure scientific ranking of countries. www.jatit.org East countries, this study applies the regression techniques with countries’ H-index as dependent variable and IDI as independent variable as follows: Although, the key role of ICT is approved in different areas such as engineering [11], education [12], health care [13], and economy [6], its effect on the degree of scientific ranking of a country is yet unexplored. Therefore, the aim of this study is to explore the impact of ICT development as measured by ICT Development Index (IDI) on H-index as a proxy of scientific progress of a country. it 0 1 it it H IDI α α ε = + + (1) (1) where 's α are parameters to be estimated, H represents H-index, IDI stands for ICT development index and subscripts i and t show the ith country in tth year, respectively. ε is the error term which is assumed to be independent of IDI. Then, for more inspections, instead of IDI, which captures the composite effect of ICT development, the current study uses three ICT sub-indices including ICT Access, ICT Use and ICT Skill as explanatory variables. In this approach, we can test the distinct impact of each sub-index on the country’s H-index. Consequently, in this step of the analysis, following equation (Equation 2) is used: In addition, in order to shed more lights on this area, present study applies three components of ICT development namely, ICT Skill, ICT Use and ICT Access to examine the distinct impact of these components on H-index as a proxy of scientific progress of a country. To this purpose, this study applies a multiple regression analysis on a panel of 14 Middle East countries over the period 1995 to 2009. The current study suggests the use of H-index provided by SCImago database [14] to measure the scientific ranking of country. Based on Hirsch [15], a scientist or a group of scientists has H-index of h when h of their papers has at least h citations. In other words, H-index is the number of articles with citation number higher or equal to h [15]. The H-index, which has been invented by Hirsch in 2005, proposes a simple and apparently strong measure to compare the scientific productivity and visibility of countries and institutions [16]. 1. INTRODUCTION However, ICT revolution and use of internet caused many structural changes [3]. As an example, the United States productivity has been revived during the late 1990s and the early 2000s because of the ICT development [4]. There are many studies introducing ICT as an important element to facilitate economic growth in both developing and developed countries [5, 6]. In addition, ICT has the potential to increase health systems through the designing new methods to prevent and detect disease [7]. Other studies confirm the dominant role of ICT in improving and modernizing the learning methods and educational systems [8, 9]. The fast progress in technology in the last two decades has reduced the cost of ICTs. In this situation, it is easier for more people to use and enjoy the advantages of ICTs [10]. However, ICT revolution and use of internet caused many structural changes [3]. As an example, the United States productivity has been revived during the late 1990s and the early 2000s because of the ICT development [4]. There are many studies introducing ICT as an important element to facilitate economic growth in both developing and developed countries [5, 6]. In addition, ICT has the potential to increase health systems through the designing new methods to prevent and detect disease [7]. Other studies confirm the dominant role of ICT in improving and modernizing the learning methods and educational systems [8, 9]. The fast progress in technology in the last two decades has reduced the cost of ICTs. In this situation, it is easier for more people to use and enjoy the advantages of ICTs [10]. In the last decades, technology has improved the life style and made easier the human activities through a new civilization. A new revolution named Information and Communication Technology (ICT) has occurred in the present world [1]. ICT is defined as a concept including information equipment as well as computers and software, auxiliary equipment that is connected to a computer (such as calculators, cash registers, photocopiers), communications equipment, and instruments [2]. In other words, ICT is a technology with the aim of connecting computers and other communication equipment to gather, produce, process, classify, manage, create, and distribute information. After the invention of machines in 1970s, industrial revolution has been started and machines have been used instead of handworks. 122 2.1. Empirical Model In order to examine the effect of ICT development on scientific degree of 14 Middle 123 Journal of Theoretical and Applied Information Technology 10th November 2013. Vol. 57 No.1 © 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org E-ISSN: 1817-3195 information-based societies. It is assumed that these countries have experienced the process of ICT development to turn into an information society. Therefore, the ITU [23] suggests the three-phase model including ICT readiness (showing the degree of networked infrastructure and ICT access), ICT intensity (showing the degree of ICT use by the society) and ICT impact (showing the result of effective and efficient ICT use). IDI is a composite indicator including ICT access, ICT skill and ICT use. Each sub-index is composed of a number of indicators explained as follow: Figure 1. Three Stages In The Evolution Towards An Information Society (Source: ITU [25]) ed Information Technology . Vol. 57 No.1 . All rights reserved. g E-ISSN: 1817-3195 Figure 1. Three Stages In The Evolution Towards An Information Society (Source: ITU [25]) Journal of Theoretical and Applied Information Technology 10th November 2013. Vol. 57 No.1 © 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 information-based societies. It is assumed that these countries have experienced the process of ICT development to turn into an information society. Therefore, the ITU [23] suggests the three-phase model including ICT readiness (showing the degree of networked infrastructure and ICT access), ICT intensity (showing the degree of ICT use by the society) and ICT impact (showing the result of effective and efficient ICT use). IDI is a composite indicator including ICT access, ICT skill and ICT use. Each sub-index is composed of a number of indicators explained as follow: information-based societies. It is assumed that these countries have experienced the process of ICT development to turn into an information society. Therefore, the ITU [23] suggests the three-phase model including ICT readiness (showing the degree of networked infrastructure and ICT access), ICT intensity (showing the degree of ICT use by the society) and ICT impact (showing the result of effective and efficient ICT use). IDI is a composite indicator including ICT access, ICT skill and ICT use. Each sub-index is composed of a number of indicators explained as follow: Figure 1. Three Stages In The Evolution Towards An Information Society (Source: ITU [25]) 1. 2.1. Empirical Model Access Sub-Index shows the availability of ICT infrastructure and individuals’ access to basic ICTs. This sub-index has five indicators including fixed telephone lines per 100 inhabitants, mobile cellular telephone subscriptions per 100 inhabitants, international Internet bandwidth (bit/s) per Internet user, proportion of households with a computer and proportion of households with Internet access at home. 2.2.2. h-index Therefore, this subgroup has three indicators including, adult literacy rate, secondary gross enrolment ratio and tertiary gross enrolment ratio. The required data for this subgroup are extracted from the UNESCO Institute for Statistics (UIS) [24]. The current study collects the required data on H-index for the panel of 14 Middle East countries over the period 1995 to 2009, from SCImago database [14]. The SCImago Journal and Country Rank website presents the journal and country scientific indicators based on the information included in the Scopus database. 2.2.2. h-index Different sources report the H-index. Some of these sources are subscription-based sources such as Scopus and Web of Science (WoS) and some of them are free access sources such as Harzing's Publish or Perish program on the basis of Google Scholar entries. It should be noted that these sources report different H-index for the same institution, country and scholar because of their different coverage. For example, WoS covers a high range of published journals while it does not cover high impact conferences. Although, the coverage of publications in Scopus is poor prior to 1996, it covers conferences properly. Documents in Google Scholar receive more citations in comparison with those in Scopus and WoS. It is argued that Google Scholar has the most coverage of journals and conferences particularly those published after 1990 [26]. 2. Use Sub-Index shows the real degree of ICTs strength of use based on the available infrastructure and access indicators. Considering the limitation on data for ICT Use at the global scale and results of the PCA, this subgroup has three indicators including Internet users per 100 inhabitants, fixed broadband Internet subscribers per 100 inhabitants and mobile broadband subscriptions per 100 inhabitants. 3. Skill Sub-Index captures the degree of ICT skill in different countries. Since the required data for many developing countries are not collected, an appropriate indicator can be the degree of literacy and education. Especially in developing countries, the poor level of education is a major obstacle to the effective use of internet and computers. With the inclusion of ICT in school courses, school attendance may offer an appropriate indicator for students’ exposure to the Internet or computers. Therefore, this subgroup has three indicators including, adult literacy rate, secondary gross enrolment ratio and tertiary gross enrolment ratio. The required data for this subgroup are extracted from the UNESCO Institute for Statistics (UIS) [24]. 3. Skill Sub-Index captures the degree of ICT skill in different countries. Since the required data for many developing countries are not collected, an appropriate indicator can be the degree of literacy and education. Especially in developing countries, the poor level of education is a major obstacle to the effective use of internet and computers. With the inclusion of ICT in school courses, school attendance may offer an appropriate indicator for students’ exposure to the Internet or computers. 3. FINDING AND DISCUSSION In this section, we first estimate Equation 1, which explores the impact of ICT development on H-index as a proxy of country’s scientific ranking. Then, applying the three ICT sub- indices, this study presents the estimation results for Equation 2, which is corresponding to the effect of ICT development sub-indices on H- index. 124 Journal of Theoretical and Applied Information Technology 10th November 2013. Vol. 57 No.1 © 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org E-ISSN: 1817-3195 Table 1. Regression Results For The Impact Of ICT Development On H-Index Dependent Variable: H-index Variables Coefficient Standard Error T IDI 31.69 4.67*** 6.79 Constant 24.16 13.35* 1.81 Observation = 210 F(1, 208) = 46.07*** R-squared = 0.1813 Adjusted R-squared = 0.1774 Note: ***, ** and * denote statistically significant at 1%, 5% and 10%, respectively. Standard errors are heteroskedasticity consistent. sample countries. The estimated coefficients show a positive and significant effect of ICT Use and ICT Skill on country’s scientific ranking. This finding asserts that increasing the application of ICTs and improving the ICT skills might significantly increase the scientific degree of a country. The results also indicate that if a country increases the ICT Use and ICT Skill indices by one unit, its H-index will raise by 133 and 407 units, respectively. As can be seen in Table 2, the coefficient explaining the effect of ICT Access on H-index is negative but not significant. Finally, based on the adjusted R- squared, ICT sub indices can explain 27.11% of variations in country’s H-index. Table 1 shows the effect of ICT development on each country’s H-index. The estimation results indicate a positive and significant effect of ICT development on country’s scientific ranking as proxied by H-index. This finding suggests that suitable policies aim at improving the level of ICT development can significantly increase the scientific rankings of a country. The results also assert that if a country raises its ICT development index by one unit, the H-index will increase by 31 units. As can be seen in Table 1, the adjusted R-squared is equal to 0.1774, which means that ICT development can explain 17.74% of variations in country’s H-index. Table 2 exhibits the impact of ICT development sub-indices on H-index of 14 Table 2. Standard errors are heteroskedasticity consistent. ***, ** and * denote statistically significant at 1%, 5% and 10%, respectively. 4. CONCLUSION AND IMPLICATIONS [7] Haridasan, K., Rangarajan, S. and Pirio, G. “Re-Inventing Health Care Training In The Developing World: The Case For Satellite Applications In Rural Environments”, Online Journal of Space Communication, 14(Winter), 2009. This paper focuses on examining the effect of ICT development on the scientific degree of 14 Middle East countries as proxied by H-index. The results of the regression analysis show that ICT development has a positive and significant effect on the H-index of these countries. Moreover, applying the ICT development sub-indices, this study finds that ICT Use and ICT Skill might have positive effects on H-index but the effect of ICT Access is not clear since its corresponding coefficient is not statistically significant. Therefore, we can conclude that ICT induces outcomes that leads to higher H-index values and raises the scientific level of sample countries. In this situation, policy makers should aim increasing the level of ICT development through increasing its indicators including fixed and mobile telephone lines, international Internet bandwidth, proportion of households with a computer and Internet access, Internet users, fixed and mobile broadband Internet subscribers, adult literacy rate, secondary and tertiary gross enrolment ratios. [8] Sharma, A., Gandhar, K., Sharma, S. and Seema, S. “Role of ICT in the Process of Teaching and Learning”, Journal of Education and Practice, Vol. 2, No. 5, 2011, pp. 1-6. [9] Sangrà, A. and González-Sanmamed, M. “The role of information and communication technologies in improving teaching and learning processes in primary and secondary schools”, Journal of Asynchronous Learning Networks, Vol. 15, No. 4, 2011, pp. 47-59. [10] International Telecommunication Union, “Measuring the information society: The ICT Development Index”, Place des Nations CH- 1211 Geneva Switzerland, 2011. [11] C.C. Aldea, A.D. Popescu, A. Draghici and G. Draghici, “ICT Tools Functionalities Analysis for the Decision Making Process of Their Implementation in Virtual Engineering Teams”, Procedia Technology, Vol. 5, 2012, pp. 649-658. [12] F. Scheuermann and F. Pedró, “Assessing the effects of ICT in education: Indicators, criteria and benchmarks for international comparisons”, Luxembourg: Publications Office of the European Union, 2009. 3. FINDING AND DISCUSSION Regression Results For The Impact Of ICT Development Sub-Indices On H-Index Dependent Variable: H-index Variables Coefficient Standard Error t ICT Access -25.46 49.80 -0.51 ICT Use 133.23 64.64** 2.06 ICT Skill 407.28 64.86*** 6.28 Constant -151.10 35.82*** -4.22 Observation = 210 F(3, 206) = 26.91*** R-squared = 0.2816 Adjusted R-squared = 0.2711 Note: ***, ** and * denote statistically significant at 1%, 5% and 10%, respectively. Standard errors are heteroskedasticity consistent. Table 2. Regression Results For The Impact Of ICT Development Sub-Indices On H-Index 125 © 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org REFERENCES: [1] R.J. Gordon, “Does the ‘New Economy’ measure up to the great inventions of the past?”, Journal of Economic Perspectives, Vol. 14, No. 4, 2000, pp. 49-74. [13] A.R. Bakker, “Health care and ICT, partnership is a must”, International Journal of Medical Informatics, Vol. 66, No. 1-3, 2002, pp. 51-57. [2] S.D. Oliner and D.E. Sichel, “Computers and output growth revisited: How big is the puzzle?”, Brookings Papers on Economic Activity, Vol. 2, 1994, pp. 273-334. [14] SCImago, SJR — SCImago Journal & Country Rank, 2007, Retrieved March 11, 2013, from http://www.scimagojr.com [3] S.S. Cohen, J.B. Delong and J. Zysman, “Tools for thought: What is new and important about the E-economy?”, BRIE Working Paper, No. 138, 2000. [15] J.E. Hirsch, “An index to quantify an individual’s scientific research output”, Proceedings of the National Academy of Science, Vol. 102, No. 46, 2005, pp. 16569- 16572. [4] D.W. Jorgenson and K.J. Stiroh, “Information technology and growth”, American Economic review Papers and Proceedings, Vol. 89, No. 2, 1999, pp. 109-115. [16] J.F. Molinari and A. Molinari, “A new methodology for ranking scientific institutions”, Scientometrics, Vol. 75, No. 1, 2008, pp. 163-174. [5] Gruber, H. and Koutroumpis, P. “Mobile telecommunications and the impact on economic development”, Economic Policy, Vol. 26, No. 67, 2011, pp. 387-426. [17] P. Ball, “Index aims for fair ranking of scientists”, Nature, Vol. 436, No. 900, 2005. doi:10.1038/436900a. [6] Farhadi, M., Ismail, R. and Fooladi, M. “Information and Communication Technology Use and Economic Growth”, PLoS ONE, Vol. 7, No. 11, 2012, e48903. doi:10.1371/journal.pone.0048903 [18] G. Saad, “Exploring the h-index at the author and journal levels using bibliometric data of productive consumer scholars and business-related journals respectively”, 126 Journal of Theoretical and Applied Information Technology 10th November 2013. 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High Prevalence of Urinary Schistosomiasis in a Desert Population: Results from an Exploratory Study Around the Ounianga Lakes in Chad
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High Prevalence of Urinary Schistosomiasis in a Desert Population: Results from an Exploratory Study Around the Ounianga Lakes in Chad High Prevalence of Urinary Schistosomiasis in a Desert Population: Results from an Exploratory Study Around the Ounianga Lakes in Chad Wendelin Moser  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Annour Aboum Batil  IRED Institut de Recherche en Elevage pour le Developpement Rebekka Ott  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Moussa Abderamane  Universite de N'Djamena Ruth Clements  Natural History Museum Rahel Wamp§er  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Sven Poppert  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Peter Steinmann  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Fiona Allan  Natural History Museum Helena Greter  (  helena.greter@swisstph.ch ) Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut https://orcid.org/0000-0003-1844-3961 Research Article Keywords: Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, Schistos bovis, Schistosoma haematobium, schistosomiasis Posted Date: October 28th 2021 Study Around the Ounianga Lakes in Chad Wendelin Moser  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Annour Aboum Batil  IRED Institut de Recherche en Elevage pour le Developpement Rebekka Ott  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Moussa Abderamane  Universite de N'Djamena Ruth Clements  Natural History Museum Rahel Wamp§er  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Sven Poppert  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Peter Steinmann  Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut Fiona Allan  Natural History Museum Helena Greter  (  helena.greter@swisstph.ch ) Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut https://orcid.org/0000-0003-1844-3961 Research Article Keywords: Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, Schisto bovis, Schistosoma haematobium, schistosomiasis ABSTRACT 23 Background: Researching a water-borne disease in the middle of the Sahara desert might 24 not seem the most relevant concern. However, nomadic Sahelian pastoralist’s health concerns 25 regarding their livestock and anecdotal reports about trematode infections of Fasciola spp and 26 Schistosoma spp in desert-raised animals justified an exploratory study focusing on the lakes 27 of Ounianga in Northern Chad. The aim was to test whether trematode parasites such as 28 Schistosoma spp occur in human populations living around the Sahara desert lakes of 29 Ounianga Kebir and Ounianga Serir in northern Chad. 30 Background: Researching a water-borne disease in the middle of the Sahara desert might 24 not seem the most relevant concern. However, nomadic Sahelian pastoralist’s health concerns 25 regarding their livestock and anecdotal reports about trematode infections of Fasciola spp and 26 Schistosoma spp in desert-raised animals justified an exploratory study focusing on the lakes 27 of Ounianga in Northern Chad. The aim was to test whether trematode parasites such as 28 Schistosoma spp occur in human populations living around the Sahara desert lakes of 29 Ounianga Kebir and Ounianga Serir in northern Chad. 30 Methods: The study comprised of three components. First, a cross sectional survey based on 31 a random sample drawn from the population to detect infections with S. haematobium and S. 32 mansoni; second, focus group discussions exploring disease priorities, access to health and 33 health seeking behaviour; and third, searching water contact sites for intermediate host snails. 34 Samples of trematode parasites and snails were confirmed on species level by molecular 35 genetics methods. 36 Methods: The study comprised of three components. First, a cross sectional survey based on 31 a random sample drawn from the population to detect infections with S. haematobium and S. 32 mansoni; second, focus group discussions exploring disease priorities, access to health and 33 health seeking behaviour; and third, searching water contact sites for intermediate host snails. 34 Samples of trematode parasites and snails were confirmed on species level by molecular 35 genetics methods. 36 Results: Among 258 participants, the overall S. haematobium prevalence using urine filtration 37 was 39.1% (95% CI 33.2% – 45.1%), with 51.5% of the infected suffering from heavy infection. 38 The intermediate host snail of S. haematobium (Bulinus truncatus) occurred at water sites near 39 both study villages, revealing the potential for local transmission. Although a positive S. Research Article Keywords: Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, Schistosoma bovis, Schistosoma haematobium, schistosomiasis Posted Date: October 28th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1016632/v1 DOI: https://doi.org/10.21203/rs.3.rs-1016632/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Infectious Diseases of Poverty on January 7th, 2022. See the published version at https://doi.org/10.1186/s40249-021-00930-4. High prevalence of urinary schistosomiasis in a desert population: results from an 1 exploratory study around the Ounianga lakes in Chad 2 3 Wendelin Moser1,2, Annour Adoum Batil3, Rebekka Ott1,2, Moussa Abderamane4, Ruth 4 Clements5, Rahel Wampfler1,2, Sven Poppert1,2, Peter Steinmann1,2, Fiona Allan5, Helena 5 Greter1,2* 6 7 1Swiss Tropical and Public Health Institute, Basel, Switzerland 8 2University of Basel, Basel, Switzerland 9 3Institut de Recherche en Elevage pour le Développement, Ndjamena, Chad 10 4University of Ndjamena, Geology Department, Ndjamena, Chad 11 5Department of Life Sciences, Natural History Museum, London, UK 12 13 *Corresponding author: 14 Helena Greter 15 Swiss Tropical and Public Health Institute 16 Socinstrasse 57, P.O. Box 17 CH-4002 Basel, Switzerland 18 Tel.: +41 61 284 87 30 19 E-mail: helena.greter@swisstph.ch 20 Orcid: 0000-0003-1844-3961 21 22 22 ABSTRACT 23 40 mansoni POC-CCA test result was obtained from 15.2% (10.6%-19.7%) of the samples no 41 intermediate host snails of S. mansoni were found, and the relevance of S. mansoni remains 42 uncertain. Qualitative findings underline the importance of morbidity caused by urinary 43 schistosomiasis, and the lack of access to diagnostics and treatment as a major health 44 concern. 45 Results: Among 258 participants, the overall S. haematobium prevalence using urine filtration 37 was 39.1% (95% CI 33.2% – 45.1%), with 51.5% of the infected suffering from heavy infection. 38 Results: Among 258 participants, the overall S. haematobium prevalence using urine filtration 37 was 39.1% (95% CI 33.2% – 45.1%), with 51.5% of the infected suffering from heavy infection. 38 Conclusion: This research revealed a high prevalence of urinary schistosomiasis in the 46 population living around the lakes of Ounianga in the Sahara, a UNESCO world heritage site 47 in Chad. Despite the high public health importance of the associated morbidity expressed by 48 the population there is no access to diagnostics and treatment. Further research is needed to 49 develop and test a context adapted intervention. 50 Conclusion: This research revealed a high prevalence of urinary schistosomiasis in the 46 population living around the lakes of Ounianga in the Sahara, a UNESCO world heritage site 47 in Chad. Despite the high public health importance of the associated morbidity expressed by 48 the population there is no access to diagnostics and treatment. Further research is needed to 49 develop and test a context adapted intervention. 50 2 2 51 Key words 52 Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, 53 Schistosoma bovis, Schistosoma haematobium, schistosomiasis 54 55 51 Key words 52 Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, 53 Schistosoma bovis, Schistosoma haematobium, schistosomiasis 54 55 51 Key words 52 55 3 3 BACKGROUND 56 Schistosome infections are listed among the 20 neglected tropical diseases (NTDs) targeted 57 by the World Health Organisation (WHO) for elimination by 2030 (1). In endemic regions, those 58 populations affected by schistosomiasis are often those living in poverty and / or in settings 59 with restricted access to clean water for their sanitation and hygiene needs (2). Worldwide, an 60 estimated 230 million people harbour an infection with Schistosoma spp (3). Occupational and 61 recreational activities in close contact with freshwater, e.g. fishing, doing laundry and bathing 62 present the main risk of infections. Highest prevalence is commonly observed among school- 63 aged children as they enjoy playing in stagnant water sites. Undetected and therewith 64 untreated urinary or intestinal schistosomiasis leads to chronic infections and serious 65 morbidities including a wide range of different pathologies as e.g. anaemia, stunted growth, 66 impaired cognition and organ damages, that negatively affect economic activities and therewith 67 maintain poverty (4, 5). The safe and effective drug, Praziquantel, is currently used for mass 68 drug administration programs in endemic settings as well as for treatment of individual acute 69 infections . However, its effectiveness is threatened by increasing resistance of the parasite 70 that is observed (6). 71 The majority of schistosomiasis cases occur in sub-Saharan Africa, and the disease is reported 72 from countries throughout the Sahel, including Mauretania, Mali, Niger, Chad and Sudan (7- 73 11). Infections are predominantly due to Schistosoma haematobium which has the ability to 74 maintain its life cycle in a semi-arid environment, including in the ecoregion of the Sahel (12). 75 Yet, there are old reports on schistosomiasis occurrence also more to the north, from within 76 the Sahara desert (7, 13). The occurrence of Schistosoma spp, a genus of water-transmitted 77 parasites belonging to the clades of digenean trematodes, and its occurrence in the hot and 78 hyper-arid desert may seem surprising but occurrence in at least two desert-specific 79 ecosystems have been described so far. These are (a) oases where schistosomiasis 80 transmission is linked to man-made irrigation systems (14, 15), and (b) areas with reclaimed 81 land for agriculture, made cultivable by artificial irrigation from deep wells (16). BACKGROUND 56 82 4 Anecdotal reports from nomadic Sahelian pastoralists on Fasciola spp, another digenean 83 trematode species, in livestock raised in the Chadian Sahara and recent reports about modern 84 and early Holocene finding of intermediate host snails pointed towards the occurrence and 85 potential ongoing transmission of schistosomiasis at the desert lakes of Ounianga, Chad (17). 86 Triggered by these information an exploratory study was conceptualized with the aim to 87 investigate whether trematode parasites such as Schistosoma spp occur in two settlements at 88 the lakes of Ounianga, Ennedi Ouest province, in Northern Chad. The study was covering 89 three aspects, namely epidemiology, malacology and the population’s health priorities, their 90 access to health care and treatment. 91 92 METHODS 93 Study site and study population 94 The study was carried out in January 2019 around the lakes and the two settlements of 95 Ounianga Kebir and Ounianga Serir, Ennedi Ouest province in Northern Chad (Figure 1). 96 97 <<Figure 1 near here>><<Fig 1. A map showing the lakes and the settlements of 98 Ounianga Kebir and Ounianga Serir in Northern Chad.>> 99 100 The official population estimates according to the latest national population census in Chad for 101 Ounianga Kebir counts around 9000 people and for Ounianga Serir about 1000 people 102 (RGPH2, 2009). In both communities, the primary schools were operational, yet not the 103 secondary schools. The only functional health centre of the Ounianga district is located in 104 Ounianga Kebir and its catchment population is estimated to include 30,000 people. Ounianga 105 Serir has no functional health centre; the population has set up a health post to provide basic 106 health services to the community members. 107 108 Epidemiological survey 109 Anecdotal reports from nomadic Sahelian pastoralists on Fasciola spp, another digenean 83 trematode species, in livestock raised in the Chadian Sahara and recent reports about modern 84 and early Holocene finding of intermediate host snails pointed towards the occurrence and 85 potential ongoing transmission of schistosomiasis at the desert lakes of Ounianga, Chad (17). 86 Triggered by these information an exploratory study was conceptualized with the aim to 87 investigate whether trematode parasites such as Schistosoma spp occur in two settlements at 88 the lakes of Ounianga, Ennedi Ouest province, in Northern Chad. BACKGROUND 56 The study was covering 89 three aspects, namely epidemiology, malacology and the population’s health priorities, their 90 access to health care and treatment. 91 Anecdotal reports from nomadic Sahelian pastoralists on Fasciola spp, another digenean 83 trematode species, in livestock raised in the Chadian Sahara and recent reports about modern 84 and early Holocene finding of intermediate host snails pointed towards the occurrence and 85 potential ongoing transmission of schistosomiasis at the desert lakes of Ounianga, Chad (17). 86 Triggered by these information an exploratory study was conceptualized with the aim to 87 investigate whether trematode parasites such as Schistosoma spp occur in two settlements at 88 the lakes of Ounianga, Ennedi Ouest province, in Northern Chad. The study was covering 89 three aspects, namely epidemiology, malacology and the population’s health priorities, their 90 access to health care and treatment. 91 <<Figure 1 near here>><<Fig 1. A map showing the lakes and the settlements of 98 Ounianga Kebir and Ounianga Serir in Northern Chad.>> 99 Parameters 111 used were “population survey” with two-sided confidence intervals of 95%, an expected 112 frequency of 50% and a population size of 10000, resulting in a sample size of 370. 113 Proportional to the total population estimates, the targeted sample size repartition was 330 114 people in Ounianga Kebir and 40 people in Ounianga Serir. At household level and at the 115 primary schools, individuals were randomly selected by applying the spatial sampling method 116 from the Expanded Programme of Immunization (EPI) of the World Health Organization as 117 previously published (18). After obtaining oral consent from each selected individual, or in case 118 of children from their caretakers, they were asked to produce a urine sample. A mobile field 119 laboratory was set up at the health centre, and health post, respectively. The urine samples 120 were analysed for haematuria by reagent strip testing (Hemastix; Siemens Healthcare 121 Diagnostics GmbH; Eschborn, Germany) and classified as negative, light and severe 122 haematuria as outlined by the testing handbook. Subsequently, 10 ml samples were subjected 123 to urine filtration, followed by microscopic screening of the filter content for the presence of S. 124 haematobium eggs. A point-of-care circulating cathodic antigen (POC-CCA) urine cassette test 125 (Rapid Medical Diagnostics; Pretoria, South Africa) was performed to screen for S. mansoni 126 infections. 127 <<Figure 1 near here>><<Fig 1. A map showing the lakes and the settlements of 98 Ounianga Kebir and Ounianga Serir in Northern Chad.>> 99 The official population estimates according to the latest national population census in Chad for 101 Ounianga Kebir counts around 9000 people and for Ounianga Serir about 1000 people 102 (RGPH2, 2009). In both communities, the primary schools were operational, yet not the 103 secondary schools. The only functional health centre of the Ounianga district is located in 104 Ounianga Kebir and its catchment population is estimated to include 30,000 people. Ounianga 105 Serir has no functional health centre; the population has set up a health post to provide basic 106 health services to the community members. 107 5 5 The resident population of Ounianga Kebir and Ounianga Serir, older than 5 years of age, were 110 eligible for participation. Sample size was calculated using Epi Info 7.1.3.3 (CDC). Parameters 111 used were “population survey” with two-sided confidence intervals of 95%, an expected 112 frequency of 50% and a population size of 10000, resulting in a sample size of 370. 113 Proportional to the total population estimates, the targeted sample size repartition was 330 114 people in Ounianga Kebir and 40 people in Ounianga Serir. At household level and at the 115 primary schools, individuals were randomly selected by applying the spatial sampling method 116 from the Expanded Programme of Immunization (EPI) of the World Health Organization as 117 previously published (18). After obtaining oral consent from each selected individual, or in case 118 of children from their caretakers, they were asked to produce a urine sample. A mobile field 119 laboratory was set up at the health centre, and health post, respectively. The urine samples 120 were analysed for haematuria by reagent strip testing (Hemastix; Siemens Healthcare 121 Diagnostics GmbH; Eschborn, Germany) and classified as negative, light and severe 122 haematuria as outlined by the testing handbook. Subsequently, 10 ml samples were subjected 123 to urine filtration, followed by microscopic screening of the filter content for the presence of S. 124 haematobium eggs. A point-of-care circulating cathodic antigen (POC-CCA) urine cassette test 125 (Rapid Medical Diagnostics; Pretoria, South Africa) was performed to screen for S. mansoni 126 infections. 127 The resident population of Ounianga Kebir and Ounianga Serir, older than 5 years of age, were 110 eligible for participation. Sample size was calculated using Epi Info 7.1.3.3 (CDC). Qualitative survey 129 In both communities, one focus group discussion (FGDs) with men and one with women were 130 organized. Additionally, one FGD was organized with the staff of the health centre in Ounianga 131 Kebir. In Ounianga Serir, an in-depth interview (IDI) was carried out with the person 132 responsible for the health post. The topics covered by the interview guides were disease 133 priorities and priority health issues, perceptions and health seeking behaviour. FGDs and IDI 134 were assisted by an interpreter who translated the conversation from Arabic to French, allowing 135 the study team to take notes. Digital recordings of the FGDs and IDI were transcribed and 136 translated into French, integrating the notes taken during the FGDs or IDI. 137 6 6 138 Malacological survey 139 Individual community members and school-aged children were asked to guide the team to 140 human-water contact sites. At each site, GPS coordinates and the water parameters 141 temperature (oC), pH, conductivity (µs/cm) and dissolved oxygen (mg/l) were recorded, using 142 a portable multimeter (Hach®, HQ40D, Loveland, USA)). For turbidity, a trubidimeter was used 143 (Formazin Nephelometric Units [FNU]; Hach®, 2100P Iso). The snail sampling was performed 144 adhering to standard protocols. In short, for 15 minutes, all aquatic snails were collected by 145 one person using a scoop or forceps to detach them from aquatic and subaquatic plants (19). 146 Subsequently, the snails were placed on wet cotton in petri dishes, and transferred to the field 147 laboratory. Snails were identified to the genus or, if possible to species level on site. At midday, 148 each collected snail identified as intermediate host species was placed in water for three hours 149 to induce cercarial shedding. The snail size (in mm) and weight (in mg) was measured using 150 a calibre and balance, respectively. Thereafter, all snail specimens were conserved in 70% 151 ethanol, and shipped to the National History Museum, London (NHM) for molecular analysis. 152 153 Molecular snail species and infections status confirmation 154 The snail samples selected for the molecular analyses represented individuals from each 155 collection site. All specimens were stored in 70% ethanol in the field. On arrival at the NHM, 156 the snail species identification was confirmed based on morphological characters and samples 157 re-spirited (absolute ethanol) for incorporation into the Schistosomiasis Collection at the 158 Natural History Museum (SCAN) (20). Photographic images were taken of the snail shells prior 159 to DNA extraction. Specimens were placed in TE buffer (10mM Tris, 0.1mM EDTA) pH 7.4 for 160 one hour in order to remove any remaining alcohol from within the tissue, which might interfere 161 with subsequent extraction steps. Total genomic DNA was isolated from head/foot tissue using 162 the DNeasy Blood and Tissue kit (Qiagen, UK) according to manufacturer’s instructions. DNA 163 was eluted into 200μl sterile water. 164 165 7 7 Checking of sequence data 171 The electropherograms produced were checked and Cox1 sequences edited using Geneious, 172 version 11.0.5 (http://www.geneious.com (24)). Sequences were compared to database 173 entries by performing BLAST searches via the National Center for Biotechnology Information 174 against GenBank and EMBL sequence databases; and aligned with reference material [3,4] 175 using Geneious version 11.0.5. 176 Amplification of Cox1 fragments of snail DNA 166 A polymerase chain reaction (PCR) amplification of a partial cytochrome oxidase 1 (Cox1) 167 sequence was performed using primers LCO1490 (5′GGTCAACAAATCATAAAG ATATTGG3′ 168 forward) and HCO2198 (5′TAAACTTCAGGGTGACCAAAAAATCA3′ reverse) (21). PCR 169 investigations and sequencing conditions were chosen as previously outlined (22, 23). 170 sequence was performed using primers LCO1490 (5′GGTCAACAAATCATAAAG ATATTGG3′ 168 forward) and HCO2198 (5′TAAACTTCAGGGTGACCAAAAAATCA3′ reverse) (21). PCR 169 investigations and sequencing conditions were chosen as previously outlined (22, 23). 170 Checking of sequence data 171 The electropherograms produced were checked and Cox1 sequences edited using Geneious, 172 version 11.0.5 (http://www.geneious.com (24)). Sequences were compared to database 173 entries by performing BLAST searches via the National Center for Biotechnology Information 174 against GenBank and EMBL sequence databases; and aligned with reference material [3,4] 175 using Geneious version 11.0.5. 176 177 Sequencing of Schistosoma spp. eggs in urine 178 Positive urine samples from Ouinanga Kebir were combined into 7 different pools of 8-12ml 179 respectively one pooled sample of 12ml for the villages of Ouinanga Serir. Samples were 180 shipped to the diagnostic center of the Swiss Tropical and Public Health Institute (Swiss TPH) 181 in Basel, Switzerland for further processing. There, each pool was centrifuged at 3000g for 10 182 minutes. Exactly 500µl of the pellet was re-suspended and transferred to a 2ml tube containing 183 garnet beads. After addition of 1 ml PBS, the sample was centrifuged 1min at 13000g and the 184 supernatant was discarded. The pellet with the garnet beads was frozen 30min at -80°C and 185 further processed as described by Barda and colleagues (25, 26). Samples were first tested 186 by simplex generic Schistosoma spp. 28S real-time PCR amplifying S. mansoni, S. 187 haematobium, S. intercalatum, S. bovis (27) and additionally S. japonicum because of 188 modifications added to the second reverse primer of the assay (table 1). The reaction mix 189 contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel, 190 Switzerland), 800nmoles of forward primer, 400nmoles of each reverse primer and 200nmoles 191 of probe. The samples were subsequently tested by a duplex real-time PCR for the presence 192 of a specific S. mansoni TRE region and of S. haematobium dra1 sequence (27, 28). Each 193 Sequencing of Schistosoma spp. eggs in urine 178 Analytical limit of detection (LOD) 204 was tested by a plasmid dilution row ranging from 107 to 10-1 plasmids/µl containing an insert 205 with the sequence of the Schistosoma real-time PCR product, and was found to be at 10 206 plasmids/µl for all assays. On each real-time PCR plate and for each target we included 207 negative and positive low-copy plasmid controls. 208 reaction mix contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel, 194 Switzerland), 800nmoles of each primer, 200nmoles each probe (table1). The thermoprofile 195 of all assays on the QuantStudio5 (ThermoFisher) consisted of 2min at 50°C, 10min at 95°C 196 followed by 45 cycles of 15s at 95°C and 1min at 58°C. The specificity of all assays was 197 previously tested on a variety of DNA from stool and blood samples including: Ascaris 198 lumbricoides, Blastocystis hominis, Cryptosporidium spp., Dientamoeba fragilis, 199 Encephalitozoon spp., Endolimax nana, Entamoeba coli, E. dispar, E. histolytica, E. 200 moshkovskii, E. polecki, Enterocytozoon bieneusi, Giardia lamblia, Hymenolepis nana, 201 Iodamoeba bütschlii, Sarcocystis spp., Taenia spp., Strongyloides stercoralis, Trichuris 202 trichiura, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, Trypanosoma cruzi, T. 203 brucei, Leishmania spp. and was found to be 100% specific. Analytical limit of detection (LOD) 204 was tested by a plasmid dilution row ranging from 107 to 10-1 plasmids/µl containing an insert 205 with the sequence of the Schistosoma real-time PCR product, and was found to be at 10 206 plasmids/µl for all assays. On each real-time PCR plate and for each target we included 207 negative and positive low-copy plasmid controls. 208 Subsequently, all samples were tested by classic PCR of the COX gene of S. haematobium 209 and S. bovis as modified from Boon and co-workers (Table 1) (29). The reaction mix contained 210 1x HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany), 800nmoles of each Primer, 5µl 211 DNA in a total reaction volume of 50µl. The thermoprofile consisted of 5min at 94°C followed 212 by 40 cycles of 40s at 94°C, 40s at 58°C and 1min at 72°C and a final step of 10min at 72°C. 213 After visualization on a 2% Agarose-Gel, the positive sample of the S. bovis-COX PCR was 214 sent for Sanger sequencing with the primers of amplification at Microsynth AG (Baldach, 215 Switzerland). The Sequence was then compared to database entries by performing BLAST 216 searches via the National Center for Biotechnology Information. Sequencing of Schistosoma spp. eggs in urine 178 Positive urine samples from Ouinanga Kebir were combined into 7 different pools of 8-12ml 179 respectively one pooled sample of 12ml for the villages of Ouinanga Serir. Samples were 180 shipped to the diagnostic center of the Swiss Tropical and Public Health Institute (Swiss TPH) 181 in Basel, Switzerland for further processing. There, each pool was centrifuged at 3000g for 10 182 minutes. Exactly 500µl of the pellet was re-suspended and transferred to a 2ml tube containing 183 garnet beads. After addition of 1 ml PBS, the sample was centrifuged 1min at 13000g and the 184 supernatant was discarded. The pellet with the garnet beads was frozen 30min at -80°C and 185 further processed as described by Barda and colleagues (25, 26). Samples were first tested 186 by simplex generic Schistosoma spp. 28S real-time PCR amplifying S. mansoni, S. 187 haematobium, S. intercalatum, S. bovis (27) and additionally S. japonicum because of 188 modifications added to the second reverse primer of the assay (table 1). The reaction mix 189 contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel, 190 Switzerland), 800nmoles of forward primer, 400nmoles of each reverse primer and 200nmoles 191 of probe. The samples were subsequently tested by a duplex real-time PCR for the presence 192 of a specific S. mansoni TRE region and of S. haematobium dra1 sequence (27, 28). Each 193 8 8 reaction mix contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel, 194 Switzerland), 800nmoles of each primer, 200nmoles each probe (table1). The thermoprofile 195 of all assays on the QuantStudio5 (ThermoFisher) consisted of 2min at 50°C, 10min at 95°C 196 followed by 45 cycles of 15s at 95°C and 1min at 58°C. The specificity of all assays was 197 previously tested on a variety of DNA from stool and blood samples including: Ascaris 198 lumbricoides, Blastocystis hominis, Cryptosporidium spp., Dientamoeba fragilis, 199 Encephalitozoon spp., Endolimax nana, Entamoeba coli, E. dispar, E. histolytica, E. 200 moshkovskii, E. polecki, Enterocytozoon bieneusi, Giardia lamblia, Hymenolepis nana, 201 Iodamoeba bütschlii, Sarcocystis spp., Taenia spp., Strongyloides stercoralis, Trichuris 202 trichiura, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, Trypanosoma cruzi, T. 203 brucei, Leishmania spp. and was found to be 100% specific. Sequencing of Schistosoma spp. eggs in urine 178 haematobium egg positive in Ounianga 230 Kebir and 54.9% (95% CI 40.8% – 69.0%) in Ounianga Serir, resulting in an overall prevalence 231 of 39.2% (95% CI 33.2% – 45.1%) (Table 1). 232 233 Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection 234 intensities in the study population, Ounianga Serir and Ounianga Kebir, Chad, 2019. 235 Ounianga Kebir Ounianga Serir Total Male Female Male Female Total number of participants 65 142 21 30 2 Participants age <18 years 47 (72%) 65 (46%) 14 (67%) 11 (37%) 137 (53 S. haematobium infection (egg positive) Total no. positive 27 (41.5%) 46 (32.4%) 11 (52.4%) 17 (56.7%) 1 (39.2 Age <18 years* 24 (51.1%) 24 (36.9%) 9 (64.3%) 8 (72.7%) 65 (47.5 Heavy S. haematobium infection Total no. positive 14 (51.9%) 21 (45.7%) 8 (72.7%) 9 (52.9%) 52 (51.5 Age <18 years* 14 (58.3%) 14 (58.3%) 8 (88.9%) 7 (87.5%) 43 (66.2 POC-CCA test results Total no. positive 14 (21.9%) 17 (13.0%) 1 (4.8%) 5 (17.9%) 37 (15.2 Age <18 years* 14 (30.4%) 10 (16.7%) 1 (7.1%) 3 (27.3%) 28 (21.4 *(%) from all participants <18 year 236 237 Descriptive statistics of epidemiological and malacological data was performed using STATA 222 version 16.0 (STATA Corp Inc., TX, USA) and ArcGIS (Version 10.7.1.; ESRI Inc. ArcMap™ 223 10.7, Redlands, CA, USA). Qualitative data analysis included full review of all transcripts, 224 followed by a descriptive and explorative thematic analysis. 225 Sequencing of Schistosoma spp. eggs in urine 178 The sequence is accessible 217 in GenBank under the number: MW937895. A table listing primers and probes is accessible in 218 the supplementary materials. 219 9 9 Descriptive statistics of epidemiological and malacological data was performed using STATA 222 version 16.0 (STATA Corp Inc., TX, USA) and ArcGIS (Version 10.7.1.; ESRI Inc. ArcMap™ 223 10.7, Redlands, CA, USA). Qualitative data analysis included full review of all transcripts, 224 followed by a descriptive and explorative thematic analysis. 225 226 RESULTS 227 Epidemiological survey 228 In both study sites, urinary schistosomiasis was highly prevalent. Indeed, 35.3% (95% CI 229 28.7% – 41.8%) of the tested participants were S. haematobium egg positive in Ounianga 230 Kebir and 54.9% (95% CI 40.8% – 69.0%) in Ounianga Serir, resulting in an overall prevalence 231 of 39.2% (95% CI 33.2% – 45.1%) (Table 1). 232 233 Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection 234 intensities in the study population, Ounianga Serir and Ounianga Kebir, Chad, 2019. 235 Ounianga Kebir Ounianga Serir Total Male Female Male Female Total number of participants 65 142 21 30 25 Participants age <18 years 47 (72%) 65 (46%) 14 (67%) 11 (37%) 137 (53% S. haematobium infection (egg positive) Total no. positive 27 (41.5%) 46 (32.4%) 11 (52.4%) 17 (56.7%) 10 (39.2% Age <18 years* 24 (51.1%) 24 (36.9%) 9 (64.3%) 8 (72.7%) 65 (47.5% Heavy S. haematobium infection Total no. positive 14 (51.9%) 21 (45.7%) 8 (72.7%) 9 (52.9%) 52 (51.5% Age <18 years* 14 (58.3%) 14 (58.3%) 8 (88.9%) 7 (87.5%) 43 (66.2% POC-CCA test results Total no. positive 14 (21.9%) 17 (13.0%) 1 (4.8%) 5 (17.9%) 37 (15.2% Age <18 years* 14 (30.4%) 10 (16.7%) 1 (7.1%) 3 (27.3%) 28 (21.4% *(%) from all participants <18 year 236 237 Descriptive statistics of epidemiological and malacological data was performed using STATA 222 version 16.0 (STATA Corp Inc., TX, USA) and ArcGIS (Version 10.7.1.; ESRI Inc. ArcMap™ 223 10.7, Redlands, CA, USA). Qualitative data analysis included full review of all transcripts, 224 followed by a descriptive and explorative thematic analysis. 225 226 RESULTS 227 Epidemiological survey 228 In both study sites, urinary schistosomiasis was highly prevalent. Indeed, 35.3% (95% CI 229 28.7% – 41.8%) of the tested participants were S. Epidemiological survey 228 For each water 243 site sampled, the abundance of the intermediate host snail Bulinus truncatus is indicated.>> 244 245 Mapping of the schistosomiasis prevalence by place of living (neighbourhood) shows a slightly 246 higher prevalence for those neighbourhoods closer to a water sites where the aquatic 247 intermediate host snail Bulinus truncates was present (Fig 2, Ounianga Kebir: Yiggybeshi, 248 Ounianga Serir: Roy). Regarding the POC-CCA testing for S. mansoni, 15.2% of the urine 249 samples showed a positive test results. 250 More than half of all participants harboured a heavy S. haematobium infection (51.5%; heavy 251 infection: >50 eggs/10 ml urine) and the burden was higher in children, whereof two third were 252 heavily infected (Table 1) (30). Overall, 10.2% of all egg-negative, 69.4% of all light and 92.3% 253 of all heavily infected participants had severe haematuria (Fig 3), with no big differences 254 between age and gender. 255 256 <<Figure 3 near here>> 257 <<Fig 3. Schistosomiasis infection intensity and haematuria stratified by sex, age 258 group and place of living.>> 259 260 Qualitative survey 261 During FGDs in both study sites, abdominal issues and blood in urine were the most frequently 262 mentioned health problems among adults and also in children. Health staff mentioned that the 263 majority of patients seeking care at the centre for any cause additionally suffers from abdominal 264 issues. Among children, diarrhoeal diseases, respiratory infections and scorpion stings were 265 <<Figure 2 near here>> 241 <<Fig 2. Map showing the prevalence and snail abundance for Ounianga Kebir and 242 Serir. The prevalence among participants is displayed by neighbourhood. For each water 243 site sampled, the abundance of the intermediate host snail Bulinus truncatus is indicated.>> 244 245 Mapping of the schistosomiasis prevalence by place of living (neighbourhood) shows a slightly 246 higher prevalence for those neighbourhoods closer to a water sites where the aquatic 247 intermediate host snail Bulinus truncates was present (Fig 2, Ounianga Kebir: Yiggybeshi, 248 Ounianga Serir: Roy). Regarding the POC-CCA testing for S. mansoni, 15.2% of the urine 249 samples showed a positive test results. 250 Mapping of the schistosomiasis prevalence by place of living (neighbourhood) shows a slightly 246 higher prevalence for those neighbourhoods closer to a water sites where the aquatic 247 intermediate host snail Bulinus truncates was present (Fig 2, Ounianga Kebir: Yiggybeshi, 248 Ounianga Serir: Roy). Regarding the POC-CCA testing for S. Epidemiological survey 228 In both study sites, urinary schistosomiasis was highly prevalent. Indeed, 35.3% (95% CI 229 28.7% – 41.8%) of the tested participants were S. haematobium egg positive in Ounianga 230 Kebir and 54.9% (95% CI 40.8% – 69.0%) in Ounianga Serir, resulting in an overall prevalence 231 of 39.2% (95% CI 33.2% – 45.1%) (Table 1). 232 Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection 34 Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection 234 intensities in the study population, Ounianga Serir and Ounianga Kebir, Chad, 2019. 235 Ounianga Kebir Ounianga Serir Total Male Female Male Female Total number of participants 65 142 21 30 258 Participants age <18 years 47 (72%) 65 (46%) 14 (67%) 11 (37%) 137 (53%) S. haematobium infection (egg positive) Total no. positive 27 (41.5%) 46 (32.4%) 11 (52.4%) 17 (56.7%) 101 (39.2%) Age <18 years* 24 (51.1%) 24 (36.9%) 9 (64.3%) 8 (72.7%) 65 (47.5%) Heavy S. haematobium infection Total no. positive 14 (51.9%) 21 (45.7%) 8 (72.7%) 9 (52.9%) 52 (51.5%) Age <18 years* 14 (58.3%) 14 (58.3%) 8 (88.9%) 7 (87.5%) 43 (66.2%) POC-CCA test results Total no. positive 14 (21.9%) 17 (13.0%) 1 (4.8%) 5 (17.9%) 37 (15.2%) Age <18 years* 14 (30.4%) 10 (16.7%) 1 (7.1%) 3 (27.3%) 28 (21.4%) *(%) from all participants <18 year 236 ab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria a 234 The S. haematobium prevalence was highest among children and adolescents below 18 years 238 in both villages (Table 1). In Ounianga Kebir, more boys than girls were infected (51.1% versus 239 36.9%), whereas in Ounianga Seker girls had a higher prevalence (72.7%). 240 The S. haematobium prevalence was highest among children and adolescents below 18 years 238 in both villages (Table 1). In Ounianga Kebir, more boys than girls were infected (51.1% versus 239 36.9%), whereas in Ounianga Seker girls had a higher prevalence (72.7%). 240 10 <<Figure 2 near here>> 241 <<Fig 2. Map showing the prevalence and snail abundance for Ounianga Kebir and 242 Serir. The prevalence among participants is displayed by neighbourhood. Epidemiological survey 228 mansoni, 15.2% of the urine 249 samples showed a positive test results. 250 More than half of all participants harboured a heavy S. haematobium infection (51.5%; heavy 251 infection: >50 eggs/10 ml urine) and the burden was higher in children, whereof two third were 252 heavily infected (Table 1) (30). Overall, 10.2% of all egg-negative, 69.4% of all light and 92.3% 253 of all heavily infected participants had severe haematuria (Fig 3), with no big differences 254 between age and gender. 255 11 A major constraint for people in both sites is the difficult access to health facilities. Accessible 269 facilities are usually underequipped; rarely have drugs available and the personnel had only 270 basic training. To obtain appropriate care and treatment, people needed to travel long 271 distances within Chad (to Faya, Abeche and N’Djamena) or abroad to Libya or Sudan. 272 Another common theme was the lack of safe drinking water as pumps are rare and open wells 273 are commonly used as water sources. Perceived water quality is low due to salty taste and 274 visible contamination. 275 The population was well aware of parasitic diseases, yet had limited knowledge on risk factors 276 and transmission. Blood in urine was linked to parasitic infections, low quality of drinking water, 277 water contact at the nearby lakes, or kidney issues. Kadi and Kouli are two local names for 278 parasite infections linked to abdominal pain. Kadi describes an intestinal worm infection 279 causing symptoms like intestinal spasms and flatulence, increased appetite with the tendency 280 of weight loss As a traditional treatment, infected people are given natron or an extract of the 281 roots of a plant called Boa to initiate diarrhoea, causing a worm with a red ‘mouth’ to leave the 282 body via the excrements. The symptoms described for Kouli correlate with symptoms of the 283 parasite Enterobius vermicularis such as persistent itching in the perianal area and sleep 284 disturbances. The traditional treatment administered to Kouli patients are eating butter or 285 drinking an extract of a medical plant called Chi. 286 Ouco is the local term to describe the health condition related to blood in urine combined with 287 pain while urinating and reduced male erectile function. In traditional medicine, the urine of the 288 animal called Nii (Fennec Fox, Vulpes zerda) is believed to have a curative effect. Epidemiological survey 228 289 The reported level of satisfaction with access to medical treatment for the above 290 mentioned health issues is mixed. Important challenges mentioned included stock outs of 291 medicines, lack of diagnostic means and non-effectiveness of the medical treatment received. 292 Especially the female FGD participants expressed a need for health education and 293 sensitization among the population. 294 Malacological survey 296 12 Among a total pf 17 different collection sites, 8 harboured fresh water snails (Tab 2, Fig 2). 297 Highest snail numbers were collected from the two intermediate host snail species Limnea 298 natalensis (n=42) and Bulinus truncatus (n=38), and two species of no medical importance, 299 Gyralus sp. and Polypylis sp. (n=42). Particularly high numbers of any snail species were 300 collected at two sites, namely Yoa 2 (n=35) in Ounianga Kebir and Agouta (n=34) in Ounianga 301 Kebir. Among all snails, only one B. truncatus was shedding cercariae (from Yoa 2). Upon 302 testing, they were recognized to not represent S. haematobium cercariae, and consequently 303 were not further studied. The average shell height of all L. natalensis specimen was 10.6mm 304 (95% confidence interval, 9.08mm - 12.11mm), 6.66 mm (95% CI, 5.97mm - 7.34mm) for B. 305 truncates, whereas all Gyralus sp. were juveniles with an average shell height of 1 mm or 306 below. 307 Among a total pf 17 different collection sites, 8 harboured fresh water snails (Tab 2, Fig 2). 297 Highest snail numbers were collected from the two intermediate host snail species Limnea 298 natalensis (n=42) and Bulinus truncatus (n=38), and two species of no medical importance, 299 Gyralus sp. and Polypylis sp. (n=42). Particularly high numbers of any snail species were 300 collected at two sites, namely Yoa 2 (n=35) in Ounianga Kebir and Agouta (n=34) in Ounianga 301 Kebir. Among all snails, only one B. truncatus was shedding cercariae (from Yoa 2). Upon 302 testing, they were recognized to not represent S. haematobium cercariae, and consequently 303 were not further studied. The average shell height of all L. natalensis specimen was 10.6mm 304 (95% confidence interval, 9.08mm - 12.11mm), 6.66 mm (95% CI, 5.97mm - 7.34mm) for B. 305 truncates, whereas all Gyralus sp. were juveniles with an average shell height of 1 mm or 306 below. 307 Across all sites where snails were found, the average water temperatures was 18.9oC 308 (standard deviation ±3.3), the average oxygen content was 4.8 mg/l (± 2.1) and a turbidity of 309 3.0 FNU (± 1.4). The sites without snails were characterized by a wide range of measured 310 water parameters, i.e. temperature of 22.1oC (range: 14.1-28.2), oxygen 5.9 mg/l (range: 1.1- 311 15.0) and turbidity 267.0 FNU (range: 1.0 - >1000.0). Malacological survey 296 Snails obviously preferred the pH range 312 between 7.0 and 8.8 compared the sites without snails with a pH varying between 7.0 and 313 10.5. Inconclusive results were found for the conductivity comparing sites with and without 314 snails with a range of 6.0 to 1941.0 µs/cm and 2.8 to >2500.0 µs/cm, respectively. 315 Table 3. Snail abundance and water parameters for each sampling site 17 Sampling sites with snails present Site* Snail species No. of snails found Temperature [oC] Conductivity [µs/cm] pH Oxygen [mg/l] Turbidity [FNU] Ounianga Kebir Yoa (Girki) 1* Gyralus sp. 1 17.5 1054.0 6.8 2.7 1.3 2* B. truncatus 28 14.5 1046.0 6.9 4.1 2.5 L. natalensis 6 Yoa (source 2) 3* L. natalensis 19 22.7 1941.0 7.1 3.2 1.9 4* L. natalensis 7 22.8 2.56 7.0 3.0 3.7 Ounianga Serir Agouta Gyralus sp. 27 14.5 8.0 8.3 4.9 3.5 L. natalensis 7 V. nilotica √ Table 3. Snail abundance and water parameters for each sampling site 13 Djara B. truncatus 4 19.7 6.0 8.5 6.6 2.4 G. ounaiangaensis √ V. nilotica √ Boku B. truncatus 6 20.8 1524.0 8.8 8.8 5.9 L. natalensis 3 G. ounaiangaensis √ Bedrim G. ounaiangaensis √ 18.7 7.3 8.4 5.2 3.0 Sampling sites without snails Ounianga Kebir Yoa 5 (hot source) 28.4 2.8 7.0 2.9 4.9 Yoa 6 (hot source) 27.7 2107.0 7.4 3.2 16.3 Yoa 7 (lake) 14.4 290.0 10.3 1.1 158.0 Uma red 27.5 >2000.0 10.1 14.2 85.8 Uma blue 18.6 >2000.0 10.5 15.0 118.0 Uma (hot spring) 28.2 4.0 9.1 3.6 14.9 Forodone 19.1 31.1 9.8 2.0 >1000.0 Ounianga Serir Telli 21.3 13.7 10.7 2.7 4.5 Edem 14.1 3.5 8.7 8.3 1.0 *All sampling sites are shown in Figure 2 318 319 Sequencing of Schistosoma spp. eggs in urine 320 All eight urine pools were positive in the generic Schistosoma spp. 28S real-time assay, in the 321 S. haematobium dra1 real-time assay and in the S. haematobium COX1 PCR consistent with 322 the presence of S. haematobium eggs in all pools. No pool was positive for S. mansoni TRE 323 real-time PCR. One pool from the village of Ouinanga Serir was positive for S. bovis COX1. 324 This result indicates the possibility of the presence of S. haematobium X bovis hybrids as 325 observed in previous studies in West Africa (29, 31). 326 327 DISCUSSION 328 Djara B. truncatus 4 19.7 6.0 8.5 6.6 2.4 G. Malacological survey 296 ounaiangaensis √ V. nilotica √ Boku B. truncatus 6 20.8 1524.0 8.8 8.8 5.9 L. natalensis 3 G. ounaiangaensis √ Bedrim G. ounaiangaensis √ 18.7 7.3 8.4 5.2 3.0 Sampling sites without snails Ounianga Kebir Yoa 5 (hot source) 28.4 2.8 7.0 2.9 4.9 Yoa 6 (hot source) 27.7 2107.0 7.4 3.2 16.3 Yoa 7 (lake) 14.4 290.0 10.3 1.1 158.0 Uma red 27.5 >2000.0 10.1 14.2 85.8 Uma blue 18.6 >2000.0 10.5 15.0 118.0 Uma (hot spring) 28.2 4.0 9.1 3.6 14.9 Forodone 19.1 31.1 9.8 2.0 >1000.0 Ounianga Serir Telli 21.3 13.7 10.7 2.7 4.5 Edem 14.1 3.5 8.7 8.3 1.0 *All sampling sites are shown in Figure 2 Sequencing of Schistosoma spp. eggs in urine 320 All eight urine pools were positive in the generic Schistosoma spp. 28S real-time assay, in the 321 S. haematobium dra1 real-time assay and in the S. haematobium COX1 PCR consistent with 322 the presence of S. haematobium eggs in all pools. No pool was positive for S. mansoni TRE 323 real-time PCR. One pool from the village of Ouinanga Serir was positive for S. bovis COX1. 324 This result indicates the possibility of the presence of S. haematobium X bovis hybrids as 325 observed in previous studies in West Africa (29, 31). 326 DISCUSSION 328 In Ounianga Serir, there are no hot springs and the two freshwater lakes (Djara 347 and Boku) are used for all water-related activities. In both, B. truncatus were present and the 348 lakes’ close proximity to the quarter Roy may explain the high urinary schistosomiasis 349 prevalence. 350 The larger of the two villages, Ounianga Kebir had an overall lower schistosomiasis prevalence 335 compared to the smaller village of Ounianga Serir (35% versus 55%). In the different 336 neighbourhoods of Ounianga Kebir the prevalence varied and ranged from 21% to 42% (Fig 337 2). This may be partly explained by the proximity to the rare freshwater sites that are used for 338 washing cloth, bathing and swimming. For example, Lake Yoa with its cold temperature and 339 high salinity is fed by numerous freshwater springs. These provide habitats for the intermediate 340 host snails, and the neighbourhood with the highest prevalence was the one closest to a 341 freshwater spring. The quarters with lower prevalence were closer to freshwater sources 342 including the two hot springs (Yoa 5 and 6). Here, the high water temperature might explain 343 the absence of snails (32). The two adjacent sampling sites Yoa 3 and 4 are cold and only 344 used for irrigating the surrounding gardens or for watering livestock. Interestingly, at these two 345 sites snails of the species Limnea natalesis were found, the intermediate host of the liver fluke 346 Fasciola spp. In Ounianga Serir, there are no hot springs and the two freshwater lakes (Djara 347 and Boku) are used for all water-related activities. In both, B. truncatus were present and the 348 lakes’ close proximity to the quarter Roy may explain the high urinary schistosomiasis 349 prevalence. 350 About half of the adult participants and two thirds of the children with a positive test suffered 351 from heavy S. haematobium infections. Our data show that the infection intensity is associated 352 with the severity of haematuria, pointing towards chronic schistosomiasis caused by long-term 353 exposure and recurrent reinfection. Hence, the major health problems reported by the local 354 population, namely abdominal issues and blood in urine, may well be due to schistosomiasis, 355 and are likely the consequences of the lacking access to diagnostics and treatment options 356 and the absence of any preventive intervention, as it has also been reported from other remote 357 areas in Chad (33). DISCUSSION 328 This exploratory study was the very first time a medical research team focused on the Sahara 329 oasis of Ounianga, Ennedi Ouest province in Chad. We were able to show for the first time the 330 high prevalence of S. haematobium in the population of both villages Ounianga Kebir and Serir. 331 Living specimens of B. truncatus were found at both sites, whereas the previous findings were 332 fossils dating back to the early Holocene (17). These findings suggest the possibility of ongoing 333 local schistosomiasis transmission in this desert oasis environment. 334 This exploratory study was the very first time a medical research team focused on the Sahara 329 oasis of Ounianga, Ennedi Ouest province in Chad. We were able to show for the first time the 330 high prevalence of S. haematobium in the population of both villages Ounianga Kebir and Serir. 331 Living specimens of B. truncatus were found at both sites, whereas the previous findings were 332 fossils dating back to the early Holocene (17). These findings suggest the possibility of ongoing 333 local schistosomiasis transmission in this desert oasis environment. 334 14 The larger of the two villages, Ounianga Kebir had an overall lower schistosomiasis prevalence 335 compared to the smaller village of Ounianga Serir (35% versus 55%). In the different 336 neighbourhoods of Ounianga Kebir the prevalence varied and ranged from 21% to 42% (Fig 337 2). This may be partly explained by the proximity to the rare freshwater sites that are used for 338 washing cloth, bathing and swimming. For example, Lake Yoa with its cold temperature and 339 high salinity is fed by numerous freshwater springs. These provide habitats for the intermediate 340 host snails, and the neighbourhood with the highest prevalence was the one closest to a 341 freshwater spring. The quarters with lower prevalence were closer to freshwater sources 342 including the two hot springs (Yoa 5 and 6). Here, the high water temperature might explain 343 the absence of snails (32). The two adjacent sampling sites Yoa 3 and 4 are cold and only 344 used for irrigating the surrounding gardens or for watering livestock. Interestingly, at these two 345 sites snails of the species Limnea natalesis were found, the intermediate host of the liver fluke 346 Fasciola spp. DISCUSSION 328 358 The study was set-up as an exploratory study with the aim to reveal the presence of the 359 Schistosoma spp. lifecycle in the desert. Its scope is therefore limited and leaves several 360 factors unaddressed at this stage. For example, the men’s main activities involve working in 361 soda extraction sites, trading using traditional caravans, and raising livestock through mobile 362 15 pastoralism. Hence, during the visit of the study team, the majority of men aged 16 to 60 years 363 were absent resulting in an over-representation of women in the study population (ratio 1:2). 364 Regarding the S. mansoni diagnostics that showed a positive POC-CCA result for 15.2% of all 365 urine samples, we cannot conclude with certainty that S. mansoni is present in the study 366 population as no stool samples were collected and hence, no parasitological proof of S. 367 mansoni infection is available. Of note, according to the tests handbook, also a heavy infection 368 with S. haematobium can lead to a positive test result (34). It is also significant that no 369 intermediate host snails of the genus Biomphalaria were found. However, the exploratory study 370 was conducted in January while snail abundance is highly seasonal (35). 371 Conclusion 373 This exploratory study presents the first modern evidence of urinary schistosomiasis among 374 the population of these oasis villages. There is clearly a need for further studies to fully 375 understand the current epidemiological situation. However, apart from further studies the main 376 problems are already evident; namely the lack of health education, diagnostics and access to 377 treatment. With a combined approach, including sensitization, mass drug administration, and 378 morbidity management the control or even elimination of urinary schistosomiasis in this 379 population might be possible. 380 Ethics approval and consent to participate 383 The study received approval from the ethics committee Northwest and Central Switzerland 384 (reference no. BASEC Nr Req-2018-0120) and the ‘Comité National de Bioéthique du Tchad’ 385 (CNBT) in N’Djamena, Chad (reference no. 134/PR/MESRI/SG/CNBT/2018). Research 386 authorization was granted by the Chadian Ministry of Health and its ‘Direction de la Lutte contre 387 la Maladie et de la Promotion de la Santé’ (reference no. 007/PR/MSP/DG/DLMPS/2018). 388 16 Upon arrival in the study villages, an assembly was organized with the community 389 representatives to discuss the study objectives and procedures. The traditional leaders, 390 together with the local authorities, discussed the study and decided about concrete 391 participation. Once a collective decision had been reached, written informed consent was 392 obtained from the community representatives. In line with high illiteracy rates among the 393 general population, individual participants consented orally. These consent procedures had 394 received approval by the respective ethics committees. Those participants with a positive test 395 result from either filtration or POC-CCA testing were invited to the health centre / health post 396 and were administered praziquantel in the adequate dose (40 mg/kg) by the study nurse. 397 Upon arrival in the study villages, an assembly was organized with the community 389 representatives to discuss the study objectives and procedures. The traditional leaders, 390 together with the local authorities, discussed the study and decided about concrete 391 participation. Once a collective decision had been reached, written informed consent was 392 obtained from the community representatives. In line with high illiteracy rates among the 393 general population, individual participants consented orally. These consent procedures had 394 received approval by the respective ethics committees. Those participants with a positive test 395 result from either filtration or POC-CCA testing were invited to the health centre / health post 396 and were administered praziquantel in the adequate dose (40 mg/kg) by the study nurse. 397 Consent for publication 398 All authors approved submission for publication of this manuscript. 399 Availability of data and materials 400 Data will be available on request by email to the corresponding author. 401 Authors' contributions 407 HG conceived and designed the study protocol with input from PS; AAB, HG, RO and WM 408 implemented the research in Chad; AAB, HG, MA, RO and WM carried out the field work and 409 the parasitological examinations, together with a medical team; HG and WM sampled the 410 snails, and FA and RC performed the genetic sequencing and analysis for species 411 confirmation. RW and SP performed the genetic sequencing and analysis of the Schistosoma 412 HG conceived and designed the study protocol with input from PS; AAB, HG, RO and WM 408 implemented the research in Chad; AAB, HG, MA, RO and WM carried out the field work and 409 the parasitological examinations, together with a medical team; HG and WM sampled the 410 snails, and FA and RC performed the genetic sequencing and analysis for species 411 confirmation. RW and SP performed the genetic sequencing and analysis of the Schistosoma 412 17 samples. AAB, FA, HG, RO and WM analysed and interpreted the epidemiological data; HG, 413 RO and WM drafted the manuscript; all authors critically revised the manuscript for intellectual 414 content and approved the final manuscript. HG is the guarantor of the paper. 415 samples. AAB, FA, HG, RO and WM analysed and interpreted the epidemiological data; HG, 413 RO and WM drafted the manuscript; all authors critically revised the manuscript for intellectual 414 content and approved the final manuscript. HG is the guarantor of the paper. 415 samples. AAB, FA, HG, RO and WM analysed and interpreted the epidemiological data; HG, 413 RO and WM drafted the manuscript; all authors critically revised the manuscript for intellectual 414 content and approved the final manuscript. HG is the guarantor of the paper. 415 Acknowledgments 416 We are deeply grateful to Dr. Baba Mallaye, director of the Centre d’Appui à la Recherche 417 (CNAR), N’djamena, Chad, who not only guided us through the desert to safely arrive at the 418 Lakes of Ounianga, but also introduced us and the purpose of our research visit to the local 419 authorities. We thank the population of Ounianga Kabir and Ounianga Serir for hosting us and 420 for their willingness to participate in the study. Special thanks go to the staff of the health 421 centers at the study sites for their support. Authors' contributions 407 Without the tireless engagement of our study nurse 422 Ali Abba Abakar, this work would not have succeeded. Nomadic pastoralists at Lake Chad 423 have triggered this study, and we like to express here our deepest thanks for their hospitality 424 and sharing of their wisdom. 425 We are deeply grateful to Dr. Baba Mallaye, director of the Centre d’Appui à la Recherche 417 (CNAR), N’djamena, Chad, who not only guided us through the desert to safely arrive at the 418 Lakes of Ounianga, but also introduced us and the purpose of our research visit to the local 419 authorities. We thank the population of Ounianga Kabir and Ounianga Serir for hosting us and 420 for their willingness to participate in the study. Special thanks go to the staff of the health 421 centers at the study sites for their support. Without the tireless engagement of our study nurse 422 Ali Abba Abakar, this work would not have succeeded. Nomadic pastoralists at Lake Chad 423 have triggered this study, and we like to express here our deepest thanks for their hospitality 424 and sharing of their wisdom. 425 18 References 428 429 1. WHO. Ending the neglect to attain the sustainable development goals. A road map for 430 neglected tropical diseases 2021–2030. Geneva: World Health Organisation; 2020. 431 2. Ehrenberg N, Ehrenberg JP, Fontes G, Gyapong M, Rocha EMM, Steinmann P, et al. 432 Neglected tropical diseases as a barometer for progress in health systems in times of COVID- 433 19. BMJ Glob Health. 2021;6(4). 434 3. Colley DG, Bustinduy AL, Secor WE, King CH. Human schistosomiasis. The Lancet. 435 2014;383(9936):2253-64. 436 4. Verjee MA. Schistosomiasis: still a cause of significant morbidity and mortality. Res 437 Rep Trop Med. 2019;10:153-63. 438 5. King CH. Parasites and poverty: the case of schistosomiasis. 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Geospatial health. 530 2014;9(1):109-18. 531 22 532 533 534 535 536 537 22 SUPPLEMENTARY MATERIAL 538 Table 1. Primers and probes 539 Parasite Name Sequence 3’-5’ Origin Generic Schistosoma spp. References 428 Schisto28S_F GTGGAGTTGAACTGCAAGC Modified from Cnops et al. 2012 Schisto28S_R1 CCATAGCAGACAGGCAGC Schisto28S_R2 GCTCAACAWTAATAGTCAAACCTG Schisto28S_P FAM- ACTGACAAGCAGACCCTCACACC- BHQl S. mansoni TRE Sman_F CCACGCTCTCGCAAATAATCTA Modified from Wichmann et al. 2013 Sman_R AAATCGTTGTATCTCCGAAACCA Sman_P YYE- ACAAACATCATAAAAATCCGTCCA- MGB-Q5 S. haematobium dra1 Shae_F GATCTCACCTATCAGACGAAAC Identical to Cnops et al. 2013 Shae_R TCACAACGATACGACCAAC Shae_P FAM- TGTTGGTGGAAGTGCCTGTTTCGCAA -BHQ1 S. haematobium COX SH_COX_F TTTTTTGGTCATCCAGAGGTGTAT Modified from Boon et al. 2018 SH_COX_R TAATAATCAATGACCCTGCAATAA S. bovis COX SB_COX_F TTTTTTGGGCATCCGGAGGTGTAT SB_COX_R CACAGGATCAGACAAACGAGTACC 540 540 23
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New vessels detected on wide-field imaging compared to two-field and seven-field imaging: implications for diabetic retinopathy screening image analysis
British journal of ophthalmology
2,015
cc-by
3,884
Copyright: This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ Talks SJ, Manjunath V, Steel DHW, Peto T, Taylor R. New vessels detected on wide-field imaging compared to two-field and seven-field imaging: implications for diabetic retinopathy screening image analysis. British Journal of Ophthalmology 2015, 99(12), 1606-1609. DOI link to article: http://dx.doi.org/10.1136/bjophthalmol-2015-306719 Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Roy Taylor4 Method A consecutive series of treatment naïve patients with DR, referred from DRSS with pre- proliferative or proliferative DR or diabetic maculopathy, were imaged with Optomap colour images, within 3 months of DRSS referral. The incidence and distribution of NVs were recorded in relation to two-field and seven-field areas. ABSTRACT Introduction Wide-field retinal imaging (Optomap), used for detecting diabetic retinopathy (DR), has been shown to compare well with seven-field early treatment diabetic retinopathy study (ETDRS) photographs. An Optomap 200° image covers 80% of the retinal surface, compared with the standard seven-field, 30° images, covering 30% of the retinal surface. In England, DR screening is performed by grading two, 45° images per eye, by the DR screening service (DRSS). eye, by t e sc ee g se ce ( SS) Purpose To assess how often retinal new vessels (NVs) are observed on Optomap imaging, outside the DRSS two fields and standard seven-field photography, in a cohort of patients referred by the DRSS. Method A consecutive series of treatment naïve patients with DR, referred from DRSS with pre- proliferative or proliferative DR or diabetic maculopathy, were imaged with Optomap colour images, within 3 months of DRSS referral. The incidence and distribution of NVs were recorded in relation to two-field and seven-field areas. Purpose To assess how often retinal new vessels (NVs) are observed on Optomap imaging, outside the DRSS two fields and standard seven-field photography, in a cohort of patients referred by the DRSS. Correspondence to Stephen James Talks, Newcastle Eye Centre, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, UK; james.talks@nuth.nhs.uk Correspondence to Stephen James Talks, Newcastle Eye Centre, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, UK; james.talks@nuth.nhs.uk Method A consecutive series of treatment naïve patients with DR, referred from DRSS with pre- proliferative or proliferative DR or diabetic maculopathy, were imaged with Optomap colour images, within 3 months of DRSS referral. The incidence and distribution of NVs were recorded in relation to two-field and seven-field areas. METHOD A Received 1 February 2015 Revised 23 March 2015 Accepted 21 May 2015 Published Online First 13 August 2015 A consecutive series of treatment naïve patients, referred from two DRSS in England, were imaged with Optomap colour images, within 3 months of referral. Referral from DRSS occurs if ‘referable’ DR is detected on analysis of two standard 45° photographs. At DRSS images are graded for the level of DR, and diabetic maculopathy (DMac): no DR is denoted as R0; mild DR as R1; pre- proliferative DR as R2; proliferative DR as R3. Potentially clinically significant DMac is repre- sented by the M1 grade. R2, R3 and M1 are then subsequently referred to hospital eye services. Results NVs were found in 102 of 1562 treatment naïve eyes (6.5%) of 781 patients. Of these, 72 were referred from DRSS as having NVs, but an additional 30 eyes (29% of NVs detected) from 25 patients were referred with a lesser degree of DR. In 25 of the 30 eyes without NVs reported on referral, NVs were located outside the standard two fields taken at DRSS, and in 12, NVs were outside the area covered on seven-field imaging (11.7% of eyes with NVs). Conclusions Wide-field imaging with Optomap detected approximately 30% more NVs than standard two-field imaging in patients referred from a UK DRSS. At the hospital eye clinic, certified medical photo- graphers took three wide-field Optomap images per eye after mydriasis, using the Optomap P2000 scan- ning laser ophthalmoscope; straight-ahead and up and down, with eye steering, which involves the patient following a fixation target (figure 1). Open Access Scan to access more free content Date deposited: Newcastle University ePrints - eprint.ncl.ac.uk group.bmj.com on April 5, 2016 - Published by http://bjo.bmj.com/ Downloaded from Clinical science Received 1 February 2015 Revised 23 March 2015 Accepted 21 May 2015 Published Online First 13 August 2015 1Newcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne, UK 2Sunderland Eye Infirmary, Sunderland and Institute of Genetic Medicine, Newcastle upon Tyne, UK 3NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK 4Magnetic renounce centre, Campus for ageing and vitality, Newcastle University, Newcastle upon Tyne, UK Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Roy Taylor4 Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Ro 1Newcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne, UK 2Sunderland Eye Infirmary, Sunderland and Institute of Genetic Medicine, Newcastle upon Tyne, UK 3NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK 4Magnetic renounce centre, Campus for ageing and vitality, Newcastle University, Newcastle upon Tyne, UK 1Newcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne, UK 2Sunderland Eye Infirmary, Sunderland and Institute of Genetic Medicine, Newcastle upon Tyne, UK 3NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK 4Magnetic renounce centre, Campus for ageing and vitality, Newcastle University, Newcastle upon Tyne, UK in a population referred from the community could not be assessed. Grading is based on the ETDRS studies that related retinal findings to the likelihood of progression of the retinopathy and is based on seven-field colour imaging. It is still unknown how often more severe DR changes are found outside the standard seven-field, in particular new vessel (NV) formation. In the English DR screening service (DRSS) two images with nominal 45° fields are taken per eye, one centred on the fovea and the other on the disc. This is said to have a sensitivity of 80.2% and specificity of 92.9% for detecting refer- able DR compared to slit lamp biomicroscopy.5 In this study, we aimed to assess how often NVs were seen with wide-field Optomap imaging when com- pared to the areas covered by DRSS’s two-field and standard seven-field photography, in a cohort of patients referred from a DRSS. ABSTRACT Introduction Wide-field retinal imaging (Optomap), used for detecting diabetic retinopathy (DR), has been shown to compare well with seven-field early treatment diabetic retinopathy study (ETDRS) photographs. An Optomap 200° image covers 80% of the retinal surface, compared with the standard seven-field, 30° images, covering 30% of the retinal surface. In England, DR screening is performed by grading two, 45° images per eye, by the DR screening service (DRSS). Purpose To assess how often retinal new vessels (NVs) are observed on Optomap imaging, outside the DRSS two fields and standard seven-field photography, in a cohort of patients referred by the DRSS. INTRODUCTION Wide-field retinal imaging (Optomap), used for detecting diabetic retinopathy (DR), has been shown to compare well with seven-field early treat- ment diabetic retinopathy study (ETDRS) photo- graphs.1–3 As it provides a wider field of view, it would not be surprising that more DR is seen; however, it is thought that most potentially sight- threatening pathology occurs between the posterior pole and retinal mid-periphery. Silva et al4 reported that 10% of a cohort of 206 eyes were given a higher DR grade on Optomap images compared to seven-field images, predominantly due to the finding of more haemorrhages per quadrant. In his study, the patients were chosen from a tertiary eye clinic to represent a range of DR severity, and so, incidence rates of previously not recorded findings The images were then graded by an independent reading centre and the number of eyes with NVs, R3, recorded. The R3 images were then further assessed to map the distribution of NVs in relation to two-field and seven-field standard images using a standard field map (figure 2). If there was more than one area of NVs and any were located inside either the two-field or seven-field areas, then they were counted as being detected by that method. In a few cases, where the distinction between haemor- rhage, intra-retinal microvascular abnormalities (IRMA) and small NVs was uncertain, a fundus fluorescein angiogram (FFA) was performed, at the examiners discretion, on a second visit. Open Access Scan to access more free content Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1606 group.bmj.com on April 5, 2016 - Published by http://bjo.bmj.com/ Downloaded from Clinical science Figure 1 Red free Optomap images of a left eye of a diabetic referred with maculopathy in the other eye, (R1, M1), the left eye being referred as, (R1, M0). (A) Up-steered, showing new vessels, (B) with zoom, (C) straight ahead showing new vessels outside two fields and on the edge of the standard seven-field images, (D) down steered, showing new vessels and (E) better seen with on zoom. Clinical science Figure 1 Red free Optomap images of a left eye of a diabetic referred with maculopathy in the other eye, (R1, M1), the left eye being referred as, (R1, M0). RESULTS d d Independent assessment of the wide-field imaging found NVs in 102 of 1562 treatment naïve eyes (6.5%) of 781 patients referred from DRSS. Of these, 72 were referred from DRSS as having NVs, but 30 eyes (29% of NVs detected) from 25 patients were referred with a lesser degree of DR: 14 were referred as R2 and 16 as R1. In these eyes, 18 had been referred with Dmac, (M1), the other 12 were graded as M0. The fellow eyes of these patients were graded as R3 (proliferative DR) in three cases, R2 in eight and R1 in nine. In nine cases both eyes were referred with R1, having been referred because of DMac. Figure 1 shows NVs outside two fields and the benefit of using up and down eye steering. Figure 2 shows NVs outside seven fields confirmed on FFA. INTRODUCTION (A) Up-steered, showing new vessels, (B) with zoom, (C) straight ahead showing new vessels outside two fields and on the edge of the standard seven-field images, (D) down steered, showing new vessels and (E) better seen with on zoom. The images were reviewed using the proprietary image review software (Optos V2 Vantage Dx Review V.2.5.0.135; Optos, Dunfermline, UK). Grading for each wide-field image involved viewing the colour composite, green-wavelength and red- wavelength images using all the available image enhancement tools, including localised optimisation and magnification. However, true NVs were recorded only if the NV was present on FFA where avail- able and also where the NV remained clearly visible on the original image once spotted on the manipulated image, where appropriate. FFAs were requested in 31 patients where the examiner had some uncertainty between R2 and R3. In 14 of these cases, the examiner had diagnosed NVs and this was confirmed in 10 cases but not found in 4 cases and in 17 cases the examiner thought they were seeing, R2 changes, this was confirmed in 9 cases, but NVs were found in 8 cases. In 25 of the 30 eyes, in which NVs were not reported on the two DRSS images, the NVs were found outside the standard two-field and in 12 of these outside seven-field (11.7% of eyes with NVs); three were just within the field of view of the two fields but had been referred as R2 with IRMA. Two had very small disc NVs but also had NVs beyond two fields. Images from 23 eyes of the referred 1562 (1.4%) were deemed ungrad- able on Optomap due to poor image quality. DISCUSSION Figure 2 Red free Optomap and fundus fluorescein angiogram of the left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1, M1 left), showing new vessels outside standard two-field (A); and seven-field (B) in the left eye. One factor that may have led us to detect this rate of NVs was the use of three images per eye, using eye steering, as less pathology is likely to be missed due to defocus or masking from eyelashes.9 The Optos camera can take a 200° image, but the resolution is best in a central band between the two arcades. The focus for the top and bottom areas of the retina is better by taking the image with the patient looking up and down. Looking at three images per eye takes extra time compared to one. Montage software is being developed to merge three pic- tures, which will help with analysis, but is not commercially available yet. A study comparing wide-field photographs, taken with undi- lated and dilated pupils, found that this did not statistically change the agreement with seven-field imaging, but reduced the ungradable rate from 4.5% to 0%.4 We had an ungradable rate of 1.4% using dilation and three images per eye. Figure 2 Red free Optomap and fundus fluorescein angiogram of the left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1, M1 left), showing new vessels outside standard two-field (A); and seven-field (B) in the left eye. Our patients with NVs not detected on DRSS images were not ‘missed’ cases, as they were correctly referred for further medical assessment. All registered patients with diabetes are offered annual DRSS photography in England and this has led to fewer patients being referred from DRSS with severe NVs, and so, our incidence figures of more peripheral pathology may be higher than in unscreened populations. It is possible that if patients had small NVs outside the two-field images they would have been eventually referred as more posterior pathology developed. patient and on the photographer to use this protocol in every- day clinical practice. In the Eurodiab paper justifying the use of 45° imaging criteria only 48 eyes were compared.6 The findings supported the use of two-field imaging as a practical method, with the agreement for correct DR between several examiners, ranging between 28 and 43 of the 48 eyes, mean of 37 eyes. DISCUSSION ETDRS seven-field colour imaging is still considered the gold standard for assessing DR; however, it is hard both on the Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1607 group.bmj.com on April 5, 2016 - Published by http://bjo.bmj.com/ Downloaded from group.bmj.com on April 5, 2016 - Published by http://bjo.bmj.com/ Downloaded from Clinical science patient and on the photographer to use this protocol in every- day clinical practice. In the Eurodiab paper justifying the use of 45° imaging criteria only 48 eyes were compared.6 The findings supported the use of two-field imaging as a practical method, with the agreement for correct DR between several examiners, ranging between 28 and 43 of the 48 eyes mean of 37 eyes Figure 2 Red free Optomap and fundus fluorescein angiogram of the left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1, M1 left), showing new vessels outside standard two-field (A); and seven-field (B) in the left eye. Our study shows that on two-field DRSS imaging there is only a small risk of missing NVs, 30/1562 (1.9%), but these repre- sented 29% of the total number of eyes graded as having NVs. The NVs were found outside even the seven-field area in 11.7%. This is a higher rate than previously reported. In a study of 206 eyes of 103 patients, 10% were given a more severe DR grade with wide-field imaging, using one image per eye.4 In relation to our findings, 46 had NVs, but only two of these were found outside the seven-field area (4% of NVs). Our study population was much larger and represents a consecutive series referred from DRSS, rather than a group from a highly specialised clinic. A study using wide-field FFA on 118 patients found a total of 22 eyes (10%) had pathology visible only outside a simulated seven-field boundary. Of those eyes, 13 had peripheral retinal non-perfusion (8%) and 9 of 54 cases (17% of NVs) had per- ipheral NV outside seven-field. While using a different technique for identification of cases with NVs, this study draws a similar conclusion to ours on the relative proportion of NVs found outside seven-field.8 In the cases where we did use FFA some changes were made in the grading and eight additional cases of NVs were found. Acknowledgements This article presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-0609-19117). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We thank the photography departments at Royal Victoria Infirmary, Newcastle upon Tyne and Sunderland Eye Infirmary for their Provenance and peer review Not commissioned; externally peer reviewed. Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/ licenses/by/4.0/ 6 Adlington SJ, Kohner EM, Meuer S, et al. Methodology for retinal photography and assessment of diabetic retinopathy: the EURODIAB IDDM complications study. Diabetologia 1995;38:437–44. g 7 Diabetic eye screening. www.diabeticeye.screening.nhs.uk 8 Wessel MM, Aaker GD, Parlitsis G, et al. Ultra-wide-field angiography improves the detection and classification of diabetic retinopathy. Retina 2012;32:785–91. 9 Rasmussen ML, Broe R, Frydkjaer U, et al. Comparison between Early Treatment Diabetic Retinopathy Study 7-field retinal photos and non-mydriatic, mydriatic and mydriatic steered widefield scanning laser ophthalmoscopy for assessment of diabetic retinopathy. J Diabetes Complications 2014;29:99–104. group.bmj.com on April 5, 2016 - Published by http://bjo.bmj.com/ Downloaded from group.bmj.com on April 5, 2016 - Published by http://bjo.bmj.com/ Downloaded from Clinical science assistance during this study. Photographers Gill O’Brien and Jade Ward prepared the figures. TP was supported by the NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. assistance during this study. Photographers Gill O’Brien and Jade Ward prepared the figures. TP was supported by the NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. examination for evaluation of diabetic retinopathy. Am J Ophthalmol 2012;154:549–59. examination for evaluation of diabetic retinopathy. Am J Ophthalmol 2012;154:549–59. 2 Kernt M, Haritoglou C, Hadi I, et al. Assessment of diabetic retinopathy using nonmydriatic ultra-widefield scanning laser ophthalmoscopy (Optomap) compared with ETDRS 7-field stereo photography. Diabetes Care 2012;35:2459–63. Contributors All the authors contributed to the design of the study. TP led the independent reading centre. SJT and VM carried out the study, recruiting and consenting the patients. All the authors contributed to writing the paper. 3 Soliman AZ, Silva PS, Aiello LP, et al. Ultra-wide field retinal imaging in detection, classification, and management of diabetic retinopathy. Semin Ophthalmol 2012;27:221–6. Funding National Institute for Health Research. Funding National Institute for Health Research. Competing interests None declared. Competing interests None declared. Competing interests None declared. 4 Silva PS, Cavallerano JD, Sun JK, et al. Peripheral lesions identified by mydriatic ultrawide field imaging: distribution and potential impact on diabetic retinopathy severity. Ophthalmology 2013;120:2587–95. Ethics approval Ethical approval was obtained from the Northern Regional Ethics Committee and the NHS Trust Research departments at the Royal Victoria Infirmary, Newcastle upon Tyne and the Sunderland Eye Infirmary. 5 Scanlon PH, Malhotra R, Greenwood RH, et al. Comparison of two reference standards in validating two field mydriatic digital photography as a method of screening for diabetic retinopathy. Br J Ophthalmol 2003;87:1258–63. Provenance and peer review Not commissioned; externally peer reviewed. DISCUSSION The kappa for interobserver and intraobserver comparisons was good at 0.83 and 0.85, respectively. Two-field imaging, where approximately 80% of patients are imaged yearly, using this protocol meets the appropriate sensitivity and specificity required for a screening programme and was therefore rolled out with scale, as shown by the England DRSS.7 This study also does not clarify how much risk there is in missing peripheral NVs, as they were not detected as a result of a patient presenting with the complications of proliferative DR, rather as a result of imaging a cohort of patients. However, if NVs are missed on DRSS images, and the patient is referred because of Dmac, appropriate management depends on the clin- ician detecting these NVs, which may not occur in a busy streamlined macular service. We would therefore advocate the use of steered wide-field images in ophthalmology clinics, and in the future hope that automated software can be developed to allow for fast, reliable and valid identification of abnormal vessels. In one study, seven-field ETDRS stereo images were ungrad- able by strict grading criteria in 31.6% and in 15.3% with a more lenient approach.5 The same paper reported good agree- ment for detecting the difference between referable and non- referable retinopathy between slit lamp biomicroscopy, 2×45° field and 7×30° field photography. However, there was only agreement on finding proliferative DR in 51/88 (58%) patients when comparing seven-field ETDRS stereo images with slit lamp examination. It is not clear how many had already had laser which may have lead to confusion on definitions between active or inactive NVs. In two cases, the clinician found NVs outside seven-field. For the comparison of two-field to seven- field only correlations between detecting referable from non- referable DR were presented. Acknowledgements This article presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-0609-19117). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We thank the photography departments at Royal Victoria Infirmary, Newcastle upon Tyne and Sunderland Eye Infirmary for their Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1608 REFERENCES 1 Silva PS, Cavallerano JD, Sun JK, et al. Nonmydriatic ultrawide field retinal imaging compared with dilated standard 7-field 35-mm photography and retinal specialist Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1609 group.bmj.com on April 5, 2016 - Published by http://bjo.bmj.com/ Downloaded from screening image analysis imaging: implications for diabetic retinopathy compared to two-field and seven-field New vessels detected on wide-field imaging doi: 10.1136/bjophthalmol-2015-306719 13, 2015 2015 99: 1606-1609 originally published online August Br J Ophthalmol http://bjo.bmj.com/content/99/12/1606 Updated information and services can be found at: #BIBL http://bjo.bmj.com/content/99/12/1606 This article cites 8 articles, 2 of which you can access for free at: http://creativecommons.org/licenses/by/4.0/ use, provided the original work is properly cited. See: others to distribute, remix, adapt and build upon this work, for commercial the Creative Commons Attribution (CC BY 4.0) license, which permits This is an Open Access article distributed in accordance with the terms of box at the top right corner of the online article. Receive free email alerts when new articles cite this article. Sign up in the Articles on similar topics can be found in the following collections (1560) Retina (1037) Epidemiology (217) Open access (99) Editor's choice Notes http://group.bmj.com/group/rights-licensing/permissions To request permissions go to: http://journals.bmj.com/cgi/reprintform To order reprints go to: http://group.bmj.com/subscribe/ To subscribe to BMJ go to: http://group.bmj.com/group/rights-licensing/permissions To request permissions go to: http://journals.bmj.com/cgi/reprintform To order reprints go to:
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https://biblio.ugent.be/publication/8701492/file/8701495.pdf
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Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites
Frontiers in microbiology
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ORIGINAL RESEARCH published: 03 February 2021 doi: 10.3389/fmicb.2021.633488 Edited by: Aleksa Obradovi ´c, University of Belgrade, Serbia A selection of bacteria was then tested for the ability to induce A/S Specialty section: This article was submitted to Microbe and Virus Interactions with Plants, a section of the journal Frontiers in Microbiology Received: 25 November 2020 Accepted: 13 January 2021 Published: 03 February 2021 Specialty section: This article was submitted to Microbe and Virus Interactions with Plants, a section of the journal Frontiers in Microbiology Received: 25 November 2020 Accepted: 13 January 2021 Published: 03 February 2021 Specialty section: This article was submitted to Microbe and Virus Interactions with Plants, Edited by: Aleksa Obradovi ´c, University of Belgrade, Serbia Reviewed by: Carolina Chiellini, Italian National Research Council, Italy Debdulal Banerjee, Vidyasagar University, India Reviewed by: Carolina Chiellini, Italian National Research Council, Italy Debdulal Banerjee, Vidyasagar University, India *Correspondence: Angélique Rat angelique.rat@ugent.be 1 Laboratory of Microbiology, Department Biochemistry and Microbiology, Faculty Sciences, Ghent University, Ghent, Belgium, 2 Laboratory of Conservation and Evaluation of Native and Floricultural Species, Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter, Thessaloniki, Greece, 3 Institut für Pflanzenkultur, Schnega, Germany, 4 Organic Chemistry Laboratory, School of Chemical Engineering, Aristotle University of Thessaloniki and Center of Interdisciplinary Research and Innovation of AUTh (CIRI-AUTh), Natural Products Research Centre of Excellence (NatPro-AUTH), Thessaloniki, Greece, 5 Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. Angélique Rat1*, Henry D. Naranjo1, Nikos Krigas2, Katerina Grigoriadou2, Eleni Maloupa2, Alicia Varela Alonso3, Carolin Schneider3, Vassilios P. Papageorgiou4, Andreana N. Assimopoulou4, Nikolaos Tsafantakis5, Nikolas Fokialakis5 and Anne Willems1 Angélique Rat1*, Henry D. Naranjo1, Nikos Krigas2, Katerina Grigoriadou2, Eleni Maloupa2, Alicia Varela Alonso3, Carolin Schneider3, Vassilios P. Papageorgiou4, Andreana N. Assimopoulou4, Nikolaos Tsafantakis5, Nikolas Fokialakis5 and Anne Willems1 Edited by: Aleksa Obradovi ´c, University of Belgrade, Serbia Reviewed by: Carolina Chiellini, Italian National Research Council, Italy Debdulal Banerjee, Vidyasagar University, India *Correspondence: Angélique Rat angelique.rat@ugent.be INTRODUCTION Soil microorganisms can also influence plant growth through the secretion of growth hormones such as indole acetic acid (IAA) or ethylene. Indeed, several studies have shown that plant inoculation with IAA-producing bacteria promote plant growth significantly (Patten and Glick, 2002; Mohite, 2013; Chandra et al., 2018). Furthermore, rhizospheric bacteria can play a role in plant defense and metabolite production (Lambers et al., 2009; Berendsen et al., 2012; Huang et al., 2019). They can first compete with pathogens for nutrients and space in the rhizosphere. For instance, siderophore-producing bacteria can contend with soil pathogens for iron uptake and consequently prevent the root colonization by these microorganisms. As iron is essential for the growth of many organisms and is involved in biofilm formation that is needed for successful root colonization, its uptake by beneficial bacteria can prevent pathogen infections (Ramey et al., 2004; Ahmed and Holmström, 2014). Microorganisms can also produce secondary metabolites with antimicrobial properties. For example, Burkholderia sp. showed antimicrobial activities against the plant pathogens Phytophthora capsici, Fusarium oxysporum, and Rhizoctonia solani through the production of pyrrolnitrin (Jung et al., 2018). In addition, some microbes can induce plant defenses. Asghari et al. (2020) demonstrated that Pseudomonas sp. Sn48 and Pantoea sp. Sa14 induce plant production of phytoalexins and polyamines resulting in a reduction of the Agrobacterium tumefaciens gall. A h i i i i i h l Studies on endophytes are indeed invaluable to the promotion of medicinal plants’ therapeutic properties. Alkanna tinctoria (family Boraginaceae) is a medicinal plant native to countries of the Mediterranean region (Strid, 2016). The plant produces quinones and phenolic compounds and has antioxidant activities. Moreover, compared to other Alkanna species, A. tinctoria produces high amounts of alkannin/shikonin (A/S) and their derivatives. These secondary metabolites are enantiomeric naphthoquinones produced by root tissues. They are sequestered to form granules in the phospholipid layer and are accumulated in the apoplastic spaces. Thus, they can be found in the cork layer of mature roots and their accumulation leads to a red or purple coloration of the root (Brigham et al., 1999; Singh et al., 2010; Tatsumi et al., 2016). The A/S derivatives are strongly involved in the antimicrobial activity of the plant and form a chemical barrier against soil-borne microorganisms. INTRODUCTION tissues of plants during the entire or part of their host’s lifecycle without causing external damage (Ryan et al., 2008; Anjum and Chandra, 2015; Orlikowska et al., 2017). Like rhizospheric microorganisms, they can promote plant growth and influence plant metabolites. In the case of medicinal plants, endophytes contribute to or are responsible for their host’s pharmaceutical properties (e.g., antioxidant and/or antimicrobial properties; Köberl et al., 2013; Brader et al., 2014; Maggini et al., 2017). The inoculation of Bacillus subtilis BERA 71 to chickpea increased the plant biomass and reduced the levels of reactive oxygen species and lipid peroxidation under salinity stress. This effect was associated with an enhancement of the activities of enzymatic and non-enzymatic antioxidants (Abd_Allah et al., 2017). Endophytes can also influence the antimicrobial activity as well as the production of secondary metabolites in medicinal plants. Pandey et al. (2018) showed that endophytic fungi increase the biomass of Withania somnifera, resulting in a higher total plant withanolide content. Moreover, the inoculation of endophytic bacteria stimulated the withanolide biosynthesis pathway by upregulating the expression of key genes within this pathway such as HMGR, DXS, and DXR (Pandey et al., 2018). Endophytes of medicinal plants also produce a wide range of bioactive secondary metabolites such as alkaloids, isoprenoids, flavonoids and indoles (Tan and Zou, 2001; Barman and Bhattacharjee, 2020; Monnanda et al., 2020). Plants communicate and interact with a wide variety of microorganisms. They release water-soluble sugars, organic acids, ionic compounds, phenolics, hormones, and other metabolites into the rhizosphere, thus providing nutrients to microorganisms. Due to the richness of nutrients in this soil region, compared to bulk soil, the rhizosphere is enriched with microorganisms, leading to increased microbial interactions (Sasse et al., 2018; Yu and Hochholdinger, 2018). Rhizospheric microorganisms influence plant growth through soil nutrient recycling and nutrient uptake. For example, bacteria such as Acidobacterium sp., Pedobacter sp., Muciliginibacter sp., and Cellulomonas sp. play an important role in the recycling of plant polysaccharides such as cellulose, pectin and lignin (López- Mondéjar et al., 2016; Poulsen et al., 2016; Belova et al., 2018). Several bacteria including those from the genera Pseudomonas, Pantoea, and Bacillus have the ability to solubilize and therefore make accessible insoluble phosphate to plants (Ghyselinck et al., 2013). Similarly, rhizobia can provide plants with combined nitrogen via biological nitrogen fixation (Mabrouk et al., 2018). Citation: Rat A, Naranjo HD, Krigas N, Grigoriadou K, Maloupa E, Alonso AV, Schneider C, Papageorgiou VP, Assimopoulou AN, Tsafantakis N, Fokialakis N and Willems A (2021) Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites. Front. Microbiol. 12:633488. doi: 10.3389/fmicb.2021.633488 February 2021 | Volume 12 | Article 633488 1 Frontiers in Microbiology | www.frontiersin.org Rat et al. Endophytic Bacteria From Alkanna tinctoria production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization. Keywords: endophytes, isolation, Alkanna tinctoria, alkannin, shikonin, hairy roots Frontiers in Microbiology | www.frontiersin.org Identification of the Isolates Dereplication and identification of the isolates obtained in the different conditions were performed using MALDI-TOF MS and 16S rRNA gene sequencing. To conduct the MALDI-TOF MS, pure 2nd generation cultures were grown. The proteins were then extracted, spotted in duplicate onto MALDI-TOF target plates and overlaid with matrix solution (10 mg/mL α-cyano-4- hydroxycinnamic acid in acetonitrile-water-trifluoroacetic acid) as previously described (Dumolin et al., 2019). The protein profiles were read with the Bruker MicroflexTM LT/SH system. The programs SPeDE (Dumolin et al., 2019) and BioNumerics (Applied Mats) were used to cluster the protein profiles. In SPeDe, each unique spectrum is assigned as a reference and clusters of all similar spectra are generated (Dumolin et al., 2019). Thus, strains from which the spectra clustered together were considered as highly similar and only the reference strains were selected for rRNA 16S gene sequencing (Jain et al., 2018). After each expedition, a bacterial isolation campaign was conducted. Three plants from each of the collections were chosen to explore the diversity of culturable bacterial endophytes. The roots were first cleaned with tap water to remove soil particles. Subsequently, they were immersed in a solution of 70% ethanol for 5 min. After rinsing with sterile water, the root was further sterilized with a solution of 1.4% of NaOCl for 20 min. The root was rinsed again and immersed in 2% Na2S2O3 for 10 min to neutralize the effect of bleach. A last rinse with sterile water was performed before testing the efficiency of the sterilization process in order to exclude non-endophytes. To do so, the external surface of the root was imprinted onto a sterile plate containing 869 agar medium (Weyens et al., 2012; Eevers et al., 2015) before incubation at 28◦C. Only roots without microbial growth on the imprints were used for the isolation of endophytic bacteria. The root material was crushed in 10 mL of sterile phosphate buffer saline (PBS) and a 10-fold dilution series was prepared. The dilutions 10−2–10−7 were then plated in triplicate. Genomic DNA was extracted by alkaline lysis according to the method of Niemann et al. (1997). Amplification of the 16S rRNA gene was performed using primers pA (3′-AGAGTTTGATCCTGGCTCAG-5′, forward) and pH (5′-AAGGAGGTGATCCAGCCGCA-3′, reverse). The PCR products were sequenced by Eurofins Genomics, Mix2seq service, with the BKL1 primer (5′-GTATTACCGCGGCTGCTGGCA- 3′, reverse) and the sequences obtained were identified with EzTaxon database (Yoon et al., 2017). Identification of the Isolates We considered two sequences as belonging to the same species when they were at least 98.7% identical and as belonging to the same genus when they were at least 94.8% identical (Moreira and López-García, 2014; Yarza et al., 2014). INTRODUCTION They are also known for wound healing, anticancer and anti-inflammatory properties (Papageorgiou et al., 1999, 2008) and they comprise the active pharmaceutical ingredients of strong wound healing medicines approved by the National Organization for Medicines in Greece. Because of these interesting traits, A. tinctoria was chosen for the production of A/S for medical applications (Papageorgiou et al., 2008; Sengul et al., 2009; Tung et al., 2013). Among the microorganisms interacting with plants, endophytes are defined as those that colonize the internal February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 2 Endophytic Bacteria From Alkanna tinctoria Rat et al. Rat et al. media were compared. Nine culture media supplemented with 50 mg/L cycloheximide were tested, including the commonly used media R2A (Difco), 1/10 TSA (Oxoid), and ISP2 (Oxoid), as well as 1/10 869 medium (Eevers et al., 2015) which is routinely used for the isolation of endophytes. In addition, 1/10 869 medium supplemented with either an infusion of dried Symphytum officinale (family Boraginaceae) roots or with allantoin was tested. To prepare the former medium supplement, dried S. officinale roots (Bioshop, Gent, Belgium) were infused in boiled water for 10 min (28 g/L). The infusion was then filtered and used to resuspend the dry ingredients of medium 1/10 869. Allantoin was added to medium 1/10 869 at four concentrations (0.32, 1.6, 9, 32 mM). Allantoin is a secondary metabolite found in the root of several members of the Boraginaceae family (Eevers et al., 2015; Dresler et al., 2017). In this first isolation campaign, plates were incubated at 28◦C for 4 days after which all distinct colony types were picked for purification. Plates of the highest dilutions were incubated for an additional 10 days at 20◦C to isolate slow growers. Despite the medicinal importance of wild-growing A. tinctoria and the potential bioactive properties of its associated bacteria, no studies have been performed so far on its endophytic bacteria and their possible interactions with the plant. The aims of this work were therefore (i) to explore the diversity of endophytic bacteria associated with A. tinctoria, (ii) to screen the isolates for a number of plant-growth promotion properties and for the production of cell-wall degrading enzymes (iii) to evaluate the effect of the A/S produced by the plant on the associated bacteria, and (iv) to test their effect on plant A/S production in a bioassay. Isolation of Root Endophytic Bacteria From Wild-Growing Alkanna tinctoria Plants Three botanical expeditions to collect wild-growing Alkanna tinctoria were undertaken. The first one was conducted in December 2017, in Northern Greece (Seich Sou area close to the Theater of the Earth, Thessaloniki, Greece), using a special collection permit obtained by the Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter (IPBGR, HAO Demeter) which is issued annually by the Greek Ministry of Environment and Energy. Samples from this expedition were used to optimize the isolation conditions. The wild plants collected were taxonomically identified and they were given the International Plant Exchange Network (IPEN) accession number GR-1-BBGK-18,6081 for their long- term ex situ conservation (including also asexual and in vitro propagation trials) at the premises of IPBGR, HAO Demeter. Two further collections were made in April and May of 2018 in Southern Greece (at the University of Athens, Campus of Zografou, Athens) and Northern Greece (Seich Sou area, Thessaloniki), respectively. Frontiers in Microbiology | www.frontiersin.org Pectinase and Cellulase Activities Depending on the objective of the test, a minimum medium (4 g/L KH2PO4, 6 g/L Na2HPO4, 2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.001 g/L FeSO4, 0.2 g/L MgSO4, 0.001 g/L CaCl2, 0.00001 g/L H3BO3, 0.00001 g/L MnSO4, 0.00007 g/L ZnSO4, 0.00005 g/L CuSO4, 0.00001 g/L MoO3, 15 g/L agar, pH 7) supplemented with 5 g/L of either pectin or carboxymethyl cellulose was used for this assay. Dense bacterial suspensions were made in sterile PBS (OD 0.5–0.6). Then, 10 µL of each bacterial suspension was spotted onto the center of the plate. The test was performed in triplicates and plates were incubated at 28◦C for 5 days. Plates were then checked for the presence of a solubilization halo. The halo was visualized by the addition of 0.01% congo red and 1% hexadecyltrimethylammonium bromide (CTAB) solutions for the cellulase and pectinase tests, respectively. The strain LMG 16323 (Cellulomonas cellasea) was used as a positive control for the cellulase assay whilst strain LMG 2404 (Pectobacterium carotovorum subsp. carotovorum) was used as a positive control for the pectinase test. Depending on the objective of the test, a minimum medium (4 g/L KH2PO4, 6 g/L Na2HPO4, 2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.001 g/L FeSO4, 0.2 g/L MgSO4, 0.001 g/L CaCl2, 0.00001 g/L H3BO3, 0.00001 g/L MnSO4, 0.00007 g/L ZnSO4, 0.00005 g/L CuSO4, 0.00001 g/L MoO3, 15 g/L agar, pH 7) supplemented with 5 g/L of either pectin or carboxymethyl cellulose was used for this assay. Dense bacterial suspensions were made in sterile PBS (OD 0.5–0.6). Then, 10 µL of each bacterial suspension was spotted onto the center of the plate. Plant Growth-Promoting Activity of Endophytic Bacteria To determine the isolation conditions (medium, incubation time and temperature) that would yield the widest bacterial diversity, during the first isolation campaign a wide selection of To test bacteria in vitro for their plant growth promoting activity, four parameters were evaluated: phosphate solubilization, February 2021 | Volume 12 | Article 633488 3 Endophytic Bacteria From Alkanna tinctoria Rat et al. siderophore production, ACC deaminase activity, and indole acetic acid production. with the culture medium free of bacteria as blanks. The test was performed in triplicate. Incubation was at 28◦C for 72 h. The strain R-42086 (Pseudomonas sp.) was used as a positive control (Ghyselinck et al., 2013). 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity The test was performed in triplicates and plates were incubated at 28◦C for 5 days. Plates were then checked for the presence of a solubilization halo. The halo was visualized by the addition of 0.01% congo red and 1% hexadecyltrimethylammonium bromide (CTAB) solutions for the cellulase and pectinase tests, respectively. The strain LMG 16323 (Cellulomonas cellasea) was used as a positive control for the cellulase assay whilst strain LMG 2404 (Pectobacterium carotovorum subsp. carotovorum) was used as a positive control for the pectinase test. DFS medium was prepared for the ACC deaminase test following the protocol from Penrose and Glick (2003). The glucose and the citric acid were filter sterilized and added to the autoclaved medium. To conduct the experiment, the bacteria were first grown in DFS medium + 2 g (NH4)2SO4 for 72 h at 28◦C, 100 rpm. Then, in a 96-well plate, for each strain 15 µL of the liquid culture was added in 135 µL of DFS medium + 3 mM ACC. Each strain was tested in triplicate. The DFS medium without any nitrogen source was used as control. As blanks, the outermost wells of the plate were filled with the culture medium free of bacteria. After 48 h at 28◦C, the OD was read at 590 nm. Then, 50 µL of the culture was re-transferred to fresh DFS + 0.3 M ACC. This procedure was repeated twice to exhaust any residual stock of nitrogen. The strain R-42058 (Pseudomonas sp.) was used as a positive control and the ACC deaminase activity was evaluated by comparison with the blank (Ghyselinck et al., 2013). Phosphate Solubilization NBRIP medium (2.5 g/L Ca3(PO4); 5 g/L MgCl2,6H2O; 0.25 g/L MgSO4,7H2O; 0.2 g/L KCl, 0.1 g/L (NH4)2SO4, and 15 g/L agar) with 10 g/L glucose was used for this test (Nautiyal, 1999). The pH of the medium was adjusted to 7 before autoclaving and small Petri dishes were used (diameter 5.5 cm). Dense bacterial suspensions (OD 0.5–0.6) were made in 1 mL of PBS. Then, 10 µL of each suspension was spotted in the middle of each plate. The test was performed in triplicate. The diameter of clear halos around the bacterial growth was measured after 2, 4, 7, and 10 days of incubation at 28◦C. The strain R-42086 (Pseudomonas sp.) was used as a positive control (Ghyselinck et al., 2013). After incubation, the plates were centrifuged at 20◦C, 3,700 rpm for 5 min. Then, 50 µL of the supernatant was transferred to flat bottom 96-wells plate and 100 µL of Salkowsky’s reagent (2% 0.5 M FeCl3 in 35% HClO4 solution) was added and mixed by pipetting. The plate was then kept in the dark for 30 min before measuring the optical density. A calibration curve was made with 0, 0.5, 1, 2, 5, 10, 15, 20, 25, 30 µg of IAA/mL. Enzymatic Activities Our isolates’ ability to recycle plant polysaccharides such as cellulose, pectin and lignin was tested through phenotypic enzymatic assays. Ligninolytic Activity This assay was conducted in two steps: a first screening on minimum medium (4 g/L KH2PO4, 6 g/L Na2HPO4, 2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.001 g/L FeSO4, 0.2 g/L MgSO4, 0.001 g/L CaCl2, 0.00001 g/L H3BO3, 0.00001 g/L MnSO4, 0.00007 g/L ZnSO4, 0.0000 5 g/L CuSO4, 0.00001 g/L MoO3, 15 g/L agar, pH 7) supplemented with 5 g/L lignin and a second test of selected bacteria on the same minimum medium with 25 mg/L of methylene blue. Siderophore Production To test the production of siderophores, the CAS (chrome azurol S) blue medium was prepared according to Louden et al. (2011). Small Petri dishes were used (diameter 5.5 cm). Dense bacterial suspensions (OD 0.5–0.6) were made in 1 mL of sterile PBS. Then, 10 µL of each suspension was spotted in the middle of each plate. The test was performed in triplicate. The halo diameter was observed and measured after 2, 4, 7, and 10 days of incubation at 28◦C. The strain R-42086 (Pseudomonas sp.) was used as a positive control (Ghyselinck et al., 2013). IAA Production Yeast malt extract medium (YM, peptone 5 g/L, yeast extract 3 g/L, malt extract 3 g/L, pH 6.2) was used to test for the production of indole acetic acid (IAA) in liquid culture (Apine and Jadhav, 2011; Mohite, 2013). Bacteria were grown in R2B medium (Difco) for 72 h at 28◦C, 100 rpm. Then, 1.5 mL of each bacterial culture was centrifuged (20◦C, 5 min, 14,000 rpm), the supernatant was removed, and the pellet was resuspended in sterile distilled water. Then, 100 µL of the suspension were inoculated in 900 µL of YM medium with and without tryptophan (1 g/L). The outermost wells of the plate were filled To screen for ligninolytic activity, dense bacterial suspensions (OD 0.5–0.6) were made in sterile PBS. Then, 10 µL of each bacterial suspension was spotted onto the middle of the plate. The experiment was conducted in triplicates and the plates were incubated at 28◦C for 5 days before checking for bacterial growth. Bacteria that were able to grow on lignin medium were transferred two more times to the same medium to ensure that survival was independent of the previous nutrient stock. After a total of three transfers, strains that retained the ability to grow were selected to check for their ability to solubilize February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 4 Endophytic Bacteria From Alkanna tinctoria Rat et al. methylene blue. Structurally, methylene blue is very similar to lignin and both compounds can be degraded by the same enzymes. When methylene blue is degraded, it loses its color and can therefore be used to identify lignin-degrading bacteria (Bandounas et al., 2011). Dense bacterial suspensions were made in sterile PBS. Then, 10 µL of each bacterial suspension was spotted onto the middle of the plate. The plates were incubated for 1 week at 28◦C and growth was checked daily. Bacteria able to metabolize methylene blue are expected to produce bright halos around their colonies. and transferred weekly (for a period of 3–4 weeks) to a new MS solid medium containing 1% sucrose, 0.8% phytoagar, and 500 mg/L of cefotaxime. The roots were then propagated in liquid MS medium supplemented with 3% sucrose (Brigham et al., 1999; Fukui et al., 1999; Kim et al., 2009). To evaluate the ability of bacteria to induce A/S production in the hairy roots of A. Susceptibility Assay Susceptibility assays were conducted to assess the sensitivity of A. tinctoria endophytic bacteria isolates to the antimicrobial effects of A/S derivatives. The diffusion assay using filter disks described by Bauer et al. (1966) was performed. The concentration tested was 50 µg per filter disk, following Brigham et al. (1999). A mixture of alkannin/shikonin and their derivatives (A/S mixture) was isolated after extraction and further fractionation steps from A. tinctoria roots, as described in Assimopoulou et al. (2008) and Tappeiner et al. (2013). The mixture was first solubilized in acetone to a concentration of 46.7 mg/mL and then diluted in sterile water to a concentration of 2.5 mg/mL. Then, 20 µL of each test solution were used to inoculate the filter disks. In total, six filter disks were used in this assay: three were inoculated with the A/S mixture (50 µg/filter disk), one with acetone-water solution (5.9% acetone; used as an A/S mixture solvent control), one with ampicillin (50 µg/filter disk; as a control antibiotic), and one with MilliQ water (as ampicillin solvent control). Dense bacterial suspensions were prepared in PBS and 100 µL of each suspension was inoculated by spreading on R2A plates. Individual filter disks containing 20 µL of each test solution were left to dry for 30 min before being transferred onto the plates. The test was performed in triplicate. Control strains were selected based on the results reported by Brigham et al. (1999): LMG 8224 (Staphylococcus aureus subsp. aureus) and LMG 7135 (Bacillus subtilis subsp. subtilis) as sensitive strains and LMG 8223 (Escherichia coli) as a resistant strain. p Among seven candidates showing an OD > 0.2 at 520 nm in the first screening, four were subsequently tested in a larger volume. Five 100 mL Erlenmeyer filled with 50 mL of MS + 1% sucrose medium were used per treatment. Pieces of young roots were then carefully added in each Erlenmeyer and incubated for 1 week at 25◦C (100 rpm). The Erlenmeyers were then randomized before being inoculated with bacteria to a final OD of 0.001. One uninoculated control (5 replicates) was also prepared. Erlenmeyers were incubated for 1 week at 25◦C (100 rpm). The roots from each Erlenmeyer were collected and lyophilized. The extraction was conducted overnight using 40 mg of dried roots in 1,250 µL of methanol. Isolation and Identification of Root Endophytic Bacteria For our study nine different populations of endogenous Alkanna tinctoria from Northern and Southern Greece have been explored. The first isolation campaign assessed the media that would allow the growth of a wide diversity of culturable bacteria and yielded 1,483 isolates. MALDI-TOF mass spectra of these isolates were compared and grouped, resulting in 914 representative strains that were selected for genotypic identification by 16S rRNA gene sequencing. This allowed identification of 142 distinct phylotypes of endophytic bacteria following the classification threshold values at genus or species level introduced by Moreira and López-García (2014) and Yarza et al. (2014). The number of isolates and phylotypes recovered for each plant sample and medium is available in Supplementary Table S1. The coverage of bacterial diversity for each medium tested was obtained by dividing the number of unique phylotypes from the selected medium by the total number of phylotypes. The media that yielded the highest diversity were medium 1/10 869 supplemented with 32 mM allantoin and IAA Production tinctoria, a first screening was performed in 6-well plates. The bacteria for this test were selected based either on their plant growth promoting properties or their enzymatic activities. Firstly, 7 mL of MS + 1% sucrose medium were added to each well, followed by the aseptic addition of young root segments. Bacteria were inoculated to a final OD of 0.001/mL. One 6-well plate was prepared per strain. In addition, an uninoculated control plate was prepared. Plates were incubated in the dark at 100 rpm agitation at 25◦C for 1 week. The roots from individual wells were pooled per plate and lyophilized. The extraction of A/S was conducted overnight with 8 mg of dried roots in 250 µL of methanol and the OD was read at 520 nm (Assimopoulou et al., 2009). Susceptibility Assay The OD was read at 520 nm and one- factor Anova followed by a Neuman-Keuls test was conducted on the results with the software R x64 3.1.0. Frontiers in Microbiology | www.frontiersin.org Plant Growth-Promoting Activity Plant Growth Promoting Activity One hundred and twenty-seven strains which represented most of the bacterial diversity recovered, were tested for their plant growth promoting activity in vitro. The results are summarized in Figure 2 (additional information is provided in the Supplementary Table S2). The strains expressing positive activity for all four tested parameters belonged to the genera Pseudomonas and Bacillus. The ones expressing positive activity for at least three of the parameters tested belonged to the genera Pseudomonas, Pantoea, Inquilinus, Rhizobium, and Bacillus. On the other hand, bacteria from the genus Stenotrophomonas seem to only be able to produce siderophores. The partial 16S sequences of these 127 tested strains are available in GenBank database under the accession numbers MW353471–MW353597 (Supplementary Table S3). Induction of A/S Production in Hairy Roots of A. tinctoria Members the genera Pseudomonas were found in eight of the nin TABLE 1 | Coverage of bacterial diversity of different media, where the number o unique phylotypes in the selected medium was divided by the total number of detected phylotypes is given in percentage (%). Medium Coverage of the diversity in 1/10 869 30.3 1/10 869 + 0.32 mM allantoin 32.6 1/10 869 + 1.6 mM allantoin 28,0 1/10 869 + 9 mM allantoin 31.8 1/10 869 + 32 mM allantoin 47.0 1/10 869 + Plant extract 8.3 ISP2 22.7 1/10 TSA 40.2 R2A 35.6 Frontiers in Microbiology | www.frontiersin.org The second and third isolation campaigns using four media only yielded 770 and 582 isolates, respectively. Together with the isolates from the first campaign, this gave 2835 primary bacterial isolates in total. Protein profiles of all these isolates were compared by MALDI-TOF MS and grouped. This allowed the selection of 1,428 representative strains, 914 of which had already been identified by 16S rRNA gene sequencing in the first isolation campaign. The remaining 514 representative strains were also identified by 16S rRNA gene sequencing. In total, among the 1,428 selected representative strains, 197 unique phylotypes were recovered, belonging to 14 genera of Actinobacteria, 6 genera of Bacteroidetes, 11 genera of Firmicutes, and 44 genera of Proteobacteria. Also, three potentially new genera and 40 potentially new species were recovered from the roots of wild A. tinctoria plants. The bacteria were distributed in four phyla, i.e., Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes. The plants of the third isolation campaign yielded relatively more Gram positive bacteria (Actinobacteria and Firmicutes) compared to the other plants. Similarly, the plants of the 1st and 2nd isolation campaigns yielded more Alphaproteobacteria and Gammaproteobacteria, respectively (Supplementary Figure S2). Most of the isolates belonged to the Proteobacteria, particularly the Gammaproteobacteria, Alphaproteobacteria, and Betaproteobacteria. Members of the genera Pseudomonas were found in eight of the nine Enzymatic Activities Enzymatic activities of the 127 strains are presented in Figure 2 (additional information is provided in Supplementary Table S2). The cellulase activity seemed to be predominant in the Rhizobiaceae family, whereas pectinase activity was abundant among the Bacteroidetes phylum. Among the strains tested, some showed two degrading activities: pectinase and ligninolytic activities were detected for R-72191 (Olivibacter sp.), R-72210 (Pseudomonas sp.), R-72249 (Pedobacter sp.), R-72394 (Pantoea sp.), R-72464 (Xanthomonas sp.), R-7264, and R-74277 (Bacillus sp.); cellulase and pectinase activities for the strain R-71830 (Brevibacillus sp.) and cellulase and ligninolytic activities for R-72157 (Rhizorhapis sp.). TABLE 1 | Coverage of bacterial diversity of different media, where the number of unique phylotypes in the selected medium was divided by the total number of detected phylotypes is given in percentage (%). Medium Coverage of the diversity in % 1/10 869 30.3 1/10 869 + 0.32 mM allantoin 32.6 1/10 869 + 1.6 mM allantoin 28,0 1/10 869 + 9 mM allantoin 31.8 1/10 869 + 32 mM allantoin 47.0 1/10 869 + Plant extract 8.3 ISP2 22.7 1/10 TSA 40.2 R2A 35.6 TABLE 1 | Coverage of bacterial diversity of different media, where the number of unique phylotypes in the selected medium was divided by the total number of detected phylotypes is given in percentage (%). Medium Coverage of the diversity in % 1/10 869 30.3 1/10 869 + 0.32 mM allantoin 32.6 1/10 869 + 1.6 mM allantoin 28,0 1/10 869 + 9 mM allantoin 31.8 1/10 869 + 32 mM allantoin 47.0 1/10 869 + Plant extract 8.3 ISP2 22.7 1/10 TSA 40.2 R2A 35.6 Frontiers in Microbiology | www frontiersin org 6 TABLE 1 | Coverage of bacterial diversity of different media, where the number of unique phylotypes in the selected medium was divided by the total number of detected phylotypes is given in percentage (%). Induction of A/S Production in Hairy Roots of A. tinctoria Hairy roots of A. tinctoria were produced with a modified protocol (Brigham et al., 1999; Fukui et al., 1999; Kim et al., 2009) as follows: Agrobacterium rhizogenes strain LMG 149 was grown in 40 mL Nutrient Broth (Difco) medium at 28◦C for 48 h in the dark and at 100 rpm agitation. The leaves of sterile A. tinctoria explants provided by IPBGR, HAO Demeter (Thessaloniki, Greece) were cut and inoculated at different spots with a syringe filled with a bacterial solution of A. rhizogenes (OD = 0.5–0.6). Each inoculated leaf was transferred onto solid Murashige and Skoog (MS, Duchefa) medium supplemented with 3% sucrose and 0.8% phytoagar (Duchefa) and kept for 1 week at 25◦C in the dark until roots appeared. As a control, non-inoculated leaves were cultivated under the same conditions. Developing roots (approximately 1 cm in length), were cut off February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 5 Endophytic Bacteria From Alkanna tinctoria Rat et al. Rat et al. medium 1/10 TSA (Table 1). Moreover, media R2A, 1/10 869 and 1/10 869 supplemented with 0.32 mM allantoin allowed the cultivation of members of additional genera including Acidovorax, Micromonospora, Kocuria, Diaminobutyricimonas, and Muciliginibacter (Supplementary Figure S1). plants studied, whereas Pantoea, Xanthomonas and Bacillus were recovered from seven A. tinctoria plants. Additionally, representatives of Stenotrophomonas were found in six of the nine plants (Figure 1). These genera were also isolated from all the tested media (1/10 869, 1/10 869 + 32 mM allantoin, 1/10 TSA and R2A). The isolates that were less than 94.8% similar to the type strain of any bacterial species represent four potentially new genera which are related to Filimonas, Bordetella, Cohnella and Paenibacillus. Moreover, 40 bacterial strains that were less than 98.7% similar to the type strain of any bacterial species probably represent potential new species. Of these, members of the Bacteroidetes are related to Chitinophaga, Flavobacterium, Mucilaginibacter, and Pedobacter. Potentially new species within the Phylum Firmicutes are related to Bacillus, Cohnella, Domibacillus, Paenibacillus, and Tumebacillus. Potentially new Proteobacteria isolates are related to Acinetobacter, Allorhizobium, Beijerinckia, Bradyrhizobium, Brevundimonas, Janthinobacterium, Massilia, Ochrobactrum, Pseudomonas, Roseomonas, Rhizobium, Rhizorhapis, Shinella, Sphingobium, Sphingomonas, and Stenotrophomonas. Finally, potentially new isolated Actinobacteria members are related to the genera Cellulomas, Conexibacter, Diaminobutyricimonas, Lysinimonas, Mycobacterium, and Nocardioides. medium 1/10 TSA (Table 1). Induction of A/S Production in Hairy Roots of A. tinctoria Moreover, media R2A, 1/10 869 and 1/10 869 supplemented with 0.32 mM allantoin allowed the cultivation of members of additional genera including Acidovorax, Micromonospora, Kocuria, Diaminobutyricimonas, and Muciliginibacter (Supplementary Figure S1). For the isolation from the second and third collection campaigns, the media R2A, 1/10 TSA, 1/10 869 and 1/10 869 + 32 mM allantoin were used to maximize the culturable diversity. Moreover, the first isolation campaign indicated that extended incubation at 20◦C led to a 15.2% increase in diversity at higher dilutions. Most of these isolates were slow-growing Gram- positive bacteria including Mycobacterium sp., Micromonospora sp., Lysinimonas sp., and Micrococcus sp. Then in further isolations, the plates were incubated at 20◦C for 2 weeks and colonies were picked after 1 and 2 week(s), respectively. positive bacteria including Mycobacterium sp., Micromonospo sp., Lysinimonas sp., and Micrococcus sp. Then in furth isolations, the plates were incubated at 20◦C for 2 weeks an colonies were picked after 1 and 2 week(s), respectively. The second and third isolation campaigns using four med only yielded 770 and 582 isolates, respectively. Togeth with the isolates from the first campaign, this gave 283 primary bacterial isolates in total. Protein profiles of a these isolates were compared by MALDI-TOF MS an grouped. This allowed the selection of 1,428 representativ strains, 914 of which had already been identified by 16 rRNA gene sequencing in the first isolation campaign. Th remaining 514 representative strains were also identifie by 16S rRNA gene sequencing. In total, among the 1,42 selected representative strains, 197 unique phylotypes we recovered, belonging to 14 genera of Actinobacteria, 6 gene of Bacteroidetes, 11 genera of Firmicutes, and 44 genera Proteobacteria. Also, three potentially new genera and 4 potentially new species were recovered from the roots wild A. tinctoria plants. The bacteria were distributed four phyla, i.e., Bacteroidetes, Proteobacteria, Actinobacteri and Firmicutes. The plants of the third isolation campaig yielded relatively more Gram positive bacteria (Actinobacter and Firmicutes) compared to the other plants. Similarly, th plants of the 1st and 2nd isolation campaigns yielded mo Alphaproteobacteria and Gammaproteobacteria, respective (Supplementary Figure S2). Most of the isolates belonge to the Proteobacteria, particularly the Gammaproteobacteri Alphaproteobacteria, and Betaproteobacteria. Susceptibility Assay Susceptibility Assay Susceptibility to the antimicrobial activity of alkannin and shikonin derivatives (in the form of a mixture of A/S pigments) February 2021 | Volume 12 | Article 633488 6 Rat et al. Endophytic Bacteria From Alkanna tinctoria was studied for the 127 strains tested for PGP and enzymatic activities. A strain was considered as sensitive to A/S derivatives when an inhibition zone of more than 1 mm was observed. An example of the assay is shown in Figure 3. A pattern could be observed in the susceptibility to the A/S mixture (50 µg): all Gram-positive bacteria tested were sensitive whereas the Gram- negative bacteria tested were resistant (Figure 2). No such pattern was observed for ampicillin which was used as a control. As shown in Figure 3, the halos observed as a result of sensitivity to A/S derivatives were always smaller in diameter than those produced as a result of ampicillin sensitivity. FIGURE 1 | Diversity of genera isolated from wild-growing Alkanna tinctoria plants and the number of plant individuals in which they were found. The number of species found per genus is specified in parentheses. Different colors correspond to specific bacterial phyla or classes. Induction of A/S Derivatives in Hairy Roots of A. tinctoria In the first screening, treatments with the following bacteria resulted in hairy root extracts showing an OD higher than the control: R-71971 (Brevibacillus sp.), R-72060 (Variovorax sp.), R-72149 (Filimonas sp.), R-72379 (Allorhizobium sp.), R-72395 (Duganella sp.), R-72401 (Shinella sp.), R-72406 (Stenotrophomonas sp.), R-73072 (Chitinophaga sp.), R-74161 (Acinetobacter sp.), and R-75348 (Micromonospora sp.) (Table 2). The seven bacteria resulting in an OD > 0.20 were repeated in five replicates in a second assay. The one-factor ANOVA conducted on the results of the second assay showed that the p-value = 0.00179 was lower than 0.05. The bacterial treatment had an influence on the A/S content. The comparison made with the test of Newman Keuls are presented Table 3. Chitinophaga sp. strain R-73072, Allorhizobium sp. strain R-72379, Massilia sp. strain R-72395, and Micromonospora sp. strain R-75348 induced significantly more A/S derivatives in the hairy roots of A. tinctoria when compared to the uninoculated control. DISCUSSION The cultivation of members of Boraginaceae with strong medicinal value such as A. tinctoria and Lithospermum spp. has proven to be difficult. For example, Lithospermum spp. germinate poorly and seedlings are highly susceptible to viral infections. Additionally, plants can only be harvested 2–3 years after germination and the in-field maintenance of these plants has also proved to be difficult (Yazaki, 2017). Applying suitable endophytes to facilitate plant growth by increasing the plant biomass and/or by inducing A/S production can be a sustainable approach toward increasing plant yield and production of these valuable compounds. As a first step toward the elucidation of the potential role of root endophytic bacteria in the production for secondary metabolites such as A/S derivatives of A. tinctoria, we isolated and characterized culturable endophytic bacteria from A. tinctoria roots. In total, 197 unique phylotypes were recovered, representing 4 phyla. Also, three potentially new genera and 40 potentially new species were recovered from the roots of wild A. tinctoria plants. Most studies to date involving the isolation of endophytic bacteria typically use one type of isolation medium to cultivate a wide range of microorganisms (e.g., nutrient agar or TSA), a set temperature of 28◦C and an incubation time between FIGURE 1 | Diversity of genera isolated from wild-growing Alkanna tinctoria plants and the number of plant individuals in which they were found. The number of species found per genus is specified in parentheses. Different colors correspond to specific bacterial phyla or classes. February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 7 Rat et al. Endophytic Bacteria From Alkanna tinctoria FIGURE 2 | Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, with Juke-Cantor model, showing the phylogenetic relationships of strains tested in vitro for plant growth, cell-wall degrading enzymes and susceptibility to the mixture of A/S. Activities detected are shown in the outer circle. Sequences were aligned using 361 CLC Main Workbench 7.9.1 (Qiagen). Subsequently, a phylogenetic maximum-likelihood tree (1000 bootstraps) was reconstructed and visualized using the iTOL software 5.6.3 (Letunic and Bork, 2019). FIGURE 2 | Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, with Juke-Cantor model, showing the phylogenetic relationships of strains tested in vitro for plant growth, cell-wall degrading enzymes and susceptibility to the mixture of A/S. Activities detected are shown in the outer circle. Sequences were aligned using 361 CLC Main Workbench 7.9.1 (Qiagen). DISCUSSION Subsequently, a phylogenetic maximum-likelihood tree (1000 bootstraps) was reconstructed and visualized using the iTOL software 5.6.3 (Letunic and Bork, 2019). 48 and 72 h (Germida et al., 1998; Mbai et al., 2013; Anjum and Chandra, 2015; Saini et al., 2015). In this study, we showed that a higher diversity of bacteria can be obtained when more than one medium is used. Nutrient-poor media such as R2A or diluted TSA seem better at capturing more diversity than a rich medium such as ISP2 (Table 1 and Supplementary Figure S1). The latter is a medium rich in nitrogen and carbon sources and has been designed for the routine cultivation of Streptomyces sp. (Shirling and Gottlieb, 1966). However, as it may favor the fast-growing bacteria, it may be less suitable to isolate slow growing ones. R2A medium was designed for slow-growing bacteria (Reasoner and Geldreich, 1985) and medium 1/10 869 for endophytes (Eevers et al., 2015), whereas TSA medium is non-selective and allows the growth of a wide range of microorganisms (Leavitt et al., 1958). The dilution of TSA increased the growth of slow- growing bacteria when compared to the non-diluted medium. Changes in the quantity and the quality of the carbon source and the nature of electrons donors (e.g., succinate, butyrate, February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 8 Endophytic Bacteria From Alkanna tinctoria Rat et al. FIGURE 3 | Susceptibility of strain R-72492 (Bacillus sp.) to the mixture of alkannin/shikonin derivatives isolated from Alkanna tinctoria roots. parameters such as the genetics of the plant (Haney et al., 2015), local soil composition (Liu et al., 2019), or the presence of neighboring plants (Schlatter et al., 2015; Essarioui et al., 2016) might also influence the diversity of bacteria recovered. Microbiome analysis as well as the study of the growth conditions of the plant in its natural environment might shed light on bacterial variation between samples. The endophytic bacteria found in most of the root samples were tested for a number of plant-growth promotion (PGP) characteristics. The most interesting species for plant growth promoting activity belong to the genera Pseudomonas, Pantoea, Inquilinus, Rhizobium, and Bacillus (Figure 2, Supplementary Table S2). Members of these genera were recovered on all media used for isolation. The ability to grow easily in vitro is promising for their application as plant growth promoting rhizobacteria (PGPRs). DISCUSSION Some bacteria showed variable results were obtained from the ACC deaminase test (Supplementary Table S2). These bacteria were colored and/or were prone to form biofilms or clumps which can lead inaccurate spectrophotometric readings. To overcome this issue, quantitative analysis might be applied by measuring the amount of α-ketobutyrate at 540 nm (Penrose and Glick, 2003). FIGURE 3 | Susceptibility of strain R-72492 (Bacillus sp.) to the mixture of alkannin/shikonin derivatives isolated from Alkanna tinctoria roots. Moreover, several bacteria produced in vitro cell-wall degrading enzymes such as cellulases, pectinases and ligninases. These properties facilitate bacterial entry and spread within the plant tissues and may contribute to the endophytic lifestyle of these bacteria. Strains belonging to Pseudomonas sp., Pantoea sp., Rhizobium sp., and Bacillus sp. expressing PGP and enzymatic activities (Figure 2 and Supplementary Table S2) might thus show stronger impact on plant growth or A/S induction due to their ability to actively colonize the plant. acetate) and acceptors (HEPES solution; Köpke et al., 2005), as well as the addition of supplements like enzymes or antibiotics to isolation media are known to generate higher microbial diversity (Alain and Querellou, 2009). For example, the nitrogen sources of TSA medium are casein and peptones whereas R2A and 869 media contain yeast extract. Moreover, TSA does not have additional carbon sources such as glucose. Poor media limit the risk that fast-growing bacteria eliminate those that grow slower by outcompeting them for nutrients or by negatively affecting the growth conditions (pH or the production of metabolites). Critically, slow-growers or species that grow poorly in vitro may be naturally abundant in planta (Alain and Querellou, 2009; Kram and Finkel, 2015). Lower temperature and longer incubation time were also more suited to recovering gram- positive bacteria such as Actinobacteria. Indeed, studies focusing on Actinobacteria demonstrated that an incubation period of 3– 4 weeks was necessary for their isolation (Araújo et al., 2000; Coombs and Franco, 2003; Misk and Franco, 2011). The resistance assay demonstrated that Gram-negative bacteria are resistant to the antimicrobial properties of A/S derivatives (Figure 2 and Supplementary Table S2). Although the activity of A/S derivatives at 50 µg seemed weak, the experiment confirmed the antimicrobial properties of these naphthoquinones. The antimicrobial activity of A/S has been linked to their ability to form semiquinone radicals by interacting with reactive oxygen species. Frontiers in Microbiology | www.frontiersin.org DISCUSSION 0.115 R-72097 Caulobacter sp. 0.163 R-72501 Inquilinus sp. 0.146 R-72135 Pseudomonas sp. 0.132 R-73072 Chitinophaga sp. 0.200 R-72149 Filimonas sp. 0.180 R-73074 Bradyrhizobium sp. 0.170 R-72160 Rhizobium sp. 0.162 R-73098 Xanthomonas sp. 0.129 R-72191 Olivibacter sp. 0.167 R-73110 Luteibacter sp. 0.124 R-72210 Pseudomonas sp. 0.157 R-74161 Acinetobacter sp. 0.223 R-72249 Pedobacter sp. 0.153 R-74611 Paenarthrobacter sp. 0.162 R-72251 Xanthomonas sp. 0.142 R-75348 Micromonospora sp. 0.231 The values in bold are higher than the OD value of the uninoculated control. 520 nm reflecting the total A/S content of hairy roots extracts of Alkanna tinctoria treated with selected bacteria. The values in bold are higher than the OD value of the uninoculated control. TABLE 3 | Comparisons of the OD 520 nm reflecting the total A/S content of hairy roots extracts of Alkanna tinctoria inoculated with the best bacterial candidates with Neuman Keuls test. Strain Identification OD 520 nm Control 0.075 b R-73072 Chitinophaga sp. 0.120 a R-72379 Allorhizobium sp. 0.113 a R-75348 Micromonospora sp. 0.108 a R-72395 Massilia sp. 0.106 a R-72060 Variovorax sp. 0.097 ab R-74161 Acinetobacter sp. 0.092 ab R-71971 Brevibacillus sp. 0.090 ab Significant differences are highlighted in bold with different letters. individuals and bacteria in the presence of alkannins and shikonins. Moreover, it has been shown that ethylene regulates the colonization of plant tissue by bacteria: the absence of ethylene in a plant or the addition of an ethylene inhibitor leads to a higher degree of colonization. Bacteria that are able to affect the ethylene level are more competent at colonizing plants (Hardoim et al., 2008; Liu et al., 2017). The modulation of plant ethylene levels by bacteria can occur by cleavage of 1-aminocyclopropane- 1-carboxylate (ACC), a precursor of ethylene or by inhibiting ACC synthase and/or β-cystathionase, both being enzymes involved in the ethylene biosynthesis pathway (Hardoim et al., 2008; Liu et al., 2017). To confirm whether bacteria can effectively colonize and positively affect the plant growth and the total A/S content, in planta tests must be performed. Significant differences are highlighted in bold with different letters. p p Chitinophaga sp. strain R-73072, Allorhizobium sp. strain R- 72379, Massilia sp. strain R-72395, and Micromonospora sp. strain R-75348 showed the ability to directly induce A/S production in A. tinctoria hairy root cultures. Hairy roots are genetically modified plant material (Gutierrez-Valdes et al., 2020). DISCUSSION They can generate endogenous superoxide anion radicals, resulting in their cytotoxicity (Papageorgiou et al., 2008, 1999; Ordoudi et al., 2011). The peptidoglycan cell wall of the Gram-positive bacteria is permeable to molecules with molecular weights in the range of 30,000–57,000 Da, and hence allows for the entry of many small antimicrobials which may partially explain their sensitivity (Lambert, 2002). On the other hand, several mechanisms might be involved in the resistance of the Gram-negative bacteria to A/S, especially through redox processes. Chemical and transcriptomic analyses may provide a better understanding of the processes involved. Most of the bacteria having a positive effect in the in vitro plant growth promoting activities tested were Gram-negative and thus resistant to A/S derivatives. This resistance may provide a competitive advantage for colonizing the plant and living in the root tissues. Consequently, the plant may select for plant growth promoting bacteria through the production of A/S derivatives. Alternatively, sensitive bacteria may colonize the plant shortly after germination when the A/S While the isolation conditions are important determining factors for the recovery of bacterial isolates, the diversity observed here between different roots (Figure 1 and Supplementary Figure S2) indicates that other factors are also important. For example, no Gammaproteobacteria such as Pseudomonas sp., Xanthomonas sp., or Pantoea sp. seemed to be present in one of the root samples (Figure 1). Bacterial isolation results can be biased by the origin and storage conditions of the biological sample or the colony picking method, in addition to the isolation conditions, which may have contributed to the variation in phylotype distribution observed between roots and isolation campaigns (Supplementary Figure S2). We studied wild-collected plants from different locations and therefore, February 2021 | Volume 12 | Article 633488 9 Endophytic Bacteria From Alkanna tinctoria Rat et al. TABLE 2 | Optical density at 520 nm reflecting the total A/S content of hairy roots extracts of Alkanna tinctoria treated with selected bacteria. Strain Identification OD 520 nm Strain Identification OD 520 nm Control 0.175 R-72269 Chitinophaga sp. 0.166 R-71838 Pseudomonas sp. 0.124 R-72367 Achromobacter sp. 0.158 R-71842 Pseudomonas sp. 0.167 R-72379 Allorhizobium sp. 0.222 R-71875 Bacillus sp. 0.163 R-72395 Massilia sp. 0.201 R-71922 Bacillus sp. 0.160 R-72401 Shinella sp. 0.192 R-71971. Brevibacillus sp. 0.244 R-72406 Stenotrophomonas sp. 0.189 R-72039 Muciliginibacter sp. 0.149 R-72433 Rhizobium sp. 0.170 R-72060 Variovorax sp. 0.207 R-72495 Variovorax sp. 0.172 R-72074 Pseudomonas sp. 0.138 R-72498 Pantoea sp. Frontiers in Microbiology | www.frontiersin.org DISCUSSION Alkannin and shikonin derivatives show antimicrobial activities and can thus be considered as plant defense compounds. these medicinal plants by increasing the total biomass. As demonstrated in our study, certain endophytes can also induce A/S production in the roots, probably through to the recognition of bacteria by the plant. These findings open-up the perspective of using a combination of endophytes with the potential for plant growth promotion and induction of secondary metabolite production as a sustainable approach toward increasing the production of secondary metabolites in selected medicinal plants. Although R-75348 did not show any cell-wall degrading activities in vitro, several studies have demonstrated that Micromonospora species do have a role in the breakdown of plant cell walls through the production of hydrolytic enzymes (Hirsch and Valdés, 2010; Trujillo et al., 2015). Our test conditions may not have been suitable for this strain. Alternatively, genome analysis might reveal the presence of such enzymes. Moreover, Micromonospora sp. is also known to produce antibiotics (Boumehira et al., 2016). Inducing the production of A/S derivatives may depend on plant-bacteria communication through such metabolites. Indeed, some molecules are described as antibiotics because of their effect on microorganisms under in vitro conditions although their function in the natural habitat can be different. Such molecules can play a role in plant-microbe interactions but can also act as Microbe-Associated Molecular Patterns (MAMPs). MAMPs are resistance-inducing stimuli recognized by specific plant receptors (Köhl et al., 2019). They can also induce pathways involved in plant defense responses. ACKNOWLEDGMENTS We would like to thank our colleague Stephanie Fordeyn for language advise and proofreading of the manuscript. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.633488/full#supplementary-material DISCUSSION The use of MS + 1% sucrose medium, which is a poor medium for bacterial culture, as well as the presence of agitation, might affect the ability of the bacteria to colonize the roots or their abilities to produce biofilm and/or metabolites. During plant colonization, the production of bacterial enzymes or metabolites may induce the production of A/S derivatives by the plant. Our hairy root system served as a general screening tool and may be not optimized for specific plant-bacteria interactions. It might therefore underestimate the number of A/S inducers. Nonetheless, in vitro whole plant systems usually employ growth medium similar to MS (Phillips and Garda, 2019) and therefore bacteria showing inducing properties in hairy roots can likely induce A/S in vitro plant system. The bacterial strains R-73072, R-72379, and R-72395 inducing A/S in our system, expressed cell-wall degrading enzymatic activities (pectinase or ligninase) derivatives content is low or they may be present in the seed and are vertically transmitted (Truyens et al., 2014). In the future, these hypotheses might be tested by inoculating isolates that are sensitive to A/S at different plant growth stages and by tracking their presence and abundance within the plant, thus establishing whether or not these strains can colonize and survive in the plant or not. Studies have shown that in the hairy roots of A. tinctoria, A/S derivatives are produced by root border cells of the growing root tips. They are then sequestered as lipid granules in the phospholipid layer of these cells and accumulate in apoplastic spaces. However, how the A/S derivatives are secreted into the environment still remains unclear. Nevertheless, it is known that this process results from plant stress and involves the actin filaments (Brigham et al., 1999; Tatsumi et al., 2016). Depending on where the bacteria colonize the plant, it is possible that they do not come into contact with A/S derivatives. Colonization assays can provide insights into the relationship between plant February 2021 | Volume 12 | Article 633488 10 Endophytic Bacteria From Alkanna tinctoria Rat et al. in vitro. Competent endophytes are able to release cell wall degrading enzymes such as cellulases, xylanases, pectinases, and endoglucanases, which facilitate bacterial entry and spread within the plant tissues (Kandel et al., 2017; Pinski et al., 2019). Nonetheless, colonization remains an invasive process and thus might induce plant defense response, resulting in the synthesis of plant antimicrobials. DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material. FUNDING We hypothesize that the endophyte’s production of cell-wall degrading enzymes and/or secondary metabolites during plant colonization may cause a plant stress resulting in the production of alkannin and shikonin derivatives. This project (Micrometabolite) has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement no. 721635. This study demonstrated the importance of the isolation conditions for the diversity of endophytic isolates recovered from plant roots of A. tinctoria. Nutrient-poor isolation media coupled with an incubation temperature of 20◦C and an incubation period of a minimum of 2 weeks allowed for a recovery of a high diversity of culturable endophytic bacteria from A. tinctoria. Although these findings should be validated in greenhouse or field conditions, the positive in vitro results found regarding potential plant growth promotion features suggest that some of these bacteria might be valuable for future in planta applications. These can be of agronomical interest to boost biomass production or crop yield. Such endophytes also have the potential to increase the yield of secondary metabolites such as A/S derivatives from AUTHOR CONTRIBUTIONS AW, CS, AAs, and NF designed the study and secured the funding. AR, HN, and AAl designed and performed the experiments. AR analyzed the data and wrote the manuscript. All authors edited, proofread, and approved the manuscript. REFERENCES 12, 53–57. doi: 10.1007/s12892-009- 0075-y Brader, G., Compant, S., Mitter, B., Trognitz, F., and Sessitsch, A. (2014). Metabolic potential of endophytic bacteria. Curr. Opin. Biotechnol. 27, 30–37. doi: 10. 1016/j.copbio.2013.09.012 Brigham, L. A., Michaels, P. J., and Flores, H. E. 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Plant Biotechnol. 34, 131–142. doi: 10.5511/plantbiotechnology.17. 0823a Copyright © 2021 Rat, Naranjo, Krigas, Grigoriadou, Maloupa, Alonso, Schneider, Papageorgiou, Assimopoulou, Tsafantakis, Fokialakis and Willems. Yu, P., and Hochholdinger, F. (2018). The role of host genetic signatures on root–microbe interactions in the rhizosphere and endosphere. Front. Plant Sci. 9:1896. doi: 10.3389/fpls.2018.01896 Frontiers in Microbiology | 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. Yoon, S.-H., Ha, S.-M., Kwon, S., Lim, J., Kim, Y., Seo, H., et al. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613–1617. doi: 10.1099/ijsem.0.001755 Yu, P., and Hochholdinger, F. (2018). The role of host genetic signatures on root–microbe interactions in the rhizosphere and endosphere. Front. Plant Sci. 9:1896. doi: 10.3389/fpls.2018.01896 February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 14
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Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
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Volume 5 Issue 1 (2021) Pages 879-890 Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini ISSN: 2549-8959 (Online) 2356-1327 (Print) Abstrak Penelitian ini bertujuan untuk mengetahui pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara. Jenis penelitian ini adalah eksperimental dengan pendekatan pre and post test without control. Populasi penelitian adalah seluruh anak prasekolah TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara yang berjumlah 16 anak. Teknik pengambilan sampel menggunakan total sampling, metode analisis menggunakan Uji Wilcoxon. Penelitian ini dilaksanakan pada bulan Juli-Agustus 2018. Hasil penelitian menunjukkan bahwa ada pengaruh pemberian bermain VEM terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara. Diharapkan kepada orang tua agar dapat memberikan gambaran tentang cara pemberian motivasi dan edukasi pada anak dengan pendekatan bermain VEM agar anak memiliki perilaku makan sayur yang baik. Kata Kunci : vegetable eating motivation; perilaku makan sayur; anak prasekolah Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah Islaeli Islaeli1, Ari Nofitasari2, Sri Wulandari3 Program Studi Keperawatan, Stikes Mandala Waluya Kendari DOI: 10.31004/obsesi.v5i1.734 py g ( ) , ,  Corresponding author : Islaeli Email Address : islaelimwkdi@gmail.com (Kendari, Sulawesi Tenggara, Indonesia) Received 29 August 2020, Accepted 4 September 2020, Published 8 September 2020 PENDAHULUAN Anak usia dini yaitu anak yang sedang berada dalam pertumbuhan dan perkembangan yang pesat baik itu fisik atau psikis serta anak-anak yang berusia dibawah 6 tahun. Jadi mulai dari anak lahir hingga ia mencapai umur 6 tahun ia akan dikategorikan sebagai anak usia dini. Menurut Piaget dalam Sumantri anak usia dini berada pada masa lima tahun pertama yang disebut The Golden Age. Masa ini merupakan masa emas perkembangan anak (Nurdin & Anhusadar, 2020). Untuk mewadahi perkembangan anak perlu lembaga Pendidikan yang menstimulus perkembangan anak sejak usia dini seperti Lembaga PAUD sehingga perkembangan anak dapat dikembangkan sesuai usia anak. Menurut Musfiroh bahwa pendidikan Anak Usia Dini merupakan pendidikan yang diselenggarakan untuk mengembangkan keterampilan yang merupakan pendidikan dasar serta mengembangkan diri secara utuh sesuai dengan asas pendidikan sedini mungkin dan sepanjang hayat. Aspek yang dikembangkan dalam pendidikan anak usia dini adalah aspek pengembangan pembiasaan meliputi sosial, emosi, kemandirian, moral, dan nilai-nilai agama, serta pengembangan kemampuan dasar yang meliputi pengembangan bahasa, kognitif, dan fisik motoric (Suriati, Kuraedah, Erdiyanti, & Anhusadar, 2019). Salah satu bentuk lembaga pendidikan anak usia dini yang berada pada jalur formal adalah taman kanak-kanak (TK) yang berusia 5-6 tahun. Tujuan pendidikan di TK untuk pencapaian perkembangan yaitu: Nilai agama dan moral, fisik motorik kasar, motorik halus, kesehatan fisik, kognitif yaitu pengetahuan umum dan sains, konsep bentuk warna ukuran dan pola, konsep bilangan lambang bilangan dan huruf, bahasa yaitu menerima bahasa, mengungkapkan bahasa, keaksaraan, sosioal emosional. Kreativitas anak usia dini dapat diupayakan melalui permainan yang dirancang oleh Pendidik di Lembaga PAUD, karena dengan permainan anak dapat mengembangkan serta mengintergrasikan semua potensinya, sehinga mereka lebih kreatif. Peran Pendidik dalam kegiatan permainan anak adalah memberikan dorongan, membimbing bermain bagi anak dan membantu anak mengembangkan potensinya, sehingga mereka menjadi anak yang kreatif (L. O. Anhusadar & Islamiyah, 2020). Usia prasekolah (3-6 tahun) disebut juga masa keemasan (the golden years), yang hanya datang sekali dan tidak dapat diulangi lagi, yang sangat menentukan untuk pengembangan kualitas manusia (Suryana, 2007). Anak prasekolah merupakan masa dimana anak mulai peka dan sensitif untuk menerima berbagai rangsangan. Pada masa inilah anak dengan mudah menerima stimulasi dari lingkungannya sebagai dasar untuk mengembangkan kemampuan kognitif, bahasa, motorik, nilai moral, dan sosial emosional pada anak usia dini (Masganti Sit, 2015). Untuk mengembangkan 5 aspek tersebut tentunya asupan nutrisi (gizi) sangat mendukung dalam pertumbuhan dan perkembangan anak (Sujiono, 2015). Abstract This study aims to determine the effect of playing vegetable eating motivation (VEM) on vegetable eating behavior in preschool children at Asma Jaya Kindergarten, Lasolo District, Konawe Utara Regency. The research is experimental with a pre and post test approach without control. The study population was all preschool children Asma Jaya Kindergarten, Lasolo District, North Konawe Regency, totaling 16 children. The sampling technique used total sampling, the method of analysis used the Wilcoxon test. This research was conducted in July-August 2018. The results showed that there was an effect of playing VEM on vegetable eating behavior in preschool children at Asma Jaya Kindergarten, Konawe Utara Regency. It is hoped that parents can provide an overview of how to provide motivation and education to children with the VEM play approach so that children have good vegetable eating behavior. Keywords: vegetable eating motivation; vegetable eating behavior; preschool children Copyright (c) 2020 Islaeli Islaeli, Ari Nofitasari, Sri Wulandari Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 879 Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 879 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 880 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 PENDAHULUAN MDG’s (Millenium Development Goals) merupakan salah satu tujuan dimana pada tahun 2015 setiap negara harus berupaya terus untuk menurunkan separuh jumlah penduduk miskin dan kelaparan yang juga terdampak pada kesehatan gizi anak-anak. Asupan makanan yang tidak sesuai dengan kebutuhan tubuh akan memicu masalah gizi yang nantinya berdampak pada rentannya tubuh terhadap penyakit dan hal ini sering terjadi di daerah atau negara berkembang. Di Indonesia pencapaian MDG’s dengan indicator indikator paling menentukan untuk memberantas kemiskinan dan kelaparan adalah prevelensi gizi kurang dan gizi buruk. Adanya penurunan untuk prevalensi gizi kurang menurun secara signifikan di tahun 1989 dari 31% menjadi 17,9% di tahun 2010. Demikian pula prevalensi gizi buruk menurun dari 12,8% (1995) menjadi 4,9% (2010). Kecenderungan ini menunjukan target penurunan prevalensi gizi kurang dan gizi buruk menjadi 15% dan 3,5% pada tahun 2015 ini dapat tercapai (Amirullah, Try, Putra, Daud, & Kahar, 2020). Menurut beberapa ahli keperawatan diantaranya Paplau H mengatakan bahwa kesehatan adalah proses yang berlangsung mengarah kepda kretifitas, konstruktif, dan produktif. Dengan demikian bahwa kesehatan adalah suatu hal yang tidak dapat dianggap 880 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 sepele keberadaannya dalam membentuk sumber daya manusia yang unggul. Bidang kesehatan perlu mendapatkan perlakuan dan perhatian khusus guna menunjang kepentingan pembangunan karakter bangsa (Udu, Anhusadar, Alias, & Ali, 2019). Kebutuhan nutrisi merupakan kebutuhan yang sangat penting dalam membantu proses pertumbuhan dan perkembangan anak serta dapat mencegah terjadinya berbagai penyakit akibat kurang nutrisi dalam tubuh. Oleh sebab itu, makanan yang dikonsumsi anak sebaiknya beraneka ragam dan mengandung berbagai vitamin. g g g Dalam pertumbuhan anak diperlukan gizi yang seimbang, supaya seluruh anggota badan dapat tumbuh secara wajar, pertumbuhan otot dan tulang dapat kuat, dan sehat. Beberapa sumber gizi yang sangat berguna tersebut terkandung di dalam sayuran (Sukamti, 1994) (Hermina & S, 2016). Sayuran merupakan sumber vitamin, mineral, dan kaya akan serat yang sangat mudah ditemukan di dalam bahan makanan. Mengkonsumsi makanan berserat sangat baik karena dapat membantu memperlancar proses pencernaan didalam tubuh, mempengaruhi peningkatan ukuran, berat, dan melunakkan feses (Kusharto, 2007). g g Karena pentingnya konsumsi buah dan sayur, organisasi pangan dan pertanian dunia Food and Agriculture Organization (FAO), merekomendasikan warga dunia untuk makan sayur dan buah secara teratur sebanyak 75 kg/kapita/tahun begitupun dengan World Health Organization (WHO) merekomendasikan agar konsumsi sayur dan buah sebanyak 400 gram setiap hari (Indira, 2015). Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 881 PENDAHULUAN Bermain adalah setiap Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 881 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 kegiatan yang dilakukan untuk kesenangan yang ditimbulkan tanpa pertimbangan hasil akhir. Melalui bermain anak akan memiliki berbagai manfaat bagi perkembangan, aspek ini saling menunjang satu sama lain dan tidak dapat dipisahkan. Apabila satu aspek tidak diberikan kesempatan untuk berkembang, maka akan terjadi ketimpangan karena bermain adalah aktivitas yang menyenangkan dan merupakan kebutuhan yang sudah melekat dalam diri anak (L. Anhusadar, 2016). Beberapa penelitian telah dilakukan untuk melihat pengaruh bermain untuk meningkatkan minat makan sayuran bagi anak usia prasekolah, salah satunya dengan bermain storytelling efektif meningkatkan pengetahuan dan sikap dalam mengkonsumsi sayur pada anak usia prasekolah (Ilya Krisnana, Yuni Sufyanti Arief, 2015). Penelitian lain menyimpulkan bahwa Kedua bermain peran mikro tersebut sama-sama memiliki pengaruh dalam kecerdasan interpersonal anak, namun bermain peran mikro bebas memberikan hasil lebih baik dalam kegiatan pembelajaran yang dilakukan dibandingkan bermain peran mikro tepimpin. Dengan demikian, hal ini merupakan bukti empiris bahwa kecerdasan interpersonal anak yang diberikan bermain peran mikro bebas lebih tinggi dibandingkan anak yang diberikan bermain peran mikro terpimpin. Maka dapat direkomendasikan bahwa bermain peran mikro bebas lebih cocok diterapkan dalam Kecerdasan interpersonal (Damayanti, Ch, & Hapidin, 2018). Penelitian yang dilakukan oleh (Wahyuni, Syukri, & Halida, 2017) yang berjudul “Peningkatan motivasi anak makan sayuran melalui metode bermain permainan cooking class pada anak usia 4-6 tahun” menemukan bahwa, melalui metode bermain permainan cooking class dapat meningkatkan motivasi anak makan sayuran pada anak usia 4-6 tahun di RA AL- Muhajirin Rasau Jaya, Kabupaten Kubu Raya, Provinsi Kalimantan Barat. Vegetable Eating Motivation (VEM) merupakan salah satu bentuk permainan yang dirancang oleh peneliti dengan tujuan memberikan motivasi dan edukasi kepada anak prasekolah dengan pendekatan bermain yang dilakukan dalam bentuk rangkaian permainan dan dilakukan selama 6 hari, dengan tujuan untuk meningkatkan perilaku makan sayuran pada anak prasekolah. Berdasarkan hasil observasi awal kepada 16 orang anak prasekolah di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara, 100 % Ibu selalu menghidangkan menu sayur setiap hari. Akan tetapi, 11 Anak diantaranya menolak untuk memakan sayur, Anak lebih memilih nasi dan lauk saja seperti telur, ikan dan ayam. PENDAHULUAN Data WHO menunjukkan bahwa kasus anak usia prasekolah underweight di dunia sebesar 15,7% dan anak usia prasekolah overweight sebanyak 6,6% (WHO, 2013). Secara nasional, prevalensi berat-kurang pada tahun 2013 adalah 19,6%, terdiri dari 5,7% gizi buruk dan 13,9% gizi kurang (Balitbang Kemenkes, 2013). Berdasarkan data Riset Kesehatan Dasar (Riskesdas) Tahun 2013 menyatakan bahwa tingkat konsumsi sayur pada anak prasekolah di Indonesia 35 kilogram per kapita per tahun. Angka itu jauh lebih rendah dengan angka konsumsi sayuran yang dianjurkan 75 kilogram per kapita per tahun (Khalimatus Sa’diya, 2016). ( y ) Menurut Soetjiningsih kebutuhan gizi, memberikan makanan yang benar pada anak harus dilihat dari banyak aspek, seperti ekonomi, sosial, budaya, agama, disamping aspek medik dari anak itu sendiri. Makanan pada anak usia dini harus serasi, selaras dan seimbang. Serasi artinya sesuai dengan tingkat tumbuh kembang anak. Selaras adalah sesuai dengan kondisi ekonomi, sosial budaya serta agama dari keluarga. Sedangkan seimbang artinya nilai gizinya harus sesuai dengan kebutuhan berdasarkan usia dan jenis bahan makanan seperti karbohidrat, protein dan lemak (Laode Anhusadar, 2020). p ( ) Salah satu penyebab rendahnya konsumsi sayur pada anak karena kurangnya pengetahuan dan sikap mengabaikan pentingnya makan sayur (Indira, 2015). Tidak efektifnya pendidikan gizi pada anak semenjak usia dini berdampak pada pengetahuan yang kurang tentang pola konsumsi makanan yang sehat dan seimbang saat dewasa, sehingga menyebabkan perilaku yang salah (Kemenkes, 2014). Masalah tersebut dapat berakibat buruk bagi tumbuh kembang anak. Anak dapat mempunyai peluang besar untuk menderita kurang gizi karena makanan yang dikonsumsi dalam jumlah sedikit sehingga tidak memenuhi kebutuhan nutrisinya (Arifin, 2016). Selain itu, anak dapat mengalami stunting atau menjadi balita pendek (KemenkesRI, 2016). Berdasarkan Riset Kesehatan Dasar tahun 2013, Indonesia dengan prevalensi anak balita pendek sebesar 35,6% merupakan negara ke-5 terbesar yang berkontribusi pada 90% stunting di dunia. Anak pendek mempunyai risiko lebih tinggi menderita diabetes, obesitas, hipertensi dan stroke pada usia dewasa (Kemenkes, 2014). Selain itu, Konstipasi juga menjadi salah satu penyakit yang akan dialami bila anak kurang mengkonsumsi sayur. Bermain secara tidak langsung akan membuat anak mengembangkan kemampuan kognitif, fisik, motorik, sosial, dan emosionalnya. Oleh karena itu memberikan stimulus dengan pendekatan permainan kepada anak usia prasekolah merupakan hal yang sangat penting yang harus dilakukan (Hasanah, 2016). Dalam Pendidikan Anak Usia dini ( PAUD ),bermain sambil belajar merupakan kegiatan yang baik untuk anak. PENDAHULUAN Tahap kerja - Memberi petunjuk pada anak cara bermain - Memperkenalkan jenis-jenis sayuran - Memberi tahu manfaat sayuran dan dampak tidak mengkonsumsi sayuran - Meminta anak untuk Memilih gambar sayuran yang ingin diwarnai - Kemudian anak di anjurkan untuk mewarnai gambar dengan warna yang disukai - Setelah selesai mewarnai gambar, Anak dibantu untuk melubangi bagian atas kertas gambar - Dipasang benang sepanjang 10 cm atau lebih pada bagian atas yang dilubangi - Memberitahukan kepada orang tua untuk mengantung gambar anak dirumah - Melakukan game tentang sayuran - Anak diminta menebak nama-nama sayuran yang mereka gambar - Dan menyebutkan manfaat serta dampaknya - Memberi pujian pada anak bila dapat melakukan - Menanyakan perasaan anak setelah bermain dan menanyakan pendapat guru dan orang tua tentang permainan. Tahap terminasi 1. Berpamitan dengan guru, orang tua, dan anak 2. Membereskan kembali alat yang telah dipakai 3. Mencuci tangan. METODOLOGI Jenis penelitian yang digunakan adalah penelitian kuantitatif dengan desain penelitia eksperimental dengan pendekatan pre and post test without control. Efektifitas perlakuan dinila dengan cara membandingkan nilai pre test dengan post test. Sampel pada penelitian in diobservasi terlebih dahulu sebelum diberi perlakuan, kemudian setelah diberi perlakua Tabel Tahapan pelaksanaan bermain Vegetable Eating Motivation (VEM) - Dipasang benang sepanjang 10 cm atau lebih pada bagian atas yang dilubangi PENDAHULUAN Anak yang menolak makan sayuran mengatakan tidak menyukai sayuran karena rasanya yang pahit, hambar, teksturnya lama dikunyah, Peneliti juga melakukan wawancara kepada 10 orang ibu dari anak prasekolah di TK Asma Jaya 8 orang ibu mengatakan anaknya sulit jika disuruh makan sayur, 2 orang ibu mengatakan anaknya mau makan sayuran. Melihat perilaku anak prasekolah yang jarang sekali suka dengan sayuran dan hari-hari mereka dipenuhi dengan bermain, maka dibutuhkanlah sebuah terapi dengan pendekatan bermain yang dapat memotivasi dan meningkatkan konsumsi sayuran pada anak prasekolah. Berdasarkan uraian diatas, peneliti tertarik untuk mengadakan penelitian tentang pengaruh bermain vegetable eating motivation (VEM) terhadap perilaku makan sayuran pada anak prasekolah di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara. p Vegetable Eating Motivation (VEM) merupakan salah satu bentuk terapi bermain yang dirancang oleh peneliti dengan tujuan memberikan motivasi dan edukasi kepada anak prasekolah dengan pendekatan bermain yang dilakukan dalam bentuk rangkaian permainan dan dilakukan selama 6 hari, dengan tujuan untuk meningkatkan perilaku makan sayuran pada anak prasekolah. Vegetable Eating Motivation (VEM) merupakan salah satu bentuk terapi bermain yang bertujuan memberikan motivasi dan edukasi kepada anak prasekolah dengan pendekatan bermain untuk meningkatan perilaku makan sayuran pada anak prasekolah. Tahapan pelaksanaan bermain Vegetable Eating Motivation (VEM) peneliti lakukan sebagai berikut: 882 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 DOI: 10.31004/obsesi.v5i1.73 Tabel Tahapan pelaksanaan bermain Vegetable Eating Motivation (VEM) Tujuan Bermain Vegetable Eating Motivation 1. Meningkatkan citra diri sebagai layaknya manusia 2. Dapat bersosialisasi dan berkomunikasi dengan teman sebaya 3. Untuk mengetahui lebih banyak tentang sayuran 4. Untuk mengetahui manfaat tentang sayuran 5. Dapat mewarnai gambar yang disukainya 6. Gerakan motorik halusnya lebih terarah Persiapan 1. Guru, orang tua, dan Anak diberitahu tujuan bermain 2. Melakukan kontrak waktu 3. Anak tidak rewel Peralatan 1. Alat mengambar (Pensil, Warna) 2. Sketsa gambar sayuran Prosedur pelaksanaan 1. Tahap Prainteraksi - Melakukan kontrak waktu - Mengecek kesiapan anak (tidak ngantuk, tidak rewel, keadaan umum membaik/kondisi yang memungkinkan) - Menyiapkan alat 2. Tahap orientasi - Memberikan salam kepada guru, orang tua, dan anak - Menjelaskan tujuan dan prosedur pelaksanaan - Menanyakan persetujuan dan kesiapan 3. Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 883 884 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 METODOLOGI Jenis penelitian yang digunakan adalah penelitian kuantitatif dengan desain penelitian eksperimental dengan pendekatan pre and post test without control. Efektifitas perlakuan dinilai dengan cara membandingkan nilai pre test dengan post test. Sampel pada penelitian ini diobservasi terlebih dahulu sebelum diberi perlakuan, kemudian setelah diberi perlakuan sampel tersebut diobservasi kembali (Notoatmodjo, 2012). Perilaku makan sayur anak prasekolah diukur dengan menggunakan FFQ (Food Frequncy Questionnaire) yang dilakukan Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 883 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 sebelum dan sesudah intervensi bermain vegetable eating motivation dan pemberian sketsa gambar sayuran. Kegiatan bermain dilakukan selama enam hari dan dilakukan pengukuran. Populasi dalam penelitian ini adalah seluruh anak prasekolah TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara yang berjumlah 16 anak dan dengan teknik total sampling. Teknis pengumpulan data adalah dokumentasi, wawancara dan observasi. Prilaku makan sayur anak prasekolah diukur sebelum dan sesudah dilakukan metode bermain vegetable eating motivation (vem) dengan metode ceramah dan dan pemberian sketsa gambar sayuran. Dengan rancangan penelitian sebagai berikut. Pre Test Perlakuan Post Test Keterangan : P1a : Perilaku makan sayur sebelum pemberian terapi vegetable eating motivation (pre test) Xa : Perlakuan (Terapi bermain vegetable eating motivation) P2a : Perilaku makan sayur sesudah pemberian terapi vegetable eating motivation (post test) Penelitian ini digambarkan dalam bentuk bagan berikut. Variabel Independen Variabel Dependen Gambar 1. Bagan Kerangka Konsep Penelitian Keterangan : Perilaku Makan Sayur : Variabel dependen Bermain vegetable eating motivation : Variabel independent HASIL DAN PEMBAHASAN Bermain vegetable eating motivation Perilaku makan sayur P1a P2a Xa Pre Test Perlakuan Post Test Keterangan : P1a : Perilaku makan sayur sebelum pemberian terapi vegetable eating motivation (pre test) Xa : Perlakuan (Terapi bermain vegetable eating motivation) P2a : Perilaku makan sayur sesudah pemberian terapi vegetable eating motivation (post test) P1a P2a Xa Keterangan : P1a : Perilaku makan sayur sebelum pemberian terapi vegetable eating motivation (pre test) Xa : Perlakuan (Terapi bermain vegetable eating motivation) P2a : Perilaku makan sayur sesudah pemberian terapi vegetable eating motivation (post test) Penelitian ini digambarkan dalam bentuk bagan berikut. Penelitian ini digambarkan dalam bentuk bagan berikut. Variabel Independen Variabel Independen Variabel Dependen Variabel Dependen Bermain vegetable eating motivation Perilaku makan sayur Gambar 1. Bagan Kerangka Konsep Penelitian ( , %) Distribusi responden menurut jenis kelamin, terlihat pada tabel berikut: Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 885 HASIL DAN PEMBAHASAN HASIL DAN PEMBAHASAN Karakteristik responden meliputi umur dan jenis kelamin responden. Setiap karakteristik responden, distribusi responden menurut umur terlihat pada tabel berikut : Tabel 1. Distribusi Kelompok Umur Responden Anak Pra Sekolah di TK Asma Jaya Kabupaten Konawe Utara No Umur N % 1 4 tahun 6 37,5 2 5 tahun 7 43,8 3 6 tahun 3 18,8 Jumlah 16 100 Sumber : Data Primer, 2018 1. Distribusi Kelompok Umur Responden Anak Pra Sekolah di TK Asma Jaya Kabupaten Konawe Utara Tabel 1. Distribusi Kelompok Umur Responden Anak Pra Sekolah di TK Asma Jaya Kabu Konawe Utara Sumber : Data Primer, 2018 Tabel 1 menunjukkan bahwa dari 16 responden, terbanyak adalah umur 5 tahun sebanyak 7 responden (43,8%) dan yang terkecil adalah umur 6 tahun yaitu sebanyak 3 responden (18,8%). p ( , ) Distribusi responden menurut jenis kelamin, terlihat pada tabel berikut: 884 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Tabel 2. Distribusi Kelompok Jenis Kelamin Responden Anak Pra Sekolah di TK Asma Jaya Kabupaten Konawe Utara No Jenis Kelamin N % 1 Laki-Laki 7 43,8 2 Perempuan 9 56,2 Jumlah 16 100 Sumber : Data Primer, 2018 l 2. Distribusi Kelompok Jenis Kelamin Responden Anak Pra Sekolah di TK Asma Jaya Kabupaten Konawe Utara Tabel 2 menunjukkan bahwa dari 16 responden terdapat 7 responden (43,8%) yang berjenis kelamin laki-laki dan terdapat 9 responden (56,2%) yang berjenis kelamin perempuan. Distribusi responden berdasarkan perilaku makan sayur pre test dapat terlihat pada Tabel 2 menunjukkan bahwa dari 16 responden terdapat 7 responden (43,8%) yang berjenis kelamin laki-laki dan terdapat 9 responden (56,2%) yang berjenis kelamin perempuan. Tabel 2 menunjukkan bahwa dari 16 responden terdapat 7 responden (43,8%) yang berjenis kelamin laki-laki dan terdapat 9 responden (56,2%) yang berjenis kelamin perempuan. Distribusi responden berdasarkan perilaku makan sayur pre test dapat terlihat pada tabel berikut : Distribusi responden berdasarkan perilaku makan sayur pre test dapat terlihat pada tabel berikut : Tabel 3. Distribusi Responden Perilaku Makan Sayur Pre Test di TK Asma Jaya Kabupaten Konawe Utara No Perilaku Makan Sayur Pre Test N % 1 Cukup 2 12,5 2 Kurang 14 87,5 Jumlah 16 100 Sumber : Data Primer, 2018 abel 3. Distribusi Responden Perilaku Makan Sayur Pre Test di TK Asma Jaya Kabupaten Konawe Utara Tabel 3. HASIL DAN PEMBAHASAN Distribusi Responden Perilaku Makan Sayur Pre Test di TK Asma Jaya Kabupa Konawe Utara Sumber : Data Primer, 2018 Tabel 3 menunjukkan bahwa sebelum diberikan terapi bermain Vegetable Eating Motivation (VEM) dari 16 responden, terdapat 2 responden (12,5%) yang memiliki perilaku makan sayur cukup dan terdapat 14 responden (87,5%) yang memiliki perilaku makan sayur kurang. Sedangkan distribusi responden berdasarkan perilaku makan sayur post test dapat terlihat pada tabel berikut : Tabel 4. Distribusi Responden Perilaku Makan Sayur Post Test di TK Asma Jaya Kabupaten Konawe Utara No Perilaku Makan Sayur Post Test N % 1 Cukup 15 93,8 2 Kurang 1 6,3 Jumlah 16 100 Sumber : Data Primer, 2018 abel 4. Distribusi Responden Perilaku Makan Sayur Post Test di TK Asma Jaya Kabupaten Konawe Utara Sumber : Data Primer, 2018 Tabel 4 menunjukkan bahwa setelah diberikan terapi bermain Vegetable Eating Motivation (VEM) dari 16 responden, terdapat 15 responden (93,8%) yang memiliki perilaku makan sayur cukup dan terdapat 1 responden (6,3%) yang memiliki perilaku makan sayur kurang. Pada uji normatitas data dalam penelitian ini adalah sesuai tebel berikut ini: Tabel 5. Uji normalitas data dengan menggunakan Shapiro-Wilk Tests of Normality Kolmogorov-Smirnova Shapiro-Wilk Statistic df Sig. Statistic Df Sig. Pre_test .278 16 .002 .871 16 .028 Post_test .204 16 .075 .906 16 .100 Tabel 5. Uji normalitas data dengan menggunakan Shapiro-Wilk Tabel 5 menunjukkan bahwa hasil uji normalitas data menggunakan uji Shapiro-Wilk pada variabel perilaku makan sayur pre test menunjukkan bahwa nilai p value (0,028) < α (0,05), dan variabel perilaku makan sayur post test menunjukkan bahwa nilai p value (0,100) Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 885 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 > α (0,05) sehingga dapat disimpulkan bahwa data berdistribusi tidak normal. Karena data berdistribusi tidak normal maka menggunakan uji Wilcoxon. > α (0,05) sehingga dapat disimpulkan bahwa data berdistribusi tidak normal. Karena data berdistribusi tidak normal maka menggunakan uji Wilcoxon. Test Statisticsa post_test - pre_test Z -3.455b Asymp. Sig. (2-tailed) .001 Berdasarkan hasil analisis uji Wilcoxon diperoleh nilai p value = 0,001 < 0,05 maka Ho ditolak dan Ha diterima berati ada pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara. Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Sayuran memiliki banyak manfaat yang baik bagi kesehatan. Mengonsumsi sayuran dan buah sangat bermanfaat untuk memenuhi kebutuhan gizi (Aswatini, Noveria, & Fitranita, 2008). Hasil penelitian menunjukkan bahwa sebelum diberikan terapi bermain Vegetable Eating Motivation (VEM) dari 16 responden, terdapat 2 responden (12,5%) yang memiliki perilaku makan sayur cukup dan terdapat 14 responden (87,5%) yang memiliki perilaku makan sayur kurang. Hal ini menunjukkan bahwa banyak anak yang kurang mengkonsumsi sayur. Hal ini sesuai dengan teori bahwa pada masa usia prasekolah anak mengalami penurunan nafsu makan dan hanya mau makan makanan yang disukai (Siti Aizah, 2003). Siswa yang kurang mengkonsumsi sayur dikarenakan beberapa faktor seperti anak tidak suka dengan rasa sayur yang kurang enak, tidak suka dengan aroma sayur, serta model penyajian sayur yang kurang disukai oleh anak. Selain itu masih banyak juga anak yang kurang mengetahui tentang manfaat mengkonsumsi sayuran. Hal ini sesuai dengan teori bahwa pada masa usia prasekolah anak mengalami penurunan nafsu makan dan hanya mau makan makanan yang disukai. Orang tua harus berusaha keras memaksa anak untuk mau makan sayuran sehingga membuat suasana makan tidak nyaman dan anak menjadi rewel. Sikap ini merupakan suatu kasus global dan merupakan suatu kesulitan bagi orang tua untuk memberi anak mereka makanan yang mengandung serat dan banyak vitamin yaitu sayur mayur (Asy’ariyah, Arief, & Krisnana, 2014). Selanjutnya setelah diberikan terapi bermain Vegetable Eating Motivation (VEM) dari 16 responden, terdapat 15 responden (93,8%) yang memiliki perilaku makan sayur cukup dan terdapat 1 responden (6,3%) yang memiliki perilaku makan sayur kurang. Hal ini menunjukkan bahwa terjadi peningkatan jumlah anak yang signifikan dalam hal perilaku mengkonsumsi sayur setelah diberikan terapi bermain VEM oleh peneliti. Hal ini dapat disebabkan oleh pemberian terapi VEM yang disukai dan diminati oleh anak prasekolah sehingga banyak siswa yang antusias dalam mengikuti kegiatan yang diadakan oleh peneliti. Anak yang antusias dikarenakan belum pernah mendapatkan kegiatan mewarnai gambar sayuran sebelumnya, serta model gambar yang menarik sehingga anak suka dengan kegiatan menggambar sayuran. Peneliti juga menjelaskan kepada siswa tentang manfaat mengkonsumsi setiap sayuran dan akibat tidak mengkonsumsi jenis sayuran yang digambar oleh siswa. Sayur dan buah merupakan pangan yang mudah diperoleh di Indonesia, dan sangat bermanfaat bagi tubuh, karena kaya akan zat gizi, seperti vitamin, mineral, dan serat untuk mengatur proses pencernaan dalam tubuh. Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 887 HASIL DAN PEMBAHASAN Uji normalitas data dilakukan untuk mengetahui distribusi data, apakah data tersebut berdistribusi normal atau tidak yang akan menentukan jenis uji yang akan digunakan dalam melakukan analisis. Untuk lebih jelasnya dapat disajikan pada tabel uji normalitas di bawah ini. Tabel 6 : Hasil Uji Normalitas Perilaku Makan Sayur Sebelum dan Sesudah Diberikan Terapi Bermain Vegetable Eating Motivation (VEM) Variabel Penelitian P value α Perilaku makan sayur pre test 0,168 0,05 Perilaku makan sayur post test 0,520 0,05 Data primer diolah 2018 bel 6 : Hasil Uji Normalitas Perilaku Makan Sayur Sebelum dan Sesudah Diberikan Terapi Bermain Vegetable Eating Motivation (VEM) Tabel 6 menunjukkan bahwa hasil uji normalitas data menggunakan uji Kolmogorov- Smirnov pada variabel perilaku makan sayur pre test menunjukkan bahwa nilai p value (0,168) > α (0,05), dan variabel perilaku makan sayur post test menunjukkan bahwa nilai p value (0,520) > α (0,05) sehingga dapat disimpulkan bahwa data berdistribusi normal. Karena data berdistribusi normal maka memenuhi syarat untuk dilakukan uji Paired T Test. Pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara. Distribusi pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara, yaitu: Tabel 7. Analisis pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara Perilaku makan sayur n Mean SD t value P value Pre Test 16 1,69 0,793 8,777 0,000 Post Tes 16 4,56 1,209 Sumber : Data Primer, 2018 Tabel 7. Analisis pengaruh pemberian bermain vegetable eating motivation (VEM) terhada perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara Tabel 8 menunjukkan bahwa perilaku makan sayur pada pre tes memiliki nilai mean 1,69 dengan nilai SD 0,793. Sedangkan pada post tes memiliki nilai mean 4,56 dengan nilai SD 1,209. Berdasarkan hasil analisis uji paired t-test diperoleh nilai t hit = 8,777 dengan taraf kepercayaan 95% (0,05) df = 15 diperoleh t tabel (1,753). Jadi t hit > t tabel dan p value = 0,000 < 0,05 maka Ho ditolak dan Ha diterima berati ada pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara. 886 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Hasil penelitian juga menunjukkan masih terdapat 1 responden (6,3%) yang tetap kurang mengkonsumsi sayuran setelah diberikan terapi VEM. Hal ini bisa disebabkan oleh beberapa hal seperti anak yang kurang serius dan antusias mengikuti kegiatan terapi bermain VEM. Anak juga kurang mendengar penjelasan yang disampaikan oleh peneliti sehingga anak tetap kurang suka mengkonsumsi sayuran. Berdasarkan hasil analisis uji paired t-test diperoleh nilai t hit = 8,777 dengan taraf kepercayaan 95% (0,05) df = 15 diperoleh t tabel (1,753). Jadi t hit > t tabel dan p value = 0,000 < 0,05 maka Ho ditolak dan Ha diterima berati ada pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara. y p p y p Hasil penelitian ini sejalan dengan penelitian menyimpulkan bahwa Hasil uji statistik didapatkan nilai rata-rata konsumsi sayur sebelum diberikan intervensi yaitu 38,22 dengan standar deviasi (SD) 18,236 dan setelah diberikan intervensi yaitu 68,11 dengan standar deviasi (SD) 18.304. Hasil analisa diperoleh p value sebesar 0,000 < α (0,05), maka dapat disimpulkan terjadi peningkatan konsumsi sayuran pada anak prasekolah setelah diberikan terapi bermain Vegetable Eating Motivation (VEM) (Noviyanti emulyani, 2017). Penelitian ini juga sejalan dengan penelitian (Burhannudin Ichsan , Bayu Hendro Wibowo, 2015) bahwa hasil penelitian dengan mengggunakan uji t (deoendent t test) dengan pendekatan pre test dan post tes diperoleh nilai p-value = 0,000 (< 0,05). Simpulannya yaitu terdapat peningkatan pengetahuan tentang pentingnya sayuran bagi anak-anak yang bermakna setelah mengikuti sesi penyuluhan. SIMPULAN Perilaku makan sayuran anak pra sekolah sebelum bermain Vegetable Eating Motivation (VEM) Di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara sebagian besar yang memiliki nafsu makan kurang. Perilaku makan sayuran anak pra sekolah sesudah bermain Vegetable Eating Motivation (VEM) Di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara sebagian besar nafsu makan cukup meningkat dibandingkan sebelum bermain Vegetable Eating Motivation. Ada pengaruh bermain Vegetable Eating Motivation (VEM) Terhadap Perilaku Makan Sayuran Pada Anak Prasekolah Di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara p<α (ρ=0,000<α=0,05). 888 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 UCAPAN TERIMA KASIH Terima kasih penulis ucapkan kepada para pendidik TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara yang telah meluangkan waktunya untuk mengisi instrument penelitian ini dan semua pihak yang telah membantu pelaksanaan penelitian dan penulisan artikel ini. Tidak lupa ucapan terima kasih kepada editor dan reviewer Jurnal Obsesi yang sudah memberikan kesempatan sehingga jurnal bisa untuk diterbitkan. Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah DOI: 10.31004/obsesi.v5i1.734 Di Indonesia menyarankan untuk mengkonsumsi sayur sebanyak 3-5 porsi dan buah-buahan 2-3 porsi dalam sehari. WHO menyarankan konsumsi sayur dan buah-buahan sebanyak 400 gram setiap hari (Rahmad & Almunadia, 2017). ) Menurut peneliti bahwa adanya peningkatan jumlah anak prasekolah setelah diberikan terapi bermain VEM disebabkan oleh beberapa faktor yaitu terapi yang diberikan secara berkelompok sehingga penerimaan informasi lebih jelas dan mudah dipahami oleh anak. Pemberian terapi VEM juga dipraktekkan langsung oleh siswa sehingga meningkatkan minat anak dalam mengkonsumsi sayuran. Kegiatan bermain VEM ini memiliki manfaat yang besar dalam menunjang perkembangan kemandirian konsumsi anak. Karena dengan bermain Vegetable eating motivation (VEM) ini menyediakan waktu dan ruang bagi anak untuk belajar memahami mamfaat mengkonsumsi sayuran. Hasil penelitian lain menyimpulkan bahwa Identifikasi hasil dari konsumsi sayur sebelum diberikan pendidikan Kesehatan dengan metode storytelling di SDN Mulyoagung 04 Dau Malang memiliki nilai rata-rata (mean) 90,21 gram perhari dengan persentase 33,3% cukup (>150 gram perhari) dan 66,7% kurang (<150 gram perhari). Identifikasi hasil dari konsumsi sayur sesudah diberikan pendidikan Kesehatan dengan metode storytelling di SDN Mulyoagung 04 Dau Malang memiliki nilai rata-rata (mean) 128,71 gram perhari dengan persentase 70,8% cukup (>150 gram perhari) dan 29,2% kurang (<150 gram perhari). Ada perbedaan konsumsi sayur sebelum dan sesudah pendidikan Kesehatan dengan metode storytelling pada anak sekolah dasar di SDN Mulyoagung 04 Dau Malang (Suryandi, Hariyanto, & Metrikayanto, 2018). Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 887 DAFTAR PUSTAKA Amirullah, A., Try, A., Putra, A., Daud, A., & Kahar, A. (2020). 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Peningkatan motivasi anak makan sayuran melalui metode bermain permainan cooking class pada anak usia 5-6 tahun, 1–11. 890 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021
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Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile
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Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile Yiting Zhang Xiamen University Xiaowen Cui Xiamen University Shuizhi Lin Xiamen University Tao Lu Xiamen University Hao Li Xiamen University Yinghua Lu Xiamen University Mingfeng Cao Xiamen University Xihuang Lin Third Institute of Oceanography, Ministry of Natural Resources Xueping Ling (  xpling@xmu.edu.cn ) Xiamen University Research Article Keywords: Schizochytrium, phospholipase D, docosahexaenoic acid, phospholipomics, acyl CoAindependent pathway Posted Date: September 28th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3376770/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/28 Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Biotechnology for Biofuels and Bioproducts on January 30th, 2024. See the published version at https://doi.org/10.1186/s13068-024-02465-w. Page 2/28 Abstract Background: The hydrolysis and transphosphatidylation of phospholipase D (PLD) play important roles in the interconversion of phospholipids (PLs), which has been shown to profoundly impact lipid metabolism in plants. In this study, the effect of the PLD1gene of Schizochytrium limacinum SR21 on lipid metabolismwas investigated. Results: PLD1knockout had little impact on cell growth and lipid production, but it significantly improved the percentage of polyunsaturated fatty acids in lipids, of which docosahexaenoic acid (DHA) content increased by 13.3% compared to the wild-type strain. Phospholipomics and qRT-PCR analysis revealed the knockout of PLD1reduced the interexchange and increasedde novo synthesis of PLs, which altered the composition of PLs, accompanied by a decrease in phosphatidylcholine (PC) and an increase in phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid levels. PLD1 knockout also increased DHA content in triglycerides (TAGs) and decreased it in PLs. Conclusions: These results indicate that PLD1 mainly performs the transphosphatidylation activity in Schizochytrium, and its knockout promotes the migration of DHA from PLs to TAGs, which is conducive to DHA accumulation and storage in TAGsvia an acyl CoA-independent pathway. This study provides a novel approach for identifying the mechanism of DHA accumulation and metabolic regulation strategies for DHA production in Schizochytrium. 1. Background Schizochytrium is a natural representative strain for the industrial production of docosahexaenoic acid (DHA) owing to its fast growth rate and high lipid content, containing more than 50% DHA [1-4]. The mechanism of DHA synthesis in Schizochytrium has been extensively studied and is known to involve two distinct biochemical pathways: the aerobic fatty acid synthesis (FAS) pathway and the anaerobic polyketide synthase (PKS) pathway [5-10], which are the main targets for regulating the synthesis of polyunsaturated fatty acids (PUFAs) in microorganisms [11-14]. Recent studies have revealed that DHA production depends not only on the pathway of DHA synthesis but also on the migration, accumulation, and assembly of DHA stored after synthesis [15-16]. Although DHA is mainly stored as DHA-triglyceride (DHA-TAG) in Schizochytrium and Thraustochytrium [17], it has been reported that the synthesized DHA might first be incorporated into phospholipids (PLs) to form DHA-PLs, before being channeled and assembled into DHA-TAG for storage [18-19]. This suggests that the final DHA yield is closely related to PL metabolism. PLs are the main components of cell membranes and play important roles in biological Page 3/28 reproduction, cell division, and membrane transport [20]. The regulation of PL metabolism in Schizochytrium may be effective for DHA production. Phospholipase D (PLD) is capable of converting different types of PLs through transphosphatidylation or hydrolyzing the polar head of PLs to produce phosphatidic acid (PA) [21], which has a significant role in cell growth and lipid metabolism [22, 23]. Currently, studies on PLD expression in the regulation of lipid metabolism mainly focus on plants [24, 25]. Overexpression of the soybean PLDγ gene (GmPLDγ) in Arabidopsis resulted in increased seed weight, a greater number of smaller lipid droplets, and significant upregulation of glycerolipid metabolism-related genes, suggesting a regulatory role for GmPLDγ in TAG synthesis and fatty acid remodelling [26]. Knockout of PLDα1 in soybean resulted in the higher unsaturation of TAG and major membrane lipids and lower unsaturation of PA and lysophospholipids during seed development. The study also found that phospholipid:diacylglycerol acyltransferase (PDAT) facilitated the conversion of acyl-CoA between phosphatidylcholine (PC) and diacylglycerol (DAG), promoting the unsaturation of TAG [27]. These results suggest that the regulation of PLD expression exerts an important influence on lipid metabolism in plants, particularly on the allocation of fatty acids between TAG and PLs. There are few publications on the function of PLD in lipid synthesis of microorganisms; therefore, it is of interest to explore the effect of PLD regulation on the patterns of PLs and DHA production in Schizochytrium, a premium engineered strain used for lipid production. In this study, we first mined two PLD genes, PLD1 and PLD2, from Schizochytrium and constructed the corresponding knockout strains, D PLD1 and D PLD2. The mutant strain with the higher DHA yield, D PLD1, was further analyzed using phospholipomics and qRT-PCR to reveal the potential regulatory mechanism of PLD on DHA production in Schizochytrium. 2. Results and Discussion 2.1 Mining of PLD genes Seventeen species of PLD corresponding to the EC3.1.4.4 enzyme classification were found in the JGI database. The sequence similarity of the PLD genes and the corresponding proteins among species was not high, indicating that the PLDs found in the JGI database were independent and non-repetitive (Fig. S1). Notably, PLD superfamily domains were found in PLD-46 and PLD-100463 proteins. Phylogenetic tree analysis of PLD-46 and PLD-100463 genes showed that they were closely related to the PLD and PLDα1 genes of Aurantiochytrium sp. FCC1311, respectively, and had 59.20% and 67.02% sequence homology, respectively (Fig. 1). Therefore, in this study, we chose to regulate the expression of PLD-46 and PLD-100463, which were named PLD1 and PLD2, respectively. Gene sequences are listed in Fig. S2 and S3. 2.2 Construction of transgenic S. limacinum SR21 with knockout of PLD1 or PLD2 Both positive transformants were screened for zeocin resistance. The PCR validations of the zeocin gene are shown in Fig. S4. The target band of the zeocin resistance gene (375 bp) appeared in the knockout Page 4/28 strains and positive control groups but not in the negative control group, indicating that the PLD1 or PLD2 gene had been successfully knocked out in S. limacinum SR21. 2.3 Effects of PLD1 or PLD2 knockout on cell growth and lipid synthesis As shown in Fig. 2a, compared with the wild type, there was no significant change in the biomass of the D PLD1 strain before 96 h, but a decrease of 11.0% (P < 0.01) and 7.4% (P<0.05) occurred at 120 h and 144 h, respectively. During the whole fermentation, the total lipid yield of D PLD1 strain showed little change when compared with the wild type. Fig. 2b shows that the D PLD1 and wild-type strains had the same glucose consumption rate throughout the fermentation process. Glucose was almost completely used up at 96 h, which was consistent with the observations of the same cell growth patterns in both strains before 96 h. The small decrease in biomass in the D PLD1 strain at the later stage was probably the result of knocking out PLD1, which slightly affected cell membrane function, resulting in minor cell damage. The lipid content in the two strains also showed no significant change during the full growth stage (Fig. 2c); however, DHA production was improved in the D PLD1 strain (Fig. 2d). The highest yield of DHA from shake flask fermentation of D PLD1 strain achieved 9.61 g/L, which was 12.3% higher than that of the wild type (P<0.01). These results suggest that knockout of PLD1 does not affect cell growth or total lipid production but enhances DHA accumulation. As shown in Fig. 3a, compared with the wild type, the biomass of the D PLD2 strain declined drastically from 72 h to 144 h, and the highest decrease was observed at 120 h (32.8%; P < 0.01). The total lipids of the D PLD2 strain showed a similar reduction as that observed for biomass, decreasing by 17.6% (P < 0.01) at 120 h. Fig. 3b shows that the glucose consumption rate in the D PLD2 strain decreased at the early stage but increased at the middle stage compared to the wild-type strain. The D PLD2 strain consumed the same amount of glucose as the wild-type strain. The lipid content of the D PLD2 strain showed no apparent change compared to that of the wild type before 96 h (Fig. 3c) but was higher at 120 and 144 h. This indicated that the two strains had the same ability to produce lipids before 96 h, and that the D PLD2 strain could improve the ability of the unit cell to produce lipids after 120 h. We postulated that the knockout of PLD2 severely impaired cell membrane function, leading to decreased cell growth and lipid yield. To maintain a certain amount of growth, cells of D PLD2 strain used glucose to synthesize other metabolites that resist cell damage and after 120 h, mature cells of the D PLD2 strain showed enhanced lipid synthesis. DHA production in the PLD2 knockout strain decreased significantly during all stages compared to the wild-type strain (Fig. 3d). These results indicated that PLD2 knockout is not conducive to cell growth, total lipid production, or DHA yield. 2.4 Effect of PLD1 or PLD2 knockout on fatty acid composition Table 1 shows that the PLD1 knockout decreased the percentage of saturated fatty acids (SFAs) and increased the proportion of PUFAs in the lipids of Schizochytrium. In the D PLD1 strain, the proportion of DHA in lipids increased by 13.3% and 12.5% at 120 h and 144 h, respectively. These results suggest that PLD1 knockout facilitates the synthesis of PUFAs, particularly DHA, in Schizochytrium. Knockdown of the Page 5/28 PLDα1 gene in soybeans resulted in higher unsaturation of TAG [27], which is similar to the results of the present study. Table 1 Fatty acid composition of wild-type strain and D PLD1 strain at late stages of fermentation Fatty acid 120 h 144 h WT D PLD1 significance WT D PLD1 significance C14:0 3.38±0.03 3.78±0.02 ** 3.20±0.07 3.78±0.08 ** C15:0 1.27±0.03 0.96±0.01 ** 1.24±0.04 0.94±0.01 ** C16:0 49.76±0.20 43.20±0.20 ** 49.05±0.34 42.96±0.99 ** C17:0 0.48±0.01 0.30±0.00 ** 0.49±0.02 0.30±0.00 ** C18:0 1.74±0.01 1.67±0.02 ** 1.85±0.03 1.63±0.01 ** EPA 0.39±0.03 0.36±0.00 0.48±0.01 0.39±0.01 ** DPA 6.92±0.03 8.32±0.01 ** 7.10±0.09 8.38±0.13 * DHA 33.11±0.16 37.52±0.18 ** 33.52±0.31 37.69±0.80 ** Others 2.94±0.12 3.89±0.11 * 3.09±0.09 3.94±0.18 ** SFAs 56.63±0.20 49.91±0.23 ** 55.81±0.48 49.60±1.10 ** PUFAs 40.43±0.16 46.19±0.19 ** 41.10±0.41 46.46±0.92 ** Table 2 shows that the PLD2 knockout had little effect on the proportion of PUFAs and SFAs in Schizochytrium. However, a decrease in the proportion of even-numbered carbon fatty acids, such as C14:0, C16:0, and C18:0, was observed, and the proportion of odd-numbered carbon fatty acids (OCFAs) increased significantly, of which C15:0 and C17:0 increased by 246.2% (P < 0.01) and 259.6% (P < 0.01), respectively. OCFA concentration in human plasma has been reported to be negatively correlated with the risk of type 2 diabetes and cardiovascular disease [14, 28, 29]; therefore, increasing the production of OCFAs helps increase the commercial potential of Schizochytrium. The above results suggest that PLD2 knockout mainly inhibited cell growth to reduce lipid yield but did not affect the synthesis pathway of PUFAs and SFAs in Schizochytrium. Table 2 Fatty acid composition of wild-type strain and △PLD2 strain at late stages of fermentation Page 6/28 Fatty acid 120 h 144 h WT △PLD2 significance WT △PLD2 significance C14:0 2.96±0.17 2.08±0.14 ** 2.83±0.46 1.92±0.07 ** C15:0 1.22±0.30 4.00±0.32 ** 1.00±0.05 3.47±0.52 ** C16:0 50.77±0.57 48.81±1.13 ** 50.51±0.55 46.54±0.75 ** C17:0 0.56±0.16 2.29±0.25 ** 0.46±0.06 2.09±0.27 ** C18:0 1.65±0.03 1.88±0.06 ** 1.74±0.12 1.89±0.03 ** EPA 0.42±0.05 0.35±0.02 0.57±0.14 0.55±0.12 ** DPA 6.90±0.35 6.64±0.21 ** 6.95±0.17 7.06±0.38 * DHA 32.48±0.69 30.63±0.26 ** 32.34±0.35 32.80±0.68 ** Others 3.04±0.13 3.32±0.39 * 3.60±0.86 3.68±0.37 ** SFAs 57.15±1.06 59.06±0.68 ** 56.54±0.79 55.91±1.53 ** PUFAs 39.80±1.09 37.62±0.48 ** 39.86±0.28 40.41±1.18 ** OCFAs 1.78±0.46 6.29±0.57 ** 1.46±0.10 5.56±0.78 ** Note: When P > 0.05, the difference is insignificant; when 0.01 < P < 0.05, the difference is significant and represented by *; when P < 0.01, the difference is extremely significant and represented by **. The regulation of PLD can influence phospholipid metabolism by affecting hydrolysis and transphosphatidylation and subsequently the function of the cell membrane and the glycerophospholipid metabolic pathway, thereby influencing the cell growth and lipid metabolism of Schizochytrium [30, 31]. The different results for the PLD1 and PLD2 knockouts may be related to their preference for hydrolysis or transphosphatidylation, allowing them to have different roles in phospholipid metabolism. PLD1 is thought to play a role in transphosphatidylation, which affects phospholipid composition and the regulation of fatty acid synthesis, whereas PLD2 mainly hydrolyzes phospholipid, which damages cell function and inhibits cell growth and lipid production. Given that the PLD1 knockout promoted DHA synthesis without a significant effect on cell growth and lipid production, whereas PLD2 knockout showed a severe reduction in cell growth and lipid synthesis compared to the wild strain, the D PLD1 strain was chosen for the subsequent experiments and analysis. 2.5 Effect of PLD1 knockout on transcriptional levels of related genes The transcriptional levels of genes involved in lipid metabolism in the wild-type and knockout strains were measured during the logarithmic growth period (60 h) and lipid transformation period (108 h) (Fig. 4). The fatty acid synthase (FAS) gene encodes a series of proteins related to the de novo synthesis of fatty acids in the form of gene clusters that can synthesize saturated 14-carbon fatty acids from acetyl Page 7/28 CoA via the traditional FAS pathway [32]. The chain length factor (CLF) gene is located in the PKS gene cluster and is responsible for chain lengthening during PUFA synthesis [33]. At 60 h, expression levels of both FAS and CLF genes were significantly upregulated in the D PLD1 strain; at 108 h, in the lipid transformation stage, the expression of CLF in the D PLD1 strain increased by 96.6%, whereas the expression of FAS did not change significantly (Fig. 4a and b). This supports the observation that PLD1 knockout enhanced DHA synthesis (Fig. 2d). Phosphatidic acid phosphatase (PAP) catalyzes the synthesis of DAG from PA, a hydrolytic product of PLD. PLD1 knockout improved the expression level of PAP, which increased by 1.71 times in the lipid transformation stage compared to the wild-type strain (Fig. 4c). This results in more PA being transformed into DAG in the D PLD1 strain, suggesting that PLD1 may play a greater role in transphosphatidylation than hydrolysis. PLD1 knockout regulates phospholipid metabolism by weakening the transphosphatidylation of phospholipids, thereby promoting the Kennedy pathway and improving TAG synthesis. CDP-diacylglycerol synthase (CDS) and phosphocholine cytidyl transferase (CCT) are the key enzymes involved in phospholipid synthesis, including that of phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and PC. At the 106 h timepoint, the expression level of CDS and CCT in the D PLD1 increased by 1.76 and 7.18 times (Fig. 4d and e), respectively, compared to the control. This indicates that PLD1 knockout increases the synthesis of phospholipids and further demonstrates that PLD1 is not responsible for hydrolysis of phospholipids in Schizochytrium. If PLD1 played a role in hydrolytic activity, the D PLD1 strain would accumulate the hydrolytic substrates of phospholipids and the hydrolytic product of PA would be reduced, thereby inhibiting the synthetic pathway of phospholipids and TAG synthesis for PA. Therefore, it is assumed that PLD1 regulates phospholipid composition by the transphosphatidylation of phospholipids, which influences fatty acid synthesis and DHA accumulation in Schizochytrium. For TAG synthesis, PDAT uses PC as an acyl donor and DAG as an acyl acceptor to transfer the acyl group of a phospholipid to DAG [27]. PLD1 knockout promoted PDAT transcription at 60 h and 108 h (Fig. 4f). Correspondingly, the transcriptional level of the diacylglycerol acyltransferase gene (DGAT), which catalyzes DAG and acyl-CoA to form TAG via the Kennedy pathway, was downregulated at both stages (Fig. 4g). These results indicate that the knockout of PLD1 increases TAG synthesis via the acyl-CoAindependent pathway of PDAT and reduces TAG synthesis via the Kennedy pathway of DGAT, which favors the production of unsaturated TAG [27]; this promotes DHA accumulation in TAG. 2.6 Effects of PLD1 knockout on phospholipid metabolism 2.6.1 Phospholipid content and fatty acid composition As shown in Fig. 5, PLD1 knockout led to an apparent increase in total PLs at 120 h, probably because PLD1 knockout reduced the transesterification activity of PLD, resulting in weakened interconversion and enhanced de novo synthesis of PLs (Fig. 4 d and e). In Thraustochytrium sp. 26185, DHA preferentially integrates into PLs rather than directly into TAG and then migrates to TAG to form DHA-TAGs at a later stage [8]. Yue [18] et al. found that DHA accumulates in both TAG and PC during the growth of Schizochytrium sp. A-2 and that DHA migrated from PC to TAG at a later stage of fermentation. The Page 8/28 increase in total phospholipids may facilitate the assembly and accumulation of DHA, which could also explain the increased DHA production caused by PLD1 knockout (Fig. 2d). Table 3 shows the main bound fatty acids in the TAGs and PLs of S. limacinum SR21. Both TAG and PLs mainly bound SFAs, which accounted for approximately 40–55%, of which C16:0 accounted for the largest proportion (>90%). However, the proportions of PUFAs in the TAG and PLs were significantly different. The proportion of PUFAs in TAGs was approximately 35%, including DHA, DPA, and EPA, whereas PUFAs in PLs only accounted for 10–15%, mainly consisting of DHA and DPA. These results indicate that PUFAs or (DHA) are mainly bound to TAG. Notably, EPA was not detected in the PLs and was thought to be at too low a level to be detected. This suggests that EPA was mostly bound to TAG, which provides a new theory for the study of EPA synthesis in S. limacinum SR21. Comparison of the fatty acid composition at different time points showed that the content of PUFAs or DHA in the TAG of both strains was higher at 120 h than at 72 h, and the content of SFAs decreased accordingly. However, the content of PUFAs or DHA in PLs at 120 h was lower than that at 72 h. It was assumed that DHA migrated from PLs to TAG in the later stages, which is consistent with previously reported results [18, 19]. Comparison of the fatty acid composition of the different strains at 72 and 120 h showed that the PLD1 knockout increased the content of DHA in TAG and decreased the content of DHA in PLs, indicating that PLD1 knockout promoted the migration of PUFAs or DHA from PLs to TAG, which is conducive to the accumulation and storage of DHA (Fig. 2d). Table 3 Main fatty acid composition in TAG and PLs of wild-type strains and △PLD1 strains Culture condition Lipid class Percent of fatty acids (%) C14:0 C16:0 C18:0 SFAs EPA DPA DHA PUFAs WT TAG 0.12 52.0 2.43 54.6 0.14 5.64 28.8 34.6 72 h PL 2.03 34.8 13.7 50.5 0.00 3.18 11.1 14.3 △PLD1 TAG 0.17 44.0 2.57 46.7 0.18 7.71 32.6 40.5 72 h PL 2.55 28.9 17.0 48.5 0.00 1.05 3.81 4.86 WT TAG 0.30 46.9 2.86 50.1 0.22 6.02 30.7 36.9 120 h PL 1.26 25.7 13.2 40.2 0.00 3.09 6.69 9.78 △PLD1 TAG 0.26 42.6 1.67 44.5 0.20 7.75 34.0 42.0 120 h PL 2.30 20.9 11.2 34.4 0.00 0.57 3.22 3.79 2.6.2 Phospholipid types and composition in S. limacinum SR21 The polar head structure of phospholipids determines the phospholipid species. As shown in Table 4, 43 phospholipid molecules were identified; including 15 PC, nine lysophosphatidylcholine (LPC), 10 PG, four phosphatidylethanolamine (PE), two PI, two PS, and one PA. The three main phospholipids in S. Page 9/28 limacinum SR21 were PC, PI, and PG; and PC accounted for half of the total PLs. PC is also the main phospholipid in other Schizochytrium strains [18, 34, 35] and is related to DHA accumulation and migration to TAG [18, 19]. PI has an inositol head group that plays an important role in cell signal transduction and metabolic regulation [36]. PG is closely related to cell growth and affects the lipid dependence of cellular stress responses and adaptation mechanisms [37]. The levels of PE, PS, and PA in S. limacinum SR21 were low (<5%); however, they also play important roles in cell membrane structure and function [30]. Table 4 Phospholipid species in S. limacinum SR21 (120 h) Page 10/28 Type Unsaturation Acyl composition Molecular formula m/z Content (μg/g) PC C30:0 C14:0/C16:0 C38H76O8NP 750.5285 6.45 C32:0 C16:0/C16:0 C40H80O8NP 778.5598 16.10 C36:5 C14:0/C22:5 C44H78O8NP 824.5442 15.53 C36:6 C14:0/C22:6 C44H76O8NP 822.5285 18.19 C37:5 C15:0/C22:5 C45H80O8NP 838.5598 6.45 C37:6 C15:0/C22:6 C45H78O8NP 836.5442 19.19 C38:5 C16:0/C22:5 C46H82O8NP 852.5755 661.44 C38:6 C16:0/C22:6 C46H80O8NP 850.5598 2076.51 C40:6 C18:0/C22:6 C48H84O8NP 878.5911 14.32 C40:7 C18:1/C22:6 C48H82O8NP 876.5755 28.22 C42:10 C20:4/C22:6 C50H80O8NP 898.5598 124.96 C42:11 C20:5/C22:6 C50H78O8NP 896.5442 270.73 C44:10 C22:5/C22:5 C52H84O8NP 926.5911 58.27 C44:11 C22:5/C22:6 C52H82O8NP 924.5755 525.61 C44:12 C22:6/C22:6 C52H80O8NP 922.5598 1518.21 C14:0 C14:0 C22H46NO7P 512.2988 4.19 C15:0 C15:0 C23H48NO7P 526.3145 1.30 C16:0 C16:0 C24H50NO7P 540.3301 59.88 C18:0 C18:0 C26H54NO7P 568.3614 1.10 C18:1 C18:1 C26H52NO7P 566.3458 2.79 C18:2 C18:2 C26H50NO7P 564.3301 2.28 C20:5 C20:5 C28H48NO7P 586.3145 2.44 C22:5 C22:5 C30H52NO7P 614.3458 109.23 C22:6 C22:6 LPC C30H50NO7P Page 11/28 612.3301 489.56 PG PI PE PS PA C30:0 C14:0/C16:0 C36H71O10P 693.4707 0.35 C31:0 C15:0/C16:0 C37H73O10P 707.4863 1.20 C32:0 C16:0/C16:0 C38H75O10P 721.502 147.72 C38:5 C16:0/C22:5 C44H77O10P 795.5176 11.97 C38:6 C16:0/C22:6 C44H75O10P 793.502 98.12 C40:6 C18:0/C22:6 C46H79O10P 821.5333 0.80 C40:7 C18:1/C22:6 C46H77O10P 819.5176 1.34 C42:10 C20:4/C22:6 C48H75O10P 841.502 3.60 C44:11 C22:5/C22:6 C50H77O10P 867.5176 2.57 C44:12 C22:6/C22:6 C50H75O10P 865.502 5.03 C38:5 C16:0/C22:5 C47H81O13P 883.5337 1069.19 C38:6 C16:0/C22:6 C47H79O13P 881.518 2005.56 C38:5 C16:0/C22:5 C43H76O8NP 764.523 74.82 C38:6 C16:0/C22:6 C43H74O8NP 762.5074 211.59 C44:11 C22:5/C22:6 C49H76O8NP 836.523 20.26 C44:12 C22:6/C22:6 C49H74O8NP 834.5074 56.60 C28:0 C14:0/C14:0 C34H66NO10P 678.4346 63.49 C44:12 C22:6/C22:6 C50H74NO10P 878.4972 12.93 C44:12 C22:6/C22:6 C47H69O8P 791.4652 180.21 The two acyl chains of PLs determine the phospholipid diversity. Fatty acids with different structures and characteristics play different roles in the structure and function of PLs. C16:0 and C22:6 were the two major fatty acids of lipids in S. limacinum SR21 (Table 4) and also the main fatty acids of acyl chains binding to PLs (Table 3). PC tended to bind to unsaturated fatty acids (UFAs), and the two most important types of PC were PC (16:0/22:6) and PC (22:6/22:6). The proportion of UFAs in PC was 73.3%, of which DHA accounted for 77.7%. Yue [18] et al. reported that in Schizochytrium sp. A-2, PC (22:6/22:6) and PC (22:5/22:6) accounted for half of the total PC. The LPC and PE in this study also showed a preference for binding UFAs. The proportions of UFAs in LPC and PE reached 90.1% and 60.6%, respectively, of which DHA accounted for 80.7% and 78.4%, respectively. PI bound to both SFAs and UFAs, of which DHA accounted for 31.6%. PG and PS tend to bind SFAs, and in this study, they accounted for 75.1% and Page 12/28 83.1% of SFAs respectively. Eriko [34] found that in Schizochytrium sp. F26-b, DHA was the main fatty acid at the acyl ends of PC, LPC, PE, and PI, whereas there was almost no PUFA at the acyl end of PS. Guang [35] also reported that in S. limacinum, PUFAs accounted for 86% and 71.6% of PC and PE, respectively, and SFAs only accounted for 11.8% and 23.5 % of PC and PE, respectively. Meanwhile, SFAs in PG reached 64.2%. These results indicate that DHA synthesized by Schizochytrium may first be incorporated into PLs, in particular PC, for storage. 2.6.3 Phospholipidomic analysis in wild-type and D PLD1 strains The proportions of various PLs in the wild-type and D PLD1 strains are shown in Fig. 6. Changes in PL content with fermentation time were compared. The percentage of PG in both strains decreased remarkably as fermentation proceeded, which may be closely related to the cell growth period. The conversion of glucose to glycerol 3-phosphate is much easier compared to that for other phospholipid polar head groups; therefore, it may be easier to synthesize PG with a sufficient carbon source to meet the membrane phospholipid supply during exponential cell production. When glucose is depleted, cells stop proliferating, and membrane PLs metabolism shifts from PL synthesis to interconversion among PLs, resulting in phospholipid composition changes. Furthermore, a decrease in PG levels increases the cellular stress response and lipid dependence of the adaptation mechanism [37]. We observed that the decrease in the proportion of PG was accompanied by a drastic increase in PI. The proportion of PI in the PLs was lower than that of PC. PI has an inositol head group, and its phosphorylation at different positions in inositol is a decisive factor in distinguishing biofilms [38, 39]. PI is also an important signaling molecule that regulates cell signal transduction and the metabolic process [36]. The increase in PI at 120 h in both strains might be the result of a more active signaling pathway after entering the lipid transformation period, especially lipid metabolism, which requires more PI for signal transduction and metabolic regulation. LPC also increased at 120 h in both strains. LPC has one less acyl chain than PC and exhibits a strong surface activity. A high proportion of LPC may cause cell membrane rupture and necrosis [40]. The conversion between PC and LPC is not only related to the acyl remodelling of phospholipids but also to the transfer of fatty acids from PC to TAG, catalyzed by PDAT [41, 42]. The increase in LPC may be due to the rupture and necrosis of cells entering the late phase of growth or might be related to the deacylation of PC to LPC. PC and PA levels showed different changes over time in both strains. In the wild-type strain at 120 h, PC increased by 9.2%, whereas PA decreased by 26.2% compared to the levels at 72 h. In the PLD1 knockout strain at 120 h, PC decreased by 20.9%, whereas PA increased by 178.9% compared to the levels at 72 h. This further proves that PLD1 mainly plays the role of transesterification rather than hydrolysis of PLs. The knockout of PLD1 reduced the interconversion among PLs, which affected their composition and strengthened the migration of DHA from PC to TAG at the lipid transformation stage (Fig. 4f). This resulted in decreased PC, increased LPC, and enhanced DHA accumulation. The changes in the PL content of the different strains were compared. For the three major PLs at 72 h, knockout of PLD1 resulted in no change in PC, a 32.9% decrease in PG, and a 20.3% increase in PL content compared to the wild-type strain. This suggests that the knockout of PLD1 mainly reduced PG Page 13/28 synthesis at the cell growth stage, which resulted in a decrease in biomass (Fig. 2a). PI not only affects the transport of substances between membranes by regulating ion channels, ion pumps, transporters, endocytosis, and exocytosis but also regulates lipid metabolism and distribution and is closely related to lipid transporters [38]. The increase in PI in the D PLD1 strain demonstrates that the knockout of PLD1 could upregulate the PI signaling pathway to increase cell metabolism. For other minor PLs at 72 h, knockout of PLD1 showed no clear change in PA and PS content compared with the wild-type strain; however, LPC and PE content showed a small increase. This increase in PE may be related to the mutual transformation of lipid types in polar lipids. In addition, the phospholipid composition significantly affects the fluidity of the cell membrane, and PE can increase the mobility of the membrane owing to its head group and anionic properties [43]. The increase in LPC was not accompanied by a decrease in PC, indicating that the GPC acylation pathway was activated to synthesize LPC; this is currently thought to be the main pathway for DHA-PC synthesis [44-46]. For PG and PI at 120 h, knockout of PLD1 resulted in similar changes to those seen at 72 h compared to the wild-type strain. However, PC showed a different change and decreased by 27.2% at 120 h compared to the wild-type strain, indicating that PC had been converted. Accordingly, LPC and PA showed a 45.0% and 275% increase, respectively, at 120 h compared with the wild-type strain, indicating that the acyl migration from PC to DAG to form TAG via the acyl CoAindependent pathway [47, 48] is increased in the D PLD1 strain (Fig. 4f). This requires that more DAG forms TAG via PDAT catalysis, thereby increasing the synthesis of PA to provide more DAG by the Kennedy pathway (Fig. 4c) and resulting in an increase in total lipids (Fig. 2a). Synthesized DHA may first be incorporated into PC to form DHA-PC, then channeled to DAG and assembled into DHA-TAG for storage [18, 19]. The results of the PL content and fatty acid composition also showed that PC tended to bind UFAs (73.3%) and that DHA accounted for 57.0% of total fatty acids in PC (Table 4). The increase of DHA production in the D PLD1 strain (Fig. 2d) combined with the upregulation of genes related to the fatty acid synthesis and acyl CoA-independent pathways (Fig. 4) and the observed changes in different PLs (Fig. 6) allows us to infer the method by which DHA is synthesized and accumulated in Schizochytrium. DHA is initially incorporated mainly into PLs, particularly PC, and then migrated to DAG to produce TAG via the acyl CoA-independent pathway depending on PDAT catalysis, which can be promoted by regulating phospholipid metabolism through PLD1. 3. Conclusion This study is the first to explore the effect of PLD expression on lipid synthesis in Schizochytrium. The genes PLD1 and PLD2 identified in S. limacinum SR21 have different effects on lipid synthesis. In S. limacinum SR21, the knockout of PLD1 demonstrated a promoting effect on PUFA synthesis and DHA accumulation without affecting biomass and total lipid production. PLD1 is presumed to play a role in transphosphatidyl activity in S. limacinum SR21, and the knockout of PLD1 reduces PL interconversion and enhances de novo synthesis of PLs, thereby improving the total PL yield and affecting their metabolism. This results in an increase in the binding of DHA to PLs and allows DAG to be assembled into DHA-TAG for storage by an acyl CoA-independent pathway. These results suggest that PLD exerts an Page 14/28 important influence on the allocation of fatty acids between TAG and PLs and is closely related to the enzyme activity of PDAT in the acyl CoA-independent pathway. Future studies should investigate its exact mechanisms and functions. 4. Materials and methods 4.1 Strains, media, and culture conditions The S. limacinum SR21 (ATCC MYA-1381) strain was purchased from the American Type Culture Collection (Manassas, VA, USA) and was used as the original strain. The seed media and fermentation broths used in this study were the same as those in our previous study [30]. The modified inorganic salt stock solution contained Na2SO4, 240 g/L; MgSO4, 40 g/L; kH2PO4, 20 g/L; (NH4)2SO4, 20 g/L; K2SO4, 13 g/L; and KCl, 10 g/L. The medium was sterilized at 115 °C for 20 min before use. The trace element and vitamin solutions were filter-sterilized using a 0.2 μm filter. The activated single colonies were cultured in seed medium at 28 °C, 200 rpm (SKY-2102C double-layer thermostatic shaker, Suzhou, China) for 24 h. After the second generation culture, the seed culture (4% v/v) was transferred to the fermentation broth and incubated at 28 °C and 200 rpm for 144 h or longer. 4.2 Mining of PLD genes PLD-related information for S. limacinum SR21 was obtained from the Joint Genome Institute (JGI) database. The similarity between the PLD gene sequence and the corresponding protein sequence was analyzed using DNAstar Megalign (Madison, WI, USA), and phylogenetic tree analysis of the selected PLD genes was performed using the NCBI database. 4.3 Construction of gene knockout strains PLD knockout strains were constructed using homologous recombination technology, as shown in Fig. S5. The up- and downstream sequences of PLD were amplified by PCR using primers from S. limacinum SR21 genomic DNA (Table S1). The PCR reagent PrimeSTAR HS (Premix) was purchased from TaKaRa Bio (TaKaRa Biotechnology Co.,Ltd, Dalian, China), and PCR primers were synthesized by Xiamen Boshang Biotechnology Company. The pBlue-zeo plasmid was previously constructed in our laboratory [30] and included an integration region with multiple cloning sites and zeocin expression cassettes for resistance screening. The plasmid pBlue-zeo-PLD was constructed by inserting the homologous arm of the PLD gene into the multiple cloning site of the vector. The disrupted fragment was PCR-amplified with primers from the constructed vectors and transformed into S. limacinum SR21 by electroporation, according to the method by Ling et al. [30]. The disrupted fragment combined the targeted gene with the homologous arms to replace it [31]. After electroporation, the cells were cultivated for 3 h at 28 °C in a seed medium containing 1 M sorbitol (Macklin Biochemical, Shanghai, China) and then recovered for 3–5 days in a solid medium containing 50 μg/mL zeocin (Sangon Biotech, Shanghai, China). The positive transformants were screened by zeocin resistance Page 15/28 plates and cultured at 28 °C and 200 rpm in a shaker (SKY-2102C double-layer thermostatic shaker) for PCR validation and fermentation experiments. 4.4 Measurement of biomass and glucose content One milliliter of fermentation broth was added to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 2 min at 28 ℃. The supernatant was collected to measure glucose concentration using the 3, 5dinitrosalicylic acid (DNS) method. The cell pellets were washed twice with normal saline and stored in a refrigerator at −20 °C. The biomass was calculated after 24 h of vacuum freeze-drying. 4.5 Lipid extraction and fatty acid composition analysis Three milliliters of fermentation broth was mixed with 4 mL 12 M HCl and incubated in a water bath at 65 °C for 45 min. Total lipids (TLs) from the mixture were extracted four times with 3 mL of n-hexane, and the lipid extract was then purified and dried by evaporation. Total fatty acid (TFA) production was calculated by subtracting unsaponifiable matter (UM) from TLs; UM was isolated from lipids by saponification [49]. The preparation of fatty acid methyl esters and analysis of fatty acid composition were performed as previously described [30]. 4.6 Real-time quantitative PCR (qRT-PCR) Total RNA was extracted from 1 mL of broth using the RNA Plant Plus reagent (TaKaRa, Japan) according to the manufacturer’s instructions, and 1 μg from each sample was reverse-transcribed using the QuantScript RT Kit reagent (TaKaRa, Japan). qRT-PCR amplification was performed using the One Step SYBR PrimeScript PLUS RT-PCR Kit (TaKaRa, Japan). Primers used are listed in Table S2. The mRNA expression level was normalized using the actin gene as the internal control, and the relative gene expression level was calculated by using 2−ΔΔCT method [50]. 4.7 Quantification of phospholipids Lipids were first extracted from S. limacinum SR21 using the Bligh–Dyer method [51] with some modifications. Five milliliters of fermentation broth was added to 2 mL of deionized water, 3 mL of chloroform, and 6 mL of methanol before ultrasonication for 30 min; 3 mL of chloroform was added for ultrasonication for 30 min. Then, 3 mL of deionized water was added, followed by incubation for 30 min. One milliliter of saturated NaCl solution was then added the solution allowed to stand for 2 h. The solutions were centrifuged at 3,000 ×g for 10 min at 28 ℃. After centrifugation, the lower layer of the lipids was transferred to a glass bottle and dried under a nitrogen stream. The obtained lipid samples were weighed and stored at −20 °C until required. Phospholipids were separated from the lipid samples using solid-phase extraction [52]. Page 16/28 Phospholipids were determined using ultra performance liquid chromatography-mass spectrometry (Waters UPLC Acquity H-Class-Xevo-G2 Q-ToF, Milford, MA, USA). The chromatographic experimental conditions were as follows: chromatographic column: ACQUITY UPLC BEH HILIC column (150 mm × 2.1 mm × 1.7 μm); mobile phase: A is acetonitrile, and B is 20 mM ammonium formate aqueous solution (0.1% formic acid added to obtain pH 3.5); the flow rate was 0.2 mL/min; injection volume: 2 μL; elution procedure: 0–4 min, 95% A and 5% B; 4–22 min, 95–60% A and 5–40% B; 22–25 min, 60 % A and 40% B; 25–25.1 min, 60–95 %A and 40–5% B; 25.1–30 min, 95% A and 5% B. The mass spectrometry experimental conditions were as follows: electrospray negative ion mode (ESI-); analyzer mode: sensitivity mode; data acquisition time: 2.5–20 min; scanning range: 250–1000 Da; scanning time: 0.5 s; capillary voltage: 2 kV; sample cone voltage: 30 V; extraction cone voltage: 4 V; ion source temperature: 100 °C; desolvation gas temperature: 350 °C; cone gas flow rate: 50 L/h; the desolvation gas flow rate was 400 L/h. 4.8 Statistical analysis Statistical significance between different strains or groups was evaluated using a t-test; 0.01 < P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely significant. Three parallel experiments were conducted. Values are expressed as the means ± SD (standard deviation). Declarations Authors' contributions XPL and YHL conceived and designed the research. YTZ and XWC conducted the experiments and wrote the manuscript. SZL contributed analytical tools. HL, TL and YTZ analyzed the data. XPL, MFC and YHL modified the manuscript. All authors read and approved the manuscript. Data availability The authors can confirm that all relevant data are included in the article and supplementary materials files. Acknowledgments We thank Editage (https://www.editage.cn) for English language editing during the preparation of this manuscript. We gratefully acknowledge the Analysis and Test Center of the Third Institute of Oceanography for its continuous support. Funding This work was supported by the Natural Science Foundation of Xiamen, China (No. 3502Z20227183), National Key Research and Development Program of China (No. 2022YFC2104600), and National Natural Science Foundation of China (No. 31871779). Page 17/28 Declaration of competing interests The authors declare no competing financial interests or personal relationships that influence the work reported in this study. 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Dhara R, Bhattacharyya DK, Ghosh M, Analysis of sterol and other components present in unsaponifiable matters of mahua, sal and mango kernel oil. Journal of Oleo Science. 2010; (59): 169-76. 50. Larionov A, Krause A, Miller W. A standard curve based method for relative real time PCR data processing. Bmc Bioinformatics. 2005; (6): 1-16. 51. Bligh EG, Dyer WJ. Extraction of lipids in solution by the method of bligh & dyer. Canadian Journal of Biochemistry and Physiology. 1959; 37: 911-7. 52. Donato P, Cacciola F, Cichello F, et al. Determination of phospholipids in milk samples by means of hydrophilic interaction liquid chromatography coupled to evaporative light scattering and mass spectrometry detection. Journal of Chromatography A. 2011; 1218(37): 6476-82. Figures Page 21/28 Figure 1 Phylogenetic tree analysis of the (a) PLD-46 and (b) PLD-100463 gene sequences of S. limacinum SR21. Bootstrap values (>50%, repeated 1,000 times) are displayed on each internal branch Page 22/28 Figure 2 (a) Biomass and total lipids, (b) glucose concentration, (c) lipid content, and (d) DHA yield of the wild-type and the ΔPLD1 strains. * and ** represent 0.01 < P < 0.05 and P < 0.01, respectively. All data are expressed as mean ± SD of three independent experiments Page 23/28 Figure 3 (a) Biomass and total lipids, (b) glucose concentration, (c) lipid content, and (d) DHA yield of the wild-type and the ΔPLD2 strains. * and ** represent 0.01 < P < 0.05 and P < 0.01, respectively. All data are expressed as mean ± SD of three independent experiments Page 24/28 Figure 4 Transcription levels of related genes in wild-type and ΔPLD1 strains: (a) fatty acid synthase (FAS), (b) chain length factor (CLF), (c) phospholipid acid phosphatase (PAP), (e) CDP-diacylglycerol synthase (CDS), (e) choline phosphate transferase (CCT), (f) phosphatidylglycerol acyltransferase (PDAT), (g) diacylglycerol acyltransferase (DGAT). There was a significant difference at the 0.01 < P < 0.05 (*) or P < Page 25/28 0.01 (**) level between the wild-type strain and the ΔPLD1 strain. All data were expressed as mean ± SD of three independent experiments Figure 5 Phospholipid content in 72 and 120 h samples of the wild-type and ΔPLD1 strains. All data were expressed as mean ± standard deviation (mean ± SD) of three independent experiments Page 26/28 Figure 6 Fractionation of total phospholipids extracted from wild strain and ΔPLD1 strains at 72 h and 120 h, respectively. PC, phosphatidylcholine; LPC, lysophosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phospholipid acid. All data were expressed as mean ± standard deviation (mean ± S.D.) of three independent experiments Page 27/28 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementarymaterialsforsubmission.pdf Page 28/28
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Electric Field Responsive Nanotransducers for Glioblastoma
bioRxiv (Cold Spring Harbor Laboratory)
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Abstract Background:  Electric field therapies such as Tumor Treating Fields (TTFields) have emerged as a bioelectronic treat- ment for isocitrate dehydrogenase wild-type and IDH mutant grade 4 astrocytoma Glioblastoma (GBM). TTFields rely on alternating current (AC) electric fields (EF) leading to the disruption of dipole alignment and induced dielec- trophoresis (DEP) during cytokinesis. Although TTFields have a favourable side effect profile, particularly compared to cytotoxic chemotherapy, survival benefits remain limited (~ 4.9 months) after an extensive treatment regime (20 hours/day for 18 months). The cost of the technology also limits its clinical adoption worldwide. Therefore, the discov- ery of new technology that can enhance both the therapeutic efficiency and efficacy of these TTFields will be of great benefit to cancer treatment and decrease healthcare costs worldwide. Methods:  In this work, we report the role of electrically conductive gold (GNPs), dielectric silica oxide ­(SiO2), and semiconductor zinc oxide (ZnO) nanoparticles (NPs) as transducers for enhancing EF mediated anticancer effects on patient derived GBM cells. Physicochemical properties of these NPs were analyzed using spectroscopic, electron microscopy, and light-scattering techniques. Results:  In vitro TTFields studies indicated an enhanced reduction in the metabolic activity of patient-derived Glioma INvasive marginal (GIN 28) and Glioma contrast enhanced core (GCE 28) GBM As per our journal style, article titles should not include capitalisedletters unless these are proper nouns/acronyms. We have therefore usedthe article title “Electric field responsive nanotransducers forglioblastoma” as opposed to “Electric Field Responsive Nanotransduc- ersfor Glioblastoma” as given in the submission system. Please check ifthis is correct.cells in groups treated with NPs vs. control groups, irrespective of NPs dielectric properties. Our results indicate the inorganic NPs used in this work enhance the intracellular EF effects that could be due to the virtue of bipolar dielectrophoretic and electrophoretic effects. Conclusions:  This work presents preliminary evidence which could help to improve future EF applications for bio- electronic medicine. Furthermore, the merits of spherical morphology, excellent colloidal stability, and low toxicity, make these NPs ideal for future studies for elucidating the detailed mechanism and efficacy upon their delivery in GBM preclinical models. Keywords:  Tumor Treating Fields, Inorganic nanoparticles, Electric fields, Glioblastoma Bioelectronic Medicine Bioelectronic Medicine Bioelectronic Medicine Jain et al. Bioelectronic Medicine (2022) 8:17 https://doi.org/10.1186/s42234-022-00099-7 Open Access © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Background While the full extent of how TTFields work is currently unclear, two proposed mechanisms explain the mode of action: dipole alignment and DEP. In the first instance, spindle formation during mitosis is affected. Microtubules are influenced by the dipole moment of the building blocks, therefore cell division is limited (Kirson et al., 2004). In the second instance, an inhomogeneous distribution of the EF within dividing cells causes molecules to become polarised. These polar molecules then move to regions with higher EF intensities, notably the cleavage furrow in mitotic cells, and thereby interfere with cytokinesis (Kir- son et al., 2007).f Despite TTFields affecting rapidly dividing cancer cells, there appears to be no effect on healthy cells with rela- tively slower cell division. Even cells that exhibit rapid cell division such as those found in bone marrow or intestine are not affected as they are protected by high impedance of the bone, and slower replication times compared to cancerous cells respectively (Blatt et al., 2021). Tight junctions between epithelial cells at the blood- brain barrier (BBB) are known to inhibit the influx and efflux of many molecules to the brain (Brightman & Reese, 1969; Reese & Karnovsky, 1967). This barrier can be overcome by taking advantage of the leaky vas- culature surrounding brain tumours which leads to the accumulation of NPs in the tumour, via the Enhanced Permeation and Retention (EPR) effect. NPs are fre- quently utilised to take advantage of this EPR effect, with studies showing that NPs of a size range of 20-100 nm are ideal to allow for maximum accumulation in the cells, tumour and longer clearance times (Perrault et al., 2009). Gold NPs (GNPs) are of particular interest for biomedical and bioelectronics applications due to During last decade, there has been much experi- ment work into the mechanism of action of TTFields, but there has been limited efforts to enhance TTFields using NPs. One example is biocompatible barium titan- ate NPs (BTNPs) with a high dielectric constant, which were investigated as breast cancer sensitisers in cells that were resistant to TTFields (Yoon et al., 2020). This study found that BTNPs accumulate within the cytoplasm when exposed to TTFields where they are then polar- ised by the inhomogeneous EF as discussed earlier, caus- ing the BTNPs to migrate to the cleavage furrow and the cells to undergo apoptosis. Background This significant improvement to OS is not accompanied by any major side effects, with the only reported effect being contact dermatitis at the site of the electrodes. their biocompatibility and tuneable properties (Shukla et  al., 2005; Perrault & Chan, 2009; Sanjuan-Alberte et al., 2019). In recent years there have been numerous clinical trials utilising GNPs to treat a range of can- cers, including glioblastoma (Libutti et  al., 2010; Gad et  al., 2012; Kumthekar et  al., 2021). ZnO NPs (semi- conductors) and ­SiO2 NPs (insulators), have also been well researched as a potential treatment for cancers, with the former showing the preferential killing of can- cer cells over normal cells, while the latter has shown advantages of being highly tuneable, allowing for tar- geted drug delivery (Hanley et  al., 2008; Wang et  al., 2009; Murugan et al., 2017). g Electric fields have been used as external stimuli for the delivery of drugs from NPs for cancer and tissue regener- ation (Kolosnjaj-Tabi et al., 2019). However, the behavior of nanoparticles under EFs in cellular environment needs further studies. Apart from the classic endocytosis medi- ated uptake of NPs in cancer cells, electric fields have shown to enhance the uptake of NPs by permeabilizing cancer cell membranes via electroporation and by modu- lating bioelectricity through voltage-gated ion channels (Aguilar et al., 2021; Chang et al., 2018). This has opened new area of research to develop new tools to study intra- cellular interaction of NPs with EFs. Apart from the well- known electrophoretic and dielectrophoretic movement of NPs, EFs have been shown to induce electrostatic induction and charge separation of nanomaterials. This electric polarization leads to generation of numerous bipolar nanoelectrodes which acts as transducer of EFs (Guo et  al., 2021). Using modelling approaches Tiwari et al. demonstrated that spherically capped gold nanow- ire enhanced EFs inside the cells. They inferred that due to the uniform and homogenous distribution of EFs over nanomaterials (due to charge separation is over a short distance ≈ nm) addition source and sinks are gener- ated. This causes a local enhancement in electric field strengths around nanomaterials in contact with cytoplas- mic entities leading to the rupture of plasma membrane and eventually apoptosis (Tiwari et al., 2009). TTFields are directional, mainly influencing cell behav- iour when the electric field and axis of cell division are parallel to one another (Kirson et  al., 2007). Background Isocitrate dehydrogenase wild-type GBM is a form of highly aggressive brain tumour accounting for 49.1% of primary malignant brain tumours with less than 7% of patients surviving after 5 years post-diagnosis (Ostrom et  al., 2021). The current standard of care is known as the ‘Stupp regimen’ and consists of surgical resection *Correspondence: Akhil.Jain@nottingham.ac.uk; Frankie. Rawson@nottingham.ac.uk 1 Bioelectronics Laboratory, Division of Regenerative Medicine and Cellular Therapies, School of Pharmacy, Biodiscovery Institute, University of Nottingham, Nottingham NG7 2RD, UK Full list of author information is available at the end of the article 1 Bioelectronics Laboratory, Division of Regenerative Medicine and Cellular Therapies, School of Pharmacy, Biodiscovery Institute, University of Nottingham, Nottingham NG7 2RD, UK Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Page 2 of 9 Jain et al. Bioelectronic Medicine followed by treatment with radiotherapy and the alkylat- ing chemotherapeutic agent, temozolomide, increasing overall survival (OS) to a median of 14.6 months (Stupp et  al., 2005). Since this finding in 2005, there has been little progression in the identification of new treatments for GBM that are United States Food and Drug Adminis- tration (FDA) approved, except for TTFields. Indeed, no molecular targeted therapeutics predicated on genome biology has shown efficacy in phase III trials to date. These low intensity (<4 V/cm), intermediate frequency (100-500 kHz) and AC EFs, have been shown to further enhance cell death when in conjunction with the Stupp regimen, increasing OS by a median of 4.9 months (Stupp et al., 2017). TTFields GIN 28 and GCE 28 cells were seeded on a 22 mm cell culture treated coverslip at a density of 3.5 × ­104 and incubated for 24 hours at 37°C and 5% ­CO2. Next, the media was replaced with media containing GNPs/SiO2/ ZnO NPs at a concentration of 5 μg/mL or 25 μg/mL and incubated at 37°C for 4 hours. Next, the coverslips were transferred to ceramic Petri dishes of the inovitro™ sys- tem (Novocure, Haifa, Israel). Finally, the TTFields were applied for a duration of 48 or 72 hours using the ino- vitro system which consists of two pairs of perpendicular transducer arrays on the outer walls of the ceramic plate containing the Petri dishes. A sinusoidal waveform gen- erator was attached to the transducer arrays producing alternating EFs set at a frequency of 300 kHz and 1V/cm intensity. TTFields of 300 KHz was chosen for this work, this is based on previous studies that used range of fre- quencies from 200-300 KHz for GBM cells (Branter et al., 1982; Linder et al., 2021). TTFields were applied bi-direc- tionally (perpendicular to each other), which switches between the two direction every second. The temperature was measured to be 37°C inside the dishes by thermistors attached to the ceramic walls. Finally, the change in met- abolic activity/ viability of GIN 28 and GCE 28 cells in response to TTFields or NP + TTFields, was determined using the ­PrestoBlueTM assay as described above. Methods Materials All the reagents were of analytical grade and were used as supplied without further purification unless speci- fied. Citrate-capped spherical GNPs, ZnO, and ­SiO2 NPs of size 50 nm were purchased from Sigma Aldrich, UK. PrestoBlue cell viability reagent and cell culture treated 22 mm coverslips (Nunc™ Thermanox™ Coverslips) were purchased from ThermoFisher Scientific, UK. In vitro toxicity In vitro toxicity HA-COR, GIN 28 and GCE 28 cells were seeded in a 96-well plate at a density of 4.5 × ­103 cells per well and incubated for 24 hours at 37°C and 5% ­CO2. Culture media was replaced with medium containing GNPs/ SiO2/ZnO NPs (concentration = 0.1, 0.5, 1, 2, 5, 10, 20, 50 or 100, 200 μg/ mL) and incubated for 4 hours. Next, the media was replaced with fresh media and cells were incu- bated for 48 or 72 hours. Finally, metabolic activity was determined using ­PrestoBlueTM. For each well, the media was replaced with media containing 10% PrestoBlue cell viability reagent and incubated at 37°C for 2 hours in an incubator before transferring the coloured metabolic product to a black-bottom 96-well plate. Finally, the fluorescence of the plate was read using a plate reader (TECAN Infinite 2000) with an excitation wavelength of 570 nm and an emission wavelength of 600 nm. Mean ± S. D values are presented relative to negative controls. Background While this study is the first example of using NPs to enhance TTFields, BTNPs are Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine Page 3 of 9 not FDA-approved, creating a barrier to translating these findings to a clinical setting. were maintained at 37°C in an incubator with humidi- fied atmosphere, containing 5% ­CO2. Cells were routinely tested for mycoplasma where they were grown in an antibiotic-free medium for one week before mycoplasma testing. All cells used were mycoplasma-free. i g g Here, we investigate the underlying mechanism with the hypothesis that conductive particles would enhance the effects of TTFields. NP-enhanced TTFields mecha- nism of action was investigated, which is of paramount importance for the discovery of new approaches that can enhance these TTFields-induced anticancer effects. The NPs chosen were gold, zinc oxide (ZnO), and silica ­(SiO2), which are FDA-approved conductive, semi- conductive, and insulating NPs respectively (Rasmus- sen et al., 2010; Zhou et al., 2018). As the two suggested modes of TTFields action are due to dipole alignment and DEP, the effects of using GNPs and ZnO NPs with different electrical conductivities were chosen to address the first mechanism, while dielectric ­SiO2 NPs have potential to enhance TTFields by the second mechanism. From a clinical application perspective, the overall toxic- ity, pharmacokinetics, and therapeutic efficacy of the NPs must be evaluated in an accurate in vitro model that can reflect the cancer heterogeneity observed in clinics. Fur- thermore, as observed in clinics, the efficacy of TTFields varies across patients which is attributed to heterogeneity in GBM. Therefore, we utilised patient-derived GIN 28 (isolated from the invasive margin) and GCE 28 (isolated from the contrast-enhanced core), which reflect GBM tumour characteristics that are observed clinically. Cell culture GIN 28 cells were isolated from the 5-aminolevulinic acid (5ALA) fluorescing infiltrative tumour margin and GCE 28 were isolated from the core central region of a GBM patient who underwent surgery at the Queen’s Medi- cal Centre, University of Nottingham (Nottingham, UK) using the method described earlier (Smith et  al., 2017; Smith et al., 2020). Low-passage patient-derived GIN 28 and GCE 28 cells were cultured in DMEM (Gibco) sup- plemented with 10% FBS, 1% Penicillin/Streptomycin, and 1% L-Glutamine. Human derived cortical astrocytes (HA-COR) were obtained from ScienCell (Cat. No. 1800, Batch No. 24490) and were cultured in astrocyte medium (AM) containing 2% FBS, 1% astrocyte growth supple- ment, 1% Penicillin/Streptomycin from ScienCell. Cells Page 4 of 9 Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine resonance of 45 nm GNPs (Haiss et al., 2007), while the 365 nm peak in ZnO arises from the intrinsic band-gap absorption due to electron transitions from the valence band to the conduction band (O → Zn ) (Khokhra et al., 2017). Furthermore, a sharp and narrow absorption peak is a characteristic of uniform dispersion of monodisperse GNPs and ZnO NPs. FTIR of ­SiO2 NPs (Fig. 1E) showed two broad absorption peaks cantered at 795 ­cm-1 and 1055 ­cm-1 corresponding to bending vibrational modes of the Si-O-Si groups (Brassard et al., 2011). To under- stand the colloidal behaviour of these NPs, we carried out ζ (Fig. 1F) and dynamic light scattering (DLS) (Fig. 1G) measurements to determine their ­hd and surface charge. Previous studies have reported that ζ and ­hd of NPs can influence their interaction with cells. For instance, NPs with smaller ­hd have higher diffusion constant but weak interaction with cells and vice versa with ζ (Villanueva- Flores et al., 2020). Furthermore, surface charge and ­hd plays a key role in determining the polarization and movement of conducting and insulating NPs under the influence of EF via electrophoresis and DEP, respectively (Zhao & Bau, 2009). Therefore, it is important to balance ­hd and ζ of the selected NPs for optimal cellular and EF interaction. In general, NPs with ζ values of ≥ - 30 mV or ≥ + 30 mV are considered to have optimal colloidal stability for biological application due to the presence of sufficient repulsive forces (Skoglund et al., 2017). Cell culture Citrate capped GNPs and ZnO NPs showed a mean ζ value of – 39.2 ± 2.2 mV and -36.5 ± 1 mV which is attributed to the charge of citrate and oxide ions, respectively, sug- gesting good physical colloidal stability. In contrast, ­SiO2 NPs showed a positive zeta potential value of +12.2 ± 0.9 mV due to the presence of -NH2 groups on the surface of these NPs indicating presence of weak repulsive forces and moderate colloidal stability. DLS analysis indicated a monodisperse sample of GNPs with a ­hd of 43.8 ± 3.2 nm. A slight increase in the size of ZnO ­(hd = 69.4 ± 6.7 nm ) and ­SiO2 ­(hd = 50.7 ± 5.5 nm) NPs compared to TEM measurements, further confirmed the agglomera- tion of these NPs in colloidal solution. Collectively, the data indicate that these inorganic NPs have optimal phys- icochemical properties for biological applications. Physicochemical Properties of NPs Physicochemical Properties of NPs Inorganic NPs such as GNPs, ­SiO2, and ZnO present several advantages for biological application such as excellent biocompatibility, wide surface conjugation chemistry, and colloidal stability (Sperling et  al., 2008; Hosseinpour et al., 2020; Jiang et al., 2018). Importantly due to a large difference in the dielectric constant ­(SiO2 > ZnO > GNPs) (Abdelhalim et al., 2011; Ahmad et al., 2019; Dutta & De, 2007), these NPs are best suited to gain further insight into the role of EF in GBM therapy. Previ- ous literature indicates that nano-bio interaction depends on size; therefore, we chose spherical ~50 nm NPs to demonstrate EF effects as this NP diameter is shown to be optimum for achieving high cell uptake (Shang et al., 2014; Zhu et al., 2013; Chithrani et al., 2006; Mittag et al., 2021). Characterizationh Collectively, the data indicate that these inorganic NPs have optimal phys- icochemical properties for biological applications. The size and morphology of the NPs were analyzed using a transmission electron microscope (JEOL 2000 FX TEM) operating at 200 kV accelerating voltage. TEM samples were prepared by dropping 15 mL of NP solu- tion on a carbon-coated copper grid (400 Mesh, Agar Scientific), where the samples were allowed to sit on the grid for at least 15 minutes before analyses. Fourier- transform infrared spectroscopy (FTIR) was carried out by drying silica NPs at 37°C for 48 hours and finally plac- ing the dry sample onto a Cary 630 FTIR spectrometer (Agilent Technologies Ltd) for the measurement of trans- mittance spectra. UV-Vis absorption spectrum of ZnO and GNPs was recorded on a Cary 3500 UV-Vis (Agilent Technologies Ltd). The hydrodynamic diameter ­(hd) and Zeta potential (ζ) of the NPs were monitored on a Mal- vern Zetasizer Nano-ZS (Malvern Instruments, UK). Statistical analysis All the statistical analyses were performed using Graph- Pad Prism v9.2.0 software (GraphPad Software, Inc). All the data are expressed as mean ± S.D., unless speci- fied. For responses that were affected by more than one variable, a two-way ANOVA with a Tukey multiple com- parison post-test was used, and a p-value of ≥ 0.05 was considered significant. Characterizationh resonance of 45 nm GNPs (Haiss et al., 2007), while the 365 nm peak in ZnO arises from the intrinsic band-gap absorption due to electron transitions from the valence band to the conduction band (O → Zn ) (Khokhra et al., 2017). Furthermore, a sharp and narrow absorption peak is a characteristic of uniform dispersion of monodisperse GNPs and ZnO NPs. FTIR of ­SiO2 NPs (Fig. 1E) showed two broad absorption peaks cantered at 795 ­cm-1 and 1055 ­cm-1 corresponding to bending vibrational modes of the Si-O-Si groups (Brassard et al., 2011). To under- stand the colloidal behaviour of these NPs, we carried out ζ (Fig. 1F) and dynamic light scattering (DLS) (Fig. 1G) measurements to determine their ­hd and surface charge. Previous studies have reported that ζ and ­hd of NPs can influence their interaction with cells. For instance, NPs with smaller ­hd have higher diffusion constant but weak interaction with cells and vice versa with ζ (Villanueva- Flores et al., 2020). Furthermore, surface charge and ­hd plays a key role in determining the polarization and movement of conducting and insulating NPs under the influence of EF via electrophoresis and DEP, respectively (Zhao & Bau, 2009). Therefore, it is important to balance ­hd and ζ of the selected NPs for optimal cellular and EF interaction. In general, NPs with ζ values of ≥ - 30 mV or ≥ + 30 mV are considered to have optimal colloidal stability for biological application due to the presence of sufficient repulsive forces (Skoglund et al., 2017). Citrate capped GNPs and ZnO NPs showed a mean ζ value of – 39.2 ± 2.2 mV and -36.5 ± 1 mV which is attributed to the charge of citrate and oxide ions, respectively, sug- gesting good physical colloidal stability. In contrast, ­SiO2 NPs showed a positive zeta potential value of +12.2 ± 0.9 mV due to the presence of -NH2 groups on the surface of these NPs indicating presence of weak repulsive forces and moderate colloidal stability. DLS analysis indicated a monodisperse sample of GNPs with a ­hd of 43.8 ± 3.2 nm. A slight increase in the size of ZnO ­(hd = 69.4 ± 6.7 nm ) and ­SiO2 ­(hd = 50.7 ± 5.5 nm) NPs compared to TEM measurements, further confirmed the agglomera- tion of these NPs in colloidal solution. In vitro Toxicity of NPs TEM analysis revealed that the mean diameter of GNPs is ~ 42 ± 4 nm (Fig. 1A) with homogenous spherical morphology, while ­SiO2 (Fig. 1B) and ZnO (Fig. 1C) were observed to agglomerate with a mean diameter of ~ 46 ± 7 nm and ~ 40 ± 11 nm, respectively. UV-Vis spectros- copy of GNPs and ZnO NPs (Fig. 1D) dispersed in phos- phate buffer saline (PBS) showed distinctive absorption peaks at 529 nm and 365 nm, respectively. The absorp- tion peak at 529 nm is attributed to surface plasmon Before investigating the effect of using NPs in conjunc- tion with TTFields, it was important to ascertain the toxicity of the different NPs on HA-COR (healthy cells), GIN 28 and GCE 28 cells. An experiment was therefore carried out to investigate the effect of increasing the NP concentration from 0 to 50 μg/ mL, by using PrestoBlue assay, which reports on the metabolic activity of cells as an indicator of cell viability (Peng et al., 2020; Xu et al., 2015). However, the limitation of this assay is that it does Jain et al. Bioelectronic Medicine (2022) 8:17 Page 5 of 9 Jain et al. Bioelectronic Medicine (2022) 8:17 Fig. 1  Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – ­SiO2). Transmission electron microscopy images of (A) GNPs, (B) ­SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of ­SiO2 NPs; F Zeta potential; and G Hydrodynamic diameter obtained using DLS. Error bars represent the standard deviation of the mean n=3; N= 3 Fig. 1  Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – ­SiO2). Transmissio images of (A) GNPs, (B) ­SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of ­S and G Hydrodynamic diameter obtained using DLS. Error bars represent the standard deviation of the mean n=3; N= 3 Fig. 1  Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – ­SiO2). Transmission electron microscopy images of (A) GNPs, (B) ­SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of ­SiO2 NPs; F Zeta potential; and G Hydrodynamic diameter obtained using DLS. TTFields and NPs Mediated Enhanced EF Effects in GBM Cells enhancement with conductive GNPs and semiconducting ZnO NPs, can be further explained by the ability of these conducting and semiconducting NPs to polarize and align themselves with the applied EF to act as bipolar nanoelec- trodes or transducers (Guo et al., 2021; Cao et al., 2018; Li & Anand, 2017). Furthermore, the observed enhanced elec- tric effects could also be due to the bipolar electrophoretic effect, as both the GNPs and ZnO are negatively charged. Moreover, TTFields are known to induce biophysical forces on charged entities, this could have triggered the intracellu- lar movement of GNPs and ZnO, thus enhancing the forces on polar intracellular structures affecting dipole alignment and disruption of mitotic spindle. In contrast, ­SiO2 is a well-known dielectric material which upon cellular uptake increases the impedance of cells. Furthermore, under the influence of TTFields (non-uniform EFs) these NPs experience dielectrophoretic forces which induces their polarization. This polarization could lead to the dielectro- phoretic movement of these particles towards the pole with higher EF intensity thus enhancing the classic intracellular TTField effects (Rominiyi et al., 2021). Cells Encouraged by the optimal biocompatibility of ZnO NPs (≤ 5 μg/mL), GNPs (≤ 50 μg/mL) and ­SiO2 (≤ 50 μg/mL), we then investigated the role of these inorganic NPs in enhancing EF effects in patient derived GBM cells. Dielec- tric properties of tissues, as well as intracellular machin- ery, play an important role in determining the efficacy of TTFields as they are known to inhibit the proliferation of cancer cells by inducing DEP of proteins involved in the cell division process (Hershkovich et  al., 2019; Wenger et  al., 2015). Therefore, we hypothesised that by intro- ducing NPs of different electrical conductivity (dielectric properties), the impedimetric properties of the cells can be modified, allowing further insights into the EF medi- ated cellular response. TTFields were delivered in GIN 28 and GCE 28 cells using the Inovitro laboratory system (Novocure, Haifa, Israel, schematic shown in Fig. 3A) that replicates the effect of the clinically used technique (the ­OptuneTM device). Before the application of TTFields, cells were incubated for 4 hours with either GNPs/ZnO/ SiO2 NPs to allow uptake. We observed that the applica- tion of TTFields alone (300 kHz and1V/cm) for 48 hours decreased the metabolic activity of both GIN 28 (Fig. 3B) and GCE 28 cells (Fig. 3C) by ~ 25% vs. untreated con- trol (p < 0.0003). In vitro Toxicity of NPs Error bars represent the standard deviation of the mean n=3; N= 3 concentration of 50 μg/ mL, and at 10 μg/ mL for GIN 28 and GCE 28 cells. This decrease in metabolic activ- ity by ZnO NPs (concentration = > 5 μg/mL) has been attributed to the generation of reactive oxygen species at higher concentrations (Liu et al., 2017). Nevertheless, the FDA has classified ZnO NPs as a “GRAS” (generally regarded as safe) at lower concentrations (Rasmussen et  al., 2010). Based on the obtained data, a concentra- tion of 5 μg/ mL of each type of NPs was chosen for the in vitro TTFields experiment, as at this concertation no significant change in the metabolic activity of GBM cells was observed for all three types of the NPs. not identify the mechanism of change in metabolic activ- ity. In the cases of GNPs and ­SiO2, there was no effect on the metabolic activity of the HA-COR (Fig. S1), GIN 28 (Fig. 2A), and GCE 28 (Fig. 2B) cells across the concentra- tion range tested. From this, we can infer GNPs and ­SiO2 are not toxic to the healthy HA-COR and patient derived GBM cells used in this study at concentrations up to 50 μg/ mL and are therefore biocompatible. In contrast, as the concentration of ZnO NPs increased, a clear effect on cellular metabolism was observed for all cell types at higher concentrations. ZnO nanoparticles significantly reduced the metabolic activity of healthy HA-COR at a Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine Page 6 of 9 Fig. 2  In vitro toxicity of inorganic NPs on patient derived GBM cells. A GIN 28, and B GCE 28 were incubated with increasing concentration of GNPs, ­SiO2 and ZnO NPs for 4 hours, before changing the media containing the NPs with fresh media. Metabolic activity was determined 48 hours after changing the media, the experiment was run in triplicate, and fluorescence at 590 nm is expressed as % of control (no NPs). Results are expressed as the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 obtained using 2-way ANOVA with a Tukey post-test Fig. 2  In vitro toxicity of inorganic NPs on patient derived GBM cells. In vitro Toxicity of NPs A GIN 28, and B GCE 28 were incubated with increasing concentration of GNPs, ­SiO2 and ZnO NPs for 4 hours, before changing the media containing the NPs with fresh media. Metabolic activity was determined 48 hours after changing the media, the experiment was run in triplicate, and fluorescence at 590 nm is expressed as % of control (no NPs). Results are expressed as the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 obtained using 2-way ANOVA with a Tukey post-test TTFields and NPs Mediated Enhanced EF Effects in GBM Cells TTFields and NPs Mediated Enhanced EF Effects in GBM Cells from triplicate or quadruplicate repeats and two independent experiments ( ld d l d ) d d h d ff Fig. 3  TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – ­SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained at 37°C and 5% ­CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/ mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar represents mean ± S.E.M. from triplicate or quadruplicate repeats and two independent experiments Fig. 3  TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – ­SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained at 37°C and 5% ­CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/ mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar represents mean ± S.E.M. from triplicate or quadruplicate repeats and two independent experiments could be due to the enhanced bipolar electrophoretic (GNPs and ZnO) and dielectrophoretic effects ­(SiO2) mediated by inorganic NPs. Further in vitro studies are required to elucidate the detailed molecular mecha- nism underlying the observed NPs mediated TTFields enhancement. TTFields and NPs Mediated Enhanced EF Effects in GBM Cells Interestingly, inorganic NPs (4 hours) + TTFields (48 hours) treated showed a ~ 40% decrease in metabolic activity compared to untreated control (p <0.0001). Importantly, this corresponds to a ~15% higher decrease in metabolic activity compared to TTFields alone (p = 0.002 for GNPs; 0.0001 for ZnO, and 0.04 for ­SiO2). f To further validate and establish the observed enhanced EF effect on GBM cells with GNPs regard- ing the conductivity of cells, we incubated GIN 28 cells with GNPs at a higher concentration (25 μg/mL) to further increase the intracellular concentration of GNPs cell conductivity. In GNP + TTFields treated group a ~52% and ~75% decrease in the metabolic activity of GIN 28 cells was observed after 48- and 72-h treatment, respectively, which was found to be signifi- cantly higher than both untreated and TTFields treated groups (Fig. 3D). Overall, the obtained data suggest that all three NPs utilized in this work enhance EF mediated anticancer effects in patient derived GBM cells. From the observed in vitro effects, we hypothesise that this Together with our previous observations and the obtained data we tentatively suggest that the inorganic NPs irrespective of their dielectric properties, could acts as EF transducers (Robinson et  al., 2021). The observed (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:1 Page 7 of 9 Jain et al. Bioelectronic Medicine could be due to the enhanced bipolar electrophoretic (GNPs and ZnO) and dielectrophoretic effects (SiO2) Conclusions In summary, conducting GNPs, semiconducting ZnO, Fig. 3  TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – ­SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained at 37°C and 5% ­CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/ mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar represents mean ± S.E.M. Conclusions In summary, conducting GNPs, semiconducting ZnO, and insulating ­SiO2 with excellent physicochemical properties, colloidal stability, and biocompatibility, were investigated as a transducer for enhancing EF activity in vitro. We demonstrated that inorganic NPs Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Page 8 of 9 Page 8 of 9 Page 8 of 9 Jain et al. Bioelectronic Medicine irrespective of dielectric permittivity, enhance the effi- ciency and efficacy of EFs in patient derived GBM cells isolated from intra-tumour regions. The in vitro efficacy was significantly enhanced by GNPs and ZnO treat- ment, which is attributed to the ability of these NPs to polarize and act as bipolar nanoelectrodes/ transduc- ers which can sense external EFs, thereby enhancing electrophoretic effects. We additionally suggest that ­SiO2 NPs may enhance EF effects by increasing the forces exerted due to DEP. Furthermore, we have dem- onstrated that the FDA approved inorganic NPs can be used as nano transducers for enhancing intracellular EF effects. This work further paves the pathway for future studies to systemically deliver these NPs across the BBB to determine the in vivo efficacy. Abbreviations l ld ad MP, Rao AV, Babu KS, Rao GN. Mater Chem Phys. 2019;224:79–84. Ahmad MP, Rao AV, Babu KS, Rao GN. Mater Chem Phys. 2019;224:79 84. Blatt R, Davidi S, Munster M, Shteingauz A, Cahal S, Zeidan A, et al. Front Oncol. 2021;11:2459. 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J Mater Sci Mater Med. 2009;20:11–22. enger C, Salvador R, Basser PJ, Miranda PC. Phys Med Biol. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? 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https://openalex.org/W4306944148
https://tckh.daihoctantrao.edu.vn/index.php/sjttu/article/download/797/767
Vietnamese
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ĐIỀU TRA, ĐÁNH GIÁ VAI TRÒ CỦA CỘNG ĐỒNG TRONG VIỆC BẢO TỒN MỘT SỐ LOÀI CÂY THUỐC QUÝ HIẾM TẠI KHU BẢO TỒN LOÀI HẠT TRẦN QUÝ HIẾM NAM ĐỘNG, TỈNH THANH HÓA
Khoa học
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Abstract: This paper introduces the research results about the role of the community in the conservation of some rare medicinal plant species in the Nam Dong Valuable Gymnosperm Conservation Area, Thanh Hoa province. As a result, we recorded 21 species belonging to the Vietnam Red Data Book (2007) as Endangered (five species) and Vulnerable (16 species). The survey evaluated 10 roles of local organizations and identify the advantages and disadvantages, opportunities and challenges of the community in the conservation and development of medicinal plants. The study also offers 2 main solutions to the conservation and development of medicinal plant resources research area. That situ Conservation (Insitu), also known as protected species live where and Conservation trans (Ex-stu) is the conservation of bonobos in artificial conditions under the supervision of the people. INVESTIGATION AND ASSESSMENT OF THE ROLE OF THE COMMUNITY IN THE CONSERVATION OF SOME RARE MEDICINAL PLANT SPECIES IN THE NAM DONG VALUABLE GYMNOSPERM CONSERVATION AREA, THANH HOA PROVINCE Nguyen Huu Cuong National University of Forestry, Vietnam Email address: nguyenhuucuong.tvr@gmail.com DOI: 10.51453/2354-1431/2022/797 Vol 8. No.3_ August 2022 Vol 8. No.3_ August 2022 TẠP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO ISSN: 2354 - 1431 http://tckh.daihoctantrao.edu.vn/ 2.1. Đối tượng, địa điểm nghiên cứu Đối tượng nghiên cứu: Cộng đồng người dân địa phương, nhà quản lý và một số loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động, tỉnh Thanh Hóa. Địa điểm nghiên cứu: Khu Bảo tồn các loài hạt trần quý hiếm Nam Động, tỉnh Thanh Hóa. Từ khóa: Bảo tồn, cây thuốc, đa dạng sinh học, khu Bảo tồn các loài hạt trần quý hiếm Nam Động, Thanh Hóa. Tóm tắt Bài báo này giới thiệu kết quả nghiên cứu về vai trò của cộng đồng trong việc bảo tồn một số loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động, tỉnh Thanh Hóa. Kết quả điều tra đã ghi nhận 21 loài cây thuốc quý hiếm theo Sách đỏ Việt Nam (2007) với 5 loài mức nguy cấp (EN) và 16 loài mức sẽ nguy cấp (VU), đánh giá 10 vai trò của các tổ chức đoàn thể địa phương và nhận định được những thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển các loài cây thuốc. Nghiên cứu cũng đưa ra được 2 giải pháp chính để bảo tồn và phát triễn nguồn cây thuốc của khu vực nghiên cứu. Đó là Bảo tồn nguyên vị (Instu) hay còn gọi là bảo vệ tại nơi loài đang sống và Bảo tồn chuyển vị (Ex-stu) là bảo tồn các cá thể của loài trong điều kiện nhân tạo dưới sự giám sát của con người. Ngày nhận bài: 21/05/2022 Ngày sửa bài: 15/07/2022 Ngày duyệt đăng: 01/08/2022 Ngày nhận bài: 21/05/2022 Ngày sửa bài: 15/07/2022 Ngày duyệt đăng: 01/08/2022 1. Đặt vấn đề việc bảo tồn cây thuốc quý hiếm có trong Khu bảo tồn. Nhằm tạo lập cơ sở khoa học cho việc đề xuất giải pháp bảo tồn một số loài cây thuốc quý hiếm trên quan điểm sinh thái nhân văn, chúng tôi đã tiến hành điều tra, đánh giá vai trò của cộng đồng trong việc bảo tồn một số loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động, tỉnh Thanh Hóa. Do có tầm quan trọng trong công tác bảo tồn đa dạng sinh học, Khu Bảo tồn các loài hạt trần quý hiếm Nam Động (thành lập theo quyết định 84/QĐ-UBND ngày 08 tháng 01 năm 2014 của Chủ tịch Ủy ban nhân dân tỉnh Thanh Hóa) được xem là một bộ phận của hệ sinh thái karst và rừng núi đá vôi đất thấp ở miền Bắc Việt Nam. Kể từ khi được thành lập các nghiên cứu ở Khu Bảo tồn các loài hạt trần quý hiếm Nam Động chỉ tập trung vào việc nghiên cứu và đánh giá hiện trạng 6 loài cây hạt trần quý hiếm: Thông pà cò (Pinus kwangtungensis), Đỉnh tùng (Cephalotaxus mannii), Dẻ tùng sọc hẹp (Amentotaxus argotaenia), Dẻ tùng sọc rộng (Amentotaxus yunnanensis), Thông đỏ đá vôi (Taxus chinensis), Thông tre lá dài (Podocarpus neriifolius) và một số nghiên cứu về khu hệ thực vật [2, 4]. Tuy nhiên chưa có bất cứ một công trình nghiên cứu cụ thể nào đề cập đến các vai trò của cộng đồng trong ĐIỀU TRA, ĐÁNH GIÁ VAI TRÒ CỦA CỘNG ĐỒNG TRONG VIỆC BẢO TỒN MỘT SỐ LOÀI CÂY THUỐC QUÝ HIẾM TẠI KHU BẢO TỒN LOÀI HẠT TRẦN QUÝ HIẾM NAM ĐỘNG, TỈNH THANH HÓA Nguyễn Hữu Cường Trường Đại học Lâm nghiệp, Việt Nam Địa chỉ email: nguyenhuucuong.tvr@gmail.com DOI: 10.51453/2354-1431/2022/797 Keywords: Conservation, medicinal plants, biodiversity, Nam Dong Valuable Gymnosperm Conservation Area, Thanh Hoa province 72| Vol 8. No.3_ August 2022 TẠP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO ISSN: 2354 - 1431 http://tckh.daihoctantrao.edu.vn/ 2.2. Phương pháp nghiên cứu Phương pháp phỏng vấn: Sử dụng một số câu hỏi cho những người được chọn, phỏng vấn tự do có thể hỏi |73 Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 bảo tồn tài nguyên cây thuốc theo Sách Đỏ Việt Nam (2007) [1], Đỗ Tất Lợi (1999) [3] và đề xuất giải pháp bảo tồn [2, 4, 5, 7] về bất cứ hoạt động nào với những câu hỏi tùy thuộc vào hoàn cảnh khi đó, thứ tự các nội dung cần hỏi có thể thay đổi tùy ý dựa trên câu trả lời của câu hỏi trước đó của người cung cấp thông tin. 3.1. Danh sách các loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động Quá trình điều tra tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động đã xác định được 21 loài cây thuốc quý hiếm (bảng 1) có tên trong Sách đỏ Việt Nam (2007), trong đó: 5 loài ở mức nguy cấp (EN), 16 loài ở mức sẽ nguy cấp (VU). Ngành Hạt kín có số lượng loài cây thuốc quý hiếm lớn nhất với 17 loài (4 loài mức EN, 13 loài mức VU) trong khi đó ngành Dương xỉ và ngành Thông chỉ có 2 đến 3 loài theo tuần tự. Kết quả này cho thấy sư đa dạng, phong phú của các loài cây thuốc quý hiếm tại khu vực. Phương pháp xử lí nội nghiệp: Sau mỗi chuyến đi điều tra thực địa, các mẫu thực vật được thu thập theo quy định và lưu giữ tại Trường Đại học Lâm nghiệp Việt Nam (VNF) và Phòng thực vật của Viện Thực vật Komarov thuộc Viện Hàn lâm Khoa học Nga (LE), các thông tin từ phiếu điều tra được chúng tôi tổng hợp thành một bảng kết quả. Tra cứu và đánh giá về giá trị Bảng 1. Danh sách các loài cây thuốc quý hiếm ở Khu Bảo tồn các loài hạt trần quý hiếm Nam Động Tt Tên khoa học Tên phổ thông SĐVN, 2007 I. Polypodiopyta Ngành Dương xỉ 1. Polypodiaceae Họ Dương xỉ  1 Drynaria bonii Christ Cốt toái bổ VU 2 Drynaria fortunei (Kunze ex Mett.) J. Sm. Tắc kè đá EN II. Pinophyta Ngành Thông 2. Cephalotaxaceae Họ Phỉ  3 Cephalotaxus mannii Hook.f. Đỉnh tùng VU 3. Taxaceae Họ Thông đỏ 4 Taxus chinensis Pilger Thông đỏ VU III. Magnoliophyta Ngành Ngọc Lan A. Magnoliopsida Lớp Ngọc Lan 4. Annonaceae Họ Na 5 Xylopia pierrei Hance Giền trắng VU 5. Apocynaceae Họ Trúc đào  6 Rauvolfia verticillata (Lour.) Baill. Ba gạc lá vòng VU 7 Rauvolfia cambodiana Pierre ex Pit. Ba gạc cam-pu-chia, Ba gạc lá to VU 6. Balanophoraceae Họ Dó đất 8 Balanophora laxiflora Hemsl. Dương hoa thưa (Nấm đất) EN 7. Bignoniaceae Họ Đinh 9 Markhamia stipulata (Wall.) Seem. Đinh VU 8. Burseraceae Họ Trám 10 Canarium tramdenum Dai. & Yakovl. Trám đen VU 9. Campanulaceae Họ Hoa chuông  11 Codonopsis javanica (Blume) Hook.f. Đảng sâm VU 10. Cucurbitaceae Họ Bầu bí ảng 1. 3. Kết quả nghiên cứu Phương pháp điều tra theo tuyến: Đã tiến hành điều tra trên 7 tuyến với chiều dài các tuyến từ 2,2– 4,1 km, trên toàn bộ khu vực nghiên cứu. Tất cả các vị trí đã được xác định bởi hệ thống định vị Garmin (gpsmap 62s, 78s). Sử dụng các phương pháp nghiên cứu thực vật truyền thống của Nguyễn Nghĩa Thìn (2007) [6]. 3.1. Danh sách các loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động 3.1. Danh sách các loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động Danh sách các loài cây thuốc quý hiếm ở Khu Bảo tồn các loài hạt trần quý hiếm Nam Động 74| Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 Tt Tên khoa học Tên phổ thông SĐVN, 2007 12 Gynostemma pentaphyllum (Thunb.) Makino Giảo cổ lam EN 11. Fabaceae Họ Đậu 13 Sophora tonkinensis Gagnep. Hoa hoè bắc bộ (sơn đậu căn) VU 12. Opiliaceae Họ Rau sắng 14 Melientha suavis Pierre Rau sắng VU 13. Polygonaceae Họ Rau răm 15 Reynoutria multiflora (Thunb.) Moldenke (Fallopia multiflora (Thunb.) Haraldson) Hà thủ ô đỏ VU 14. Primulaceae (Myrsinaceae) Họ Anh thảo (Đơn nem) 16 Ardisia silvestris Pit. Lá khôi VU B. Liliopsida Lớp Hành 15. Arecaceae Họ Cau  17 Calamus nambariensis Becc. (Calamus platyacanthus Warb. ex Becc.) Song mật (Mây gai dẹt) VU 16. Asparagaceae (Convalariaceae) Họ Măng tây (Mạch môn) 18 Disporopsis longifolia Craib Trúc căn thất, Hoàng tinh cách VU 19 Peliosanthes teta Andrews Sâm cau (Sơn mộc đá) VU 17. Orchidaceae Họ Lan  20 Anoectochilus setaceus Blume Lan gấm trung bộ (Lan kim tuyến) EN 18. Trilliaceae Họ Trọng lâu 21 Paris polyphylla Smith Bảy lá một hoa EN Ghi chú: EN - Nguy cấp; VU - Sẽ nguy cấp 3.2. Đánh giá vai trò của cộng đồng địa phương trong công tác bảo tồn một số loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động STT Các tổ chức, đoàn thể địa phương có quan tâm đến các loài cây thuốc trong Khu Bảo tồn các loài hạt trần quý hiếm Nam Động 7 Cộng đồng khác 8 Chính quyền xã 9 Ban quản lý Khu Bảo tồn các loài hạt trần quý hiếm Nam Động 10 Hạt Kiểm lâm huyện huyện Quan Hóa Kết quả điều tra, thảo luận với người dân địa phương trong Khu Bảo tồn các loài hạt trần quý hiếm Nam Động về việc quản lý và sử dụng tài nguyên thiên nhiên, trong đó có các loài cây thuốc quý hiếm, đã xác định có 10 tổ chức đoàn thể địa phương quan tâm (bảng 2). Các tổ chức có quan tâm đến sử dụng tài nguyên cây thuốc của khu vực được xắp xếp theo thứ tự từ 1 đến 10. Thuận lợi (S) - Đất đai màu mỡ, khí hậu lạnh ẩm, phù hợp cho sự phát triển của các loài cây thuốc. - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động có diện tích tự nhiên 647 ha, với trên 502 ha rừng nguyên sinh rất thuận lợi cho việc quản lý các loài cây thuốc. - Nhà nước đang có nhiều chính sách phát triển kinh tế nông thôn để nâng cao đời sống nhân dân, từ đó sẽ giảm bớt các tác động đến nguồn tài nguyên rừng, đặc biệt là các loài cây thuốc quý hiếm. gạ - Thường xuyên xảy ra các hoạt động khai thác lâm sản, lâm sản ngoài gỗ và các loài cây thuốc để buôn bán, - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động khá đa dạng về thực vật làm thuốc. Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức Thuận lợi (S) Khó khăn (W) - Đất đai màu mỡ, khí hậu lạnh ẩm, phù hợp cho sự phát triển của các loài cây thuốc. - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động có diện tích tự nhiên 647 ha, với trên 502 ha rừng nguyên sinh rất thuận lợi cho việc quản lý các loài cây thuốc. - Nguồn nhân lực sẵn có tại địa phương. - Cộng đồng địa phương có kinh nghiệm trong việc sử dụng và gây trồng một số loài cây thuốc như: Khôi nhung, Giảo cổ lam, Thất diệp nhất chi hoa,… - Có các chương trình, dự án đầu tư và phát triển kinh tế nông thôn xóa đói giảm nghèo, tại địa phương. - Nhu cầu của thị trường về cây thuốc là rất lớn - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động nằm trên huyện vùng sâu vùng xa, kinh tế - xã hội chậm phát triển, dân trí thấp, tỷ lệ mù chữ còn cao. - Địa hình trong khu vực khá hiểm trở với nhiều núi cao, bị chia cắt mạnh, giao thông đi lại trong vùng còn gặp nhiều khó khăn. - Một số cấp ủy, chính quyền còn chưa thật sự quan tâm đến công tác xóa đói giảm nghèo cho cộng đồng. - Đời sống nhân dân chủ yếu dựa vào sản xuất nông nghiệp, khai khai thác lâm sản và các loài lâm sản ngoài gỗ. - Nguồn cây thuốc ngoài tự nhiên ngày càng khan hiếm, đặc biệt là một số loài có giá trị cao và một số loài quý hiếm. Cơ hội (O) Thách thức (T) - Hiện nay thị trường về thực vật làm thuốc đang được mở rộng. - Nhà nước đang có nhiều chính sách phát triển kinh tế nông thôn để nâng cao đời sống nhân dân, từ đó sẽ giảm bớt các tác động đến nguồn tài nguyên rừng, đặc biệt là các loài cây thuốc quý hiếm. 3.4. Đề xuất một số giải pháp bảo tồn và phát triễn nguồn cây thuốc của khu vực nghiên cứu - Thường xuyên tuyên truyền và giáo dục cho quần chúng nhân dân, đặc biệt là các thôn bản xa trung tâm về tầm quan trọng của rừng đối với con người, trong đó có các loài cây thuốc. Từ những phân tích về điểm mạnh, điểm yếu, cơ hội và thách thức nêu ở trên, chúng tôi xin đề xuất hai giải pháp chính nhằm bảo tồn và phát triển nguồn thực vật làm thuốc tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động như sau: - Cần nghiêm cấm hoạt động chăn thả gia súc trong Khu bảo tồn. Theo điều 18 của quy chế quản lý rừng (2006) đã chỉ rõ: Nghiêm cấm mọi hoạt động chăn thả gia súc, gia cầm trong Phân khu bảo vệ nghiêm ngặt và Phân khu Phục hồi sinh thái của các VQG hoặc các KBTTN. 3.4.1. Bảo tồn nguyên vị (Instu) hay còn gọi là bảo vệ tại nơi loài đang sống 3.1. Danh sách các loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động Trong số đó, chính quyền thôn, cộng đồng thôm, các hộ gia đình, người khai thác và buôn bán là những tổ chức và cá nhân quan tâm trước tiên nhất đến các loài cây thuốc trong khu vực. Khi xét về ảnh hưởng thì cộng đồng thôn là tổ chức có ảnh hưởng nhiều nhất đến các loài cây thuốc của khu vực. Tuy nhiên, khi đánh giá về vai trò của các tổ chức đến việc quản lý, bảo tồn và sử dụng các loài cây thuốc của khu vực thì Ban quản lý Khu Bảo tồn các loài hạt trần quý hiếm Nam Động, Hạt kiểm lâm huyện Quan Hóa, Chính quyền thôn, người khai thác và buôn bán là các tổ chức và cá nhân có ảnh hưởng mạnh mẽ nhất. Bảng 2. Các tổ chức, đoàn thể địa phương có quan tâm đến các loài cây thuốc trong Khu Bảo tồn các loài hạt trần quý hiếm Nam Động STT Các tổ chức, đoàn thể địa phương có quan tâm đến các loài cây thuốc trong Khu Bảo tồn các loài hạt trần quý hiếm Nam Động 1 Cộng đồng thôn 2 Chính quyền thôn 3 Tổ an ninh thôn 4 Đoàn thể 5 Hộ gia đình 6 Người khai thác và buôn bán các loài cây thuốc |75 Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 3.3. Đánh giá thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển các loài cây thuốc quý hiếm Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức - Ngày nay, xu thế chữa bệnh bằng các loại thuốc có nguồn gốc từ thực vật và chữa bệnh trực tiếp bằng những cây thuốc sẵn có đang được nhiều người quan tâm. - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động khá đa dạng về thực vật làm thuốc. - Thiếu nguồn vốn đầu tư và khoa học kỹ thuật cho việc phát triển nguồn thực vật làm thuốc của khu vực. - Việc truyền đạt các thông tin, kiến thức khoa học cho họ về bảo tồn và phát triển các loài cây thuốc gặp nhiều khó khăn, trở ngại. - Thường xuyên xảy ra các hoạt động khai thác lâm sản, lâm sản ngoài gỗ và các loài cây thuốc để buôn bán, Thuận lợi (S) 3.3. Đánh giá thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển các loài cây thuốc quý hiếm 3.3. Đánh giá thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển các loài cây thuốc quý hiếm Để bảo tồn và phát triển các loài cây thuốc quý hiếm ở khu vực này chúng ta cần đánh giá được những điểm mạnh, điểm yếu, cơ hội và thách thức của vùng để đề xuất các biện pháp bảo tồn và phát triển hợp lý. Kết quả phân tích, tổng hợp được như sau: Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức REFERENCES ngoài tự nhiên đặc biệt là các loài quý hiếm, làm giảm sự tác động vào rừng tự nhiên. [1]. Ban, N.T. (Ed.) 2007. Vietnam Red Data Book, Part 2 - Plants. Natural Science and Technology Publishing House, Hanoi, Vietnam. - Áp dụng các tiến bộ khoa học kỹ thuật hiện đại vào việc nhân giống, gây trồng và chăm sóc các loài cây thuốc. [2]. Center for Environment and Sustainable Forestry Development (2006), The result of the biodiversity survey in Nam Dong Conservation Area, Thanh Hoa, Vietnam. - Cần đầu tư nguồn vốn, giống và các vật tư cần thiết cho những HGĐ gây trồng, chăm sóc các loài cây thuốc đặc biệt là các loài quý hiếm tại các vườn thuốc. [3]. Loi, D.T. (1999), Vietnam medicinal plants and herbs. Science and Technology Publishing House, Hanoi. Ngoài ra, sau khi gây trồng và bảo tồn các loài cây thuốc nhà nước cần phải mở rộng thị trường tiêu thụ, tăng cường quan hệ hợp tác với mục đích tạo đầu ra cho các sản phẩm cây thuốc. [4]. Cuong, N.H., Averyanov L., Egorov A., Doi, B.T. (2020), Conservation status of conifers in Nam Dong Conservation Area (Thanh Hoa Province, northern Vietnam), IOP Conf. Series: Earth and Environmental Science 574 (2020) 012012. doi:10.1088/1755- 1315/574/1/012012 3.4.1. Bảo tồn nguyên vị (Instu) hay còn gọi là bảo vệ tại nơi loài đang sống Với giải pháp bảo tồn này chúng ta cần thực hiện như sau: 3.4.2. Bảo tồn chuyển vị (Ex-stu) là bảo tồn các cá thể của loài trong điều kiện nhân tạo dưới sự giám sát của con người - Ban quản lý Khu Bảo tồn các loài hạt trần quý hiếm Nam Động và chính quyền các cấp trong khu vực cần phải thực hiện tốt công tác quản lý, bảo vệ nguồn tài nguyên rừng, đặc biệt là nguồn cây thuốc quý hiếm. Đối với giải pháp này ta thực hiện như sau: - Cần phải xây dựng các vườn cây thuốc theo quy mô lớn như Công ty dược liệu và quy mô nhỏ trong các Hộ gia đình. Việc trông cây thuốc không những đáp ứng được cho nhu cầu sử dụng tại chỗ của nhân dân, mà còn làm giảm sự phụ thuộc và nguồn nguyên liệu thuốc - Cần nghiêm cấm các hoạt động khai thác và buôn bán trái phép tài nguyên cây thuốc trong Khu bảo tồn. - Cần tăng cường các lực lượng bảo vệ rừng: Kiểm Lâm, Tổ, Đội tuần tra bảo vệ rừng để đảm bảo cho các loài cây thuốc được phát triển tốt. 76| Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 [7]. Cuong,N.H., Egorov A.A. (2021), Medicinal plants of forests in the mountain areas of Nam Dong Reserve (Quan Hoa district, Thanh Hoa province, northern Vietnam). Collection of materials of the international conference in the natural and human sciences, St. Petersburg, 2021. 4. Kết luận Kết quả điều tra đã ghi nhận được 21 loài cây thuốc quý hiếm theo Sách đỏ Việt Nam (2007) với 5 loài mức nguy cấp (EN) và 16 loài sẽ nguy cấp (VU), đánh giá được 10 vai trò của các tổ chức đoàn thể địa phương (Cộng đồng thôn, Chính quyền thôn, Tổ an ninh thôn, Đoàn thể, Hộ gia đình, Người khai thác và buôn bán các loài cây thuốc, Cộng đồng khác, Chính quyền xã, Ban quản lý Khu Bảo tồn các loài hạt trần quý hiếm Nam Động, Hạt Kiểm lâm huyện Quan Hóa,) và nhận định được những thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển các loài cây thuốc. Từ đó, nghiên cứu cũng đưa ra được 2 giải pháp chính để bảo tồn và phát triễn nguồn cây thuốc của khu vực nghiên cứu. Đó là Bảo tồn nguyên vị (Instu) hay còn gọi là bảo vệ tại nơi loài đang sống và Bảo tồn chuyển vị (Ex-stu) là bảo tồn các cá thể của loài trong điều kiện nhân tạo dưới sự giám sát của con người. [5]. Cuong,N.H., Averyanov L., Egorov A., Doi, B.T., Quyet, P.T. (2021), Traditional knowledge on non-medicinal plants used by the tribal people in Nam Dong Commune, Quan Hoa district, Thanh Hoa provice, nothern Vietnam, IOP Conf. Series: Earth and Environmental Science 876 (2021) 012053. doi:10.1088/1755-1315/876/1/012053 [6]. Thin, N.N. (1997), Manual for biodiversity research: Agricultural Publishing House, Hanoi. [7]. Cuong,N.H., Egorov A.A. (2021), Medicinal plants of forests in the mountain areas of Nam Dong Reserve (Quan Hoa district, Thanh Hoa province, northern Vietnam). Collection of materials of the international conference in the natural and human sciences, St. Petersburg, 2021. |77
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English
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Correlated Inter-Domain Motions in Adenylate Kinase
PLOS computational biology/PLoS computational biology
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cc-by
7,524
Introduction DPQ~- m0cPcQh 8p3rPQ3 X ij SijcoswPQ i coswPQ j ð1Þ ð1Þ Conformational heterogeneity as a consequence of dynamics is an intrinsic feature of proteins linked to biological function.[1] An important aspect for our understanding of protein dynamics is a molecular characterization of the structural states that are populated and of how correlated conformational changes can mediate biological functions such as allostery and signal transduc- tion.[2,3] The characterization of correlated conformational changes requires a description of how local structural heteroge- neity translates into global conformational changes through collective motions. Recent developments in the analysis of various NMR parameters at atomic resolution, such as spin relaxation rates[4] and residual dipolar couplings (RDCs),[5–7] have enabled the description of local structural heterogeneity. However, inferences of collective conformational changes from local correlated motions have relied on force fields or motional models.[7–9] In Eq. 1, for two nuclei P and Q, Qi PQ is the angle between the inter-nuclear vector and axis i of the molecular frame, Sij is an element of the alignment tensor (S), rPQ is the distance between P and Q, cX is the gyromagnetic ratio of nucleus X, m0 is the magnetic susceptibility of vacuum and h is Planck’s constant. The alignment tensor S is given by the alignment mechanism and, under steric alignment conditions, can be computed accurately and is closely related to the shape of the aligned macromole- cule.[12–14] RDCs measured in steric alignment combine the local information contained in NMR parameters with the shape information contained in relaxation rates[15–17] and small angle X-ray scattering (SAXS).[18] Since inter-domain motions alter the shape of proteins RDCs measured in steric alignment have, as we will show, the potential to act as reporters of inter-domain structural heterogeneity. RDCs are NMR parameters that report on both the local and global structural properties of weakly aligned macromolecules and, under the assumption that alignment does not alter the properties of the protein, can be used to study the amplitude of dynamics, especially when combined with simulations.[5,10] Alignment can be induced by steric and/or electrostatic interactions of the macromolecule with external media. Specif- ically, for a given conformation, RDCs depend on the geometrical properties of the environment of the nuclei in the molecular frame and on the direction and degree of align- ment[11,12] (Eq. 1, Fig. 1). Abstract Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes. Citation: Esteban-Martı´n S, Fenwick RB, A˚de´n J, Cossins B, Bertoncini CW, et al. (2014) Correlated Inter-Domain Motions in Adenylate Kinase. PLoS Comput Biol 10(7): e1003721. doi:10.1371/journal.pcbi.1003721 Editor: Helmut Grubmu¨ller, Max Planck Institute for Biophysical Chemistry, Germany d February 5, 2014; Accepted May 28, 2014; Published July 31, Copyright:  2014 Esteban-Martı´n et al. This is an open-access article distributed under the terms of the Creative Commons Attribu unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ban-Martı´n 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. Funding: This work was supported by IRB (RBF, CWB, XS), the BSC (SEM, BC, VG), the Swedish Research Council (621-2010-5247 to MWW), ‘‘Umea˚ University Young researcher award’’ (MWW), ICREA (XS) MICINN (CTQ2009-08850-BQU to XS), MINECO (FIS2008-00114 to VG and BIO2012-31043 to XS), Marato´ de TV3 (102030 to XS) and the Juan de la Cierva (SEM) and Ramo´n y Cajal (CWB) programs. The authors acknowledge the use of computational resources of the Red Espan˜ola de Supercomputacio´n (RES). Spanish ministry of education and science MICINN CTQ2009-08850, MINECO FIS2008-00114, MINECO BIO2012-31043 (http://www.mecd.gob.es) Marato´ de TV3 102030. Swedish Research Council (http://www.vr.se/) Grant Number 621-2010-5247. 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. * Email: santiago.esteban.martin@gmail.com (SEM); xavier.salvatella@irbbarcelona.org (XS) Correlated Inter-Domain Motions in Adenylate Kinase Santiago Esteban-Martı´n1*, Robert Bryn Fenwick2, Jo¨ rgen A˚ de´n3, Benjamin Cossins1, Carlos W. Bertoncini2, Victor Guallar1,4, Magnus Wolf-Watz3, Xavier Salvatella2,4* 1 Joint BSC CRG IRB Research Programme in Computational Biology Barcelona Supercomputing Center BSC Barcelona Spain 2 Joint BSC CRG IRB Research Programme Santiago Esteban-Martı´n1*, Robert Bryn Fenwick2, Jo¨ rgen A˚ de´n3, Benjamin Cossins Carlos W. Bertoncini2, Victor Guallar1,4, Magnus Wolf-Watz3, Xavier Salvatella2,4* 1 Joint BSC-CRG-IRB Research Programme in Computational Biology, Barcelona Supercomputing Center - BSC, Barcelona, Spain, 2 Joint BSC-CRG-IRB Research Programme in Computational Biology, Institute for Research in Biomedicine – IRB Barcelona, Barcelona, Spain, 3 Department of Chemistry, Chemical Biological Centre, Umea˚ University, Umea˚, Sweden, 4 Institucio´ Catalana de Recerca i Estudis Avanc¸ats - ICREA, Barcelona, Spain PLOS Computational Biology | www.ploscompbiol.org July 2014 | Volume 10 | Issue 7 | e1003721 Correlated Inter-Domain Motions in Adenylate Kinase An ensemble size of 4 to 64 accounted for the data and equivalent distributions were observed regardless of ensemble size and method used to calculate the RDCs (Figs S3 to S5). It is worth noting that the LID domain of AKe is in equilibrium with a locally unfolded state with a population of ca. 5% at 37uC;[32] under the conditions used in this study (25uC) this state has a low population (,1%). Similarly, cracking of the AMPbd along the closing mechanism may play a role.[26,33] However, cracking occurs at the transition state, and therefore has a very low population. The states obtained for AKe are shown in Fig. 2b. An analysis of the results indicated that substrate free AKe samples three major states corresponding to i) a closed-like state (hLID ,105u, hAMPbd ,60u, population ,0.560.1), where the LID is closed and the AMPbd slightly opened ii) the open state (hLID ,146u, hAMPbd ,74u, population ,0.2560.1), where both nucleotide binding domains are open, and iii) conformations in which the degree of domain opening is intermediate between that of the fully closed and fully open. The estimate of the fraction closed AKe from our analysis is 0.5, which is in agreement with numbers from single molecule FRET studies where fractions of 0.6[29] and 0.3[28] has been enumerated. Results and Discussion Our approach is based on the determination of ensembles that collectively agree with RDCs. The generation of the ensemble is divided in two steps (see Methods and Supporting Information). First, an enumeration (ca. 105) of inter-domain orientations is performed using unrestrained simulations. Secondly, the ensemble of minimal size that best fits the RDCs is identified by a genetic algorithm (see Figs. S1 and S2).[23] To maximize the coverage of conformational space in the first step we used PELE, an all atom Monte Carlo simulation algorithm with a move set designed to explore normal modes (see Methods and Supporting Information).[24] The RDCs of each conformer are calculated via Eq. 1 using two independent methods to compute the S from knowledge of the structure.[12,13] In this case we used only the steric tensor because it is related to shape but it is principle possible to use also the electrostatic tensor, particularly when conformational changes modulate the charge distribution. An analysis of the conformational ensemble allowed us to assess the presence and degree of inter-domain correlation, an important property of AKe. As shown in Fig. 2b, the ATP and AMP domain movements are correlated, disfavouring conformations where the LID is open and the AMPbd is closed. Our findings agree with free energy calculations[34], molecular dynamics simulations[35] as well as with normal mode analysis.[33,36] Although it is impossible to derive the closure pathway from equilibrium data, the conformational states observed favour a step-wise mechanism in which LID closure takes place before AMPbd closure. Such a mechanism would be beneficial because the initial closure of the LID, together with the substantially higher affinity of this domain for its substrate (ATP) as compared to AMP (50 vs 1700 mM),[30] would reduce the probability of non-productive binding of AMP to the LID.[37] AKe is an essential enzyme that catalyses the reversible conversion of ATP and AMP into two ADP molecules. The role of AKe is to maintain the energy balance in the cell, which is essential. AKe is a modular enzyme composed of three sub- domains: a CORE domain responsible for thermal stability[25,26] and two flexible substrate-binding domains referred to as LID and AMPbd (Fig 2a). Author Summary Most proteins contain several domains, and inter-domain motions play important roles in their biological functions. Describing the various inter-domain orientations that multi-domain proteins adopt at equilibrium is challenging, but key for understanding the relationship between protein structure and function. When more than two domains are present in a protein, correlated domain motions can be of fundamental importance for biological function. This type of behaviour is typical of molecular machines but is extremely challenging to characterize both from experimental and theoretical viewpoints. In this paper, we present a hybrid experimental/computational approach to address this problem by exploiting the information on molecular shape contained in nuclear magnetic resonance experiments to determine accurate conformation ensembles for the multi-domain enzyme adenylate kinase with help of advanced simulation methods. the various conformations in fast exchange that contribute to the measured RDC can have different alignment tensors.[20,22] Here we show that for a molecule undergoing conformational changes in two distal sites the RDCs depend on the degree of correlation of such changes. We exploit this to characterize the degree of correlation of the inter-domain conformational changes occurring in the substrate-free state of E. coli Adenylate Kinase (AKe), an enzyme that undergoes conformational changes involving shape changes. We performed additional control simulations to assess the robustness of the distributions obtained. Scrambling the RDCs lead to a list of restraints that could not be fit to any distribution (Fig. S6), showing that the distribution obtained is encoded in the data and not biased by the population of the conformers in the pool. The distribution shown in Fig. 2a was found to be robust to various sources of error, including errors in the experimental RDCs (Fig. S7) and in the prediction of the alignment tensor (Fig. S8). We also performed computational experiments to ensure that a single set of RDCs suffices to distinguish between ensembles with a distinct number of states and identify correlated structural changes (see Figs. S9 to S13). Correlated Inter-Domain Motions in Adenylate Kinase state was better than that of the free state, which we interpreted as evidence for inter-domain dynamics in the substrate-free enzyme. To identify major conformational states sampled by substrate- free AKe we used backbone NH RDCs measured in steric alignment to select ensembles of conformations from a pool derived from simulations started from the open and closed X-ray structures[27],[31] (see Methods). The ensembles collectively agree with the RDCs (Q<0.26) even though the structures they contain do not (Q.0.6) when considered individually. An ensemble size of 4 to 64 accounted for the data and equivalent distributions were observed regardless of ensemble size and method used to calculate the RDCs (Figs S3 to S5). It is worth noting that the LID domain of AKe is in equilibrium with a locally unfolded state with a population of ca. 5% at 37uC;[32] under the conditions used in this study (25uC) this state has a low population (,1%). Similarly, cracking of the AMPbd along the closing mechanism may play a role.[26,33] However, cracking occurs at the transition state, and therefore has a very low population. The states obtained for AKe are shown in Fig. 2b. An analysis of the results indicated that substrate free AKe samples three major states corresponding to i) a closed-like state (hLID ,105u, hAMPbd ,60u, population ,0.560.1), where the LID is closed and the AMPbd slightly opened ii) the open state (hLID ,146u, hAMPbd ,74u, population ,0.2560.1), where both nucleotide binding domains are open, and iii) conformations in which the degree of domain opening is intermediate between that of the fully closed and fully open. The estimate of the fraction closed AKe from our analysis is 0.5, which is in agreement with numbers from single molecule FRET studies where fractions of 0.6[29] and 0.3[28] has been enumerated. state was better than that of the free state, which we interpreted as evidence for inter-domain dynamics in the substrate-free enzyme. state was better than that of the free state, which we interpreted as evidence for inter-domain dynamics in the substrate-free enzyme. To identify major conformational states sampled by substrate- free AKe we used backbone NH RDCs measured in steric alignment to select ensembles of conformations from a pool derived from simulations started from the open and closed X-ray structures[27],[31] (see Methods). The ensembles collectively agree with the RDCs (Q<0.26) even though the structures they contain do not (Q.0.6) when considered individually. Introduction RDCs can be used to study the structural heterogeneity of globular and disordered proteins by analysing the effect of (sub-ms) motional averaging on this NMR parameter. The effect of fast (sub-ns) local motions that do not directly alter the magnitude and main directions of alignment can be analysed in the molecular frame defined by the alignment tensor of the average struc- ture.[19,20] The analysis of motions that change the shape of the protein, that are typically slower than the timescale of alignment (0.5 to 5 ns) [21] needs on the contrary to explicitly consider that July 2014 | Volume 10 | Issue 7 | e1003721 1 July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org Correlated Inter-Domain Motions in Adenylate Kinase RDC calculation NH RDCs used in this work for free AKe were measured in stretched polyacrylamide gels.[30] Two independent methods, PALES [12] and ALMOND[13], [13]were used to calculate the alignment tensor using Eq. 1. They gave equivalent distributions as shown in Figs. S3–S5. In Figs. 2 we provide the distributions that lead the best agreement between calculated and experimental RDCs [43] (Tab. S1). For AKe it was obtained using ALMOND. The agreement between calculated and experimental (or refer- ence) RDCs was assessed by the quality factor Q [43] defined in Eq. 2. Here we have used the dependence of RDCs on global molecular shape via S to determine ensembles reporting on the amplitude and degree of correlation of inter-domain motions. Although RDCs are uniquely suited for this, this is a challenging endeavour due to their intrinsic degeneracies.[39] It is therefore relevant to discuss the factors that allowed the approach used in this work to alleviate them. The first factor is that our approach accounts for how shape changes alter the value of the RDC by computing S for each inter-domain orientation i.e. two inter- domain orientations that would have degenerate RDCs when the tensor is assumed to be constant can be differentiated if they have sufficiently large differences in shape (Fig. S14).[39] A second factor is the presence of structural constraints due to the covalent linkage, where steric clashes between domains restrict the available conformational space;[40,41] AKe is a well-structured proteins where this effect is particularly important but for multi-domain proteins with flexible linker sequences RDCs measured in multiple alignment media will be required. Alternatively, RDCs could be complemented with structural restraints derived from smFRET or SAXS data. Q~ rms(Dexp{Dcalc) rms(Dexp) ð2Þ ð2Þ The calculated RDCs were scaled, after averaging across the ensemble, to minimize Q to account for the difficulty of predicting the absolute concentration of alignment medium. We used RDCs corresponding to structured regions in the protein (see Fig. S15). For AKe, we found that RDCs in loop regions were more difficult to fit and therefore we decided to exclude them. However, we note that the ensemble derived including RDCs in the loop regions remains unaffected. We also evaluated the impact of the alignment media by monitoring changes in chemical shifts induced by the presence of the stretched polyacrylamide gel. Results and Discussion Crystallographic structures of AKe have been solved for the open (inactive) and closed (active) states.[27] Substrate-free AKe has been shown to sample a closed-like state using single-molecule Fo¨ster resonance energy transfer (smFRET) experiments.[28,29] We previously reported an analysis of AKe using RDCs in the substrate-free (open) and substrate-bound (closed) states where we fit the RDCs to the corresponding crystallographic structures.[30] We found that the fit of the closed AKe is an essential enzyme that catalyses the reversible conversion of ATP and AMP into two ADP molecules. The role of AKe is to maintain the energy balance in the cell, which is essential. AKe is a modular enzyme composed of three sub- domains: a CORE domain responsible for thermal stability[25,26] and two flexible substrate-binding domains referred to as LID and AMPbd (Fig 2a). Crystallographic structures of AKe have been solved for the open (inactive) and closed (active) states.[27] Substrate-free AKe has been shown to sample a closed-like state using single-molecule Fo¨ster resonance energy transfer (smFRET) experiments.[28,29] We previously reported an analysis of AKe using RDCs in the substrate-free (open) and substrate-bound (closed) states where we fit the RDCs to the corresponding crystallographic structures.[30] We found that the fit of the closed Single-molecule FRET[28,29] and NMR[28] have shown that the open and closed states of AKe are in equilibrium and that nucleotide binding shifts it towards the closed state.[30] Of particular interest are smFRET experiments, as these can resolve conformational states and provide a qualitative validation of the ensemble. Two different reaction coordinates, corresponding to distances between residues in the LID and the AMPbd (Aquifex July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org 2 Correlated Inter-Domain Motions in Adenylate Kinase Figure 1. RDCs report on molecular shape. RDCs depend on the geometry of the relevant nuclei (Qi) and on the degree and direction of alignment (S) of the molecule with respect to the laboratory frame. S is a 363 symmetric and traceless matrix, which contains information on the shape of the molecule. Qi is the angle between the inter-nuclear vector and axis i of the molecular reference frame where S is defined. The direction and magnitude of alignment are shown as eigenvectors (eˆi, i = 1, 2, 3) and eigenvalues (vector size, not in scale) for AKe in the open (left) and closed (right) states. doi:10.1371/journal.pcbi.1003721.g001 Figure 1. RDCs report on molecular shape. Results and Discussion RDCs depend on the geometry of the relevant nuclei (Qi) and on the degree and direction of alignment (S) of the molecule with respect to the laboratory frame. S is a 363 symmetric and traceless matrix, which contains information on the shape of the molecule. Qi is the angle between the inter-nuclear vector and axis i of the molecular reference frame where S is defined. The direction and magnitude of alignment are shown as eigenvectors (eˆi, i = 1, 2, 3) and eigenvalues (vector size, not in scale) for AKe in the open (left) and closed (right) states. doi:10 1371/journal pcbi 1003721 g001 g doi:10.1371/journal.pcbi.1003721.g001 AK)[28] and between residues in the LID and the CORE domain (AKe),[29] have been studied. In the former the authors resolved two states in which open conformations were populated to ca. 70%, whereas in the latter the authors found that the open conformation was disfavoured, with a population of ca. 40%. These results can be rationalized based on the ensemble. Following the approach of Beckstein et al.,[38] where distances corresponding to the open and closed states are mapped in domain angle space, we estimated open populations along the LID- AMPbd and LID-CORE smFRET reaction coordinates .50% and ,30%, respectively (for LID-AMPbd large uncertainties are found due to difficulties in assigning open and closed states as quantified by smFRET). Overall, the ensemble is able to qualitatively reconcile two apparently contradictory smFRET studies, which also suggests that steric alignment does not significantly alter the structural heterogeneity of AKe. AK)[28] and between residues in the LID and the CORE domain (AKe),[29] have been studied. In the former the authors resolved two states in which open conformations were populated to ca. 70%, whereas in the latter the authors found that the open conformation was disfavoured, with a population of ca. 40%. These results can be rationalized based on the ensemble. Following the approach of Beckstein et al.,[38] where distances corresponding to the open and closed states are mapped in domain angle space, we estimated open populations along the LID- AMPbd and LID-CORE smFRET reaction coordinates .50% and ,30%, respectively (for LID-AMPbd large uncertainties are found due to difficulties in assigning open and closed states as quantified by smFRET). Overall, the ensemble is able to qualitatively reconcile two apparently contradictory smFRET studies, which also suggests that steric alignment does not significantly alter the structural heterogeneity of AKe. binding allostery. Results and Discussion Our results, therefore, reinforce that correlated inter-domain motions in proteins can mediate important bio- chemical processes. PLOS Computational Biology | www.ploscompbiol.org RDC calculation As shown in the Supporting Figure S16, no significant chemical shift perturbations were observed when apo AKe was immersed into the anisotropic phase. As a reference the chemical shift perturbations induced by binding of the inhibitor Ap5A to AKe are also displayed. A thorough analysis of the robustness of the procedure to various source of error is provided as Supporting Information. To select the optimum ensemble size we monitored the agreement with RDCs used to guide the genetic algorithm and to RDCs left out of this process or free RDCs. Ten sets of randomly removed RDCs (20%) or free RDCs sets were used and 20 independent calculations were run for each set of randomly removed RDCs. Correlated motions in proteins are thought to mediate biochemical functionality.[42] Recently, we have identified weak long-range correlated motions in a surface patch of ubiquitin involved in molecular recognition.[7] Here, we move a step forward and find correlated domain movements in a representa- tive multi-domain enzyme. The observed inter-domain correlation suggests functional roles in allowing ligand access, in adopting the inter-domain orientation necessary for catalysis as well as in July 2014 | Volume 10 | Issue 7 | e1003721 3 Correlated Inter-Domain Motions in Adenylate Kinase Figure 2. Determination of correlated inter-domain conformational heterogeneity in AKe from RDCs. (A) X-ray structure of substrate free open AKe (pdb code 4ake). The CORE (yellow), LID (red) and AMPbd (cyan) domains are shown. The angles used to describe inter-domain conformational changes (see methods) are shown. (B) Inter-domain orientation distribution for AKe. The robustness of the distributions against various sources of error is discussed in the Supporting Information. (C) Representative conformations for each basin are shown together with angles and their estimated populations; little structural heterogeneity occurs within basins. The uncertainties in the populations due to estimated experimental in the measurements of the RDCs and computational errors are estimated to 10%. doi:10.1371/journal.pcbi.1003721.g002 Figure 2. Determination of correlated inter-domain conformational heterogeneity in AKe from RDCs. (A) X-ray structure of substrate free open AKe (pdb code 4ake). The CORE (yellow), LID (red) and AMPbd (cyan) domains are shown. The angles used to describe inter-domain conformational changes (see methods) are shown. (B) Inter-domain orientation distribution for AKe. The robustness of the distributions against various sources of error is discussed in the Supporting Information. Reference pool of conformations An extensive set of inter-domain conformations (ca. 105) was generated for AKe. The X-ray structures 1ake [31] and 4ake [27] were used as seeds. Trajectories were generated where either the AMP or ATP binding domain open (or closed). From each state along the trajectory a second trajectory was generated where the other domain was forced to open (or to close). These simulations were performed with the molecular simulation package CHARMM c35.[44] A time step of 1.0 fs was used. Simulations were performed at 300K. A shape-term (biasing) potential on the backbone atoms was used. The CHARMM commands "CON- Straint HARmonic BEStfit COMParison" and the IC table tool (CONStraint IC BOND ANGL IMPR DIHE) were used to sample inter-domain orientations between open and closed RDC calculation (C) Representative conformations for each basin are shown together with angles and their estimated populations; little structural heterogeneity occurs within basins. The uncertainties in the populations due to estimated experimental in the measurements of the RDCs and computational errors are estimated to 10%. doi:10.1371/journal.pcbi.1003721.g002 The ensemble was qualitatively validated against two independent smFRET experiments (see results and discussion). conformations. The initial force was set to 0.1 and exponentially increased by a factor of 1.05 during 200 cycles of 100 steps each. The degree of opening ranged between ,30–90u and ,90–160u for the AMP- and ATP-binding domains (see angle definitions for AKe). The angles sampled covered the range observed by known X-ray structures of AKe and related proteins. [38] An advanced sampling strategy based on the PELE [24] method was used (see Protein Energy Landscape Exploration, see Supporting Informa- tion). This allowed identifying high-energy conformations which are unlikely to be of sufficiently low energy to be present in the crystalline state or sampled by conventional MD. Searching algorithm An in-house genetic algorithm GA was developed to efficiently search within the pool of structures used to determine the experimental inter-domain orientation distributions of T4L and AKe. Initially 1000 ensembles were generated with structures July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org 4 Correlated Inter-Domain Motions in Adenylate Kinase randomly selected from a pool of ca. 105 conformations (see reference pool of conformations above). Ensembles of size N = 1, 2, 4, 8, 16, 32 and 64 were used. The 103 ensembles of size N were submitted to evolution through 1000 steps. At each step two new sets of 103 ensembles were generated by mutation and crossing operations, leaving 36103 ensembles. At each step the best 103 ensembles, based on the value of Q (see below), were retained. After 1000 iterations the best ensemble was saved and the complete procedure was repeated 200 times. Because too high mutation and recombination rates may lead to loss of good solutions and to premature convergence of the GA, these parameters evolved during the calculations. Initially, the mutation and crossing rates were set to 100% and 2%, respectively. At each step new mutation and crossing rates were determined by dividing/multiplying them by a factor of 1.001, respectively. In Figs. S1 and S2 a scheme illustrating the GA and the convergence of the method are shown. The error in the population of the states observed were determined from the influence of RDC experi- mental uncertainty by performing 200 calculations with random Gaussian error added to each experimental RDC. We used a standard deviation of 1.0 Hz for AKe, three times the estimated experimental error. iterations. b, Mutation and crossing operation rates applied at each step of the ensemble refinement. (TIF) iterations. b, Mutation and crossing operation rates applied at each step of the ensemble refinement. (TIF) Figure S3 Fitting of NH RDCs for AKe and determina- tion of the optimum ensemble size by monitoring the agreement with RDCs used to guide the genetic algorithm and RDCs left out of the calculation (20%). The results obtained using two independent methods to calculate the alignment tensor, ALMOND and PALES, are shown. The Qfactor for working (Qwork; restrained) and free (Qfree; unrestrained) RDCs are shown (see Supporting Text S1). For each ensemble size 200 independent calculations were performed and the results pooled. Inter-domain angle definitions The definition used by Beckstein et al. [38] was adopted in this work. Briefly, the angle formed between the AMP-binding domain (AMPbd) and the core domain (CORE) was determined as the angle formed by two vectors: Vector 1 connects the centers of mass (Ca atoms) of CORE residues 90–100 with residues 115–125 (‘‘hinge region’’ between the CORE and LID domains). Vector 2 connects the centers of mass of CORE residues 90–100 and AMPbd residues 35–55. The angle formed between the ATP- binding domain (LID) and the CORE was defined equivalently using the angle formed between two vectors: Vector 1 connects the centers of mass of residues 115–125 (‘‘hinge region’’ CORE–LID domains) with CORE residues 179–185. Vector 2 connects the centers of mass for residues 115–125 (‘‘hinge region’’ CORE–LID) with LID residues 125–153. Figure S5 Experimental AKe inter-domain orientation distributions obtained for ensembles of several sizes (see Supporting Text S1). The distributions were obtained using PALES to calculate the alignment tensor (see Fig. S4 for distributions obtained using the ALMOND method). For each ensemble size 200 independent calculations were performed and the results pooled. The x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. (TIF) Figure S6 Distributions obtained for AKe after random- ly scrambling the experimental RDCs to obtain incor- rect lists of restraints (see Supporting Text S1). The Q factor obtained in all cases was .0.9, indicating that it was not possible to fit the incorrect data to any physically possible distribution of inter-domain orientations; for each ensemble size a different list of randomly scrambled RDCs was used. For AKe, the x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. (TIF) Searching algorithm Figure S4 Experimental AKe inter-domain orientation distributions obtained for ensembles of several sizes (see Supporting Text S1). The distributions were obtained using the ALMOND method to calculate the alignment tensor (see Fig. S5 for distributions obtained using PALES). For each ensemble size 200 independent calculations were performed and the results pooled. The x and y axis correspond to the hLID and hAMPbd angles (in degrees) shown in Fig. 3a. (TIF) 5. Clore GM, Schwieters CD (2004) How much backbone motion in ubiquitin is required to account for dipolar coupling data measured in multiple alignment media as assessed by independent cross-validation? J Am Chem Soc 126: 2923– 2938. Supporting Information Figure S1 Schematic flow-chart showing the steps involved in the calculation of inter-domain conforma- tional heterogeneity from RDCs. In step 1 a pool of conformations is generated. In step 2 an algorithm for searching ensembles of structures that match experimental data is used (see Methods in the main text). The algorithm randomly selects 103 ensembles of size N from a pool of ca. 105 conformations generated in the first step. For each value of N the agreement of the ensemble with the experimental RDCs is optimized by substituting ensemble members with structures of the pool as well as by swapping structures between ensembles. The best 103 ensembles generated in the second step are retained and the process is repeated until agreement between calculated and experimental RDCs is reached. The optimal value of N is determined by monitoring the agreement with RDCs used to guide the genetic algorithm (Qwork) and RDCs left out of the calculation (free RDCs, Qfree). (TIF) Figure S7 Impact of error in the experimentally mea- sured RDCs of AKe. The fitting of RDCs (Qwork) as well as the distributions obtained are shown. Random Gaussian error was added to the RDCs prior to ensemble calculations. The results shown were obtained using the ALMOND method to calculate the alignment tensor (see Supporting Text S1). For AKe, The x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. Figure S8 Experimental distributions of AKe as a function of error in the prediction of the alignment magnitude (see Supporting Text S1). The RDCs of each conformation were scaled by a number which depended on its position along the RC(s) used in this work. The error was increased exponentially along the RC(s) reaching at the end points of the RC a value of 2, 3, 10, 20 and 50 fold. The x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. Figure S2 Genetic algorithm. a, Convergence of the algorithm developed in this work to select ensembles of structures that match RDCs. Text S1 Supporting methods. (DOCX) Text S1 Supporting methods. (DOCX) Figure S13 Agreement with RDCs left out of the calculations (20%) for AKe protein for the synthetic distributions shown in Figs. S11 and S12 (see Supporting Text S1). The Qfactor for working (Qwork; restrained) and free (Qfree; unrestrained) RDCs is shown. Distributions rebuilt with added random Gaussian error with standard deviations of 1% (first row), 3% (second row) and 6% (third row) of error with respect the maximum coupling are shown. 6. Lange OF, Lakomek N-A, Fare`s C, Schro¨der GF, Walter KFA, et al. (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320: 1471–1475. 7. Fenwick RB, Esteban-Martı´n S, Richter B, Lee D, Walter KFA, et al. (2011) Weak Long-Range Correlated Motions in a Surface Patch of Ubiquitin Involved in Molecular Recognition. J Am Chem Soc 133: 10336–10339. Author Contributions Conceived and designed the experiments: SEM XS. Performed the experiments: SEM RBF JA˚ BC. Analyzed the data: SEM RBF VG MWW. Contributed reagents/materials/analysis tools SEM RBF JA˚ BC VG MWW. Wrote the paper: SEM RBF CWB MWW XS. Conceived and designed the experiments: SEM XS. Performed the experiments: SEM RBF JA˚ BC. Analyzed the data: SEM RBF VG MWW. Contributed reagents/materials/analysis tools SEM RBF JA˚ BC VG MWW. Wrote the paper: SEM RBF CWB MWW XS. g 8. Clore GM, Schwieters CD (2004) Amplitudes of protein backbone dynamics and correlated motions in a small alpha/beta protein: correspondence of dipolar Supporting Information Both data sets, restrained (Qwork) and unrestrained RDCs (Qfree), reached a plateau region after 400 July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org 5 Correlated Inter-Domain Motions in Adenylate Kinase Figure S9 Reconstruction of a synthetic (computer designed) unimodal distribution represented by a magenta box with an increasing number of conforma- tions, N = 1, 8 and 32. Ensembles with 8 or more members fit equally well the synthetic RDCs and best predicted RDCs (20%) left out of the calculations (see Supporting Text S1). The x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. (TIF) Figure S14 Maximum deviation in the fitted popula- tions as a function of the alignment tensor prediction error for AKe protein for computer designed distribu- tions (see Supporting Text S1). The target distributions covered closed/open states whose populations varied between 0% to 100% in steps of 1 percent. For each degree of random error added 100 independent runs were performed. (TIF) Figure S15 Sequence (first raw) and secondary struc- ture of AKe in the open (pdb code 4ake; second raw) and closed (pdb code 1ake; third raw) states. $ indicates that the RDC was used in the calculations. Residues labeled with * were excluded. The label – indicates that the RDC was not available. The AMPbd (residues 28–72) and LID (113–176) domains are highlighted in red and yellow, respectively. Residues 1–27, 73–112 and 176–214 correspond to the CORE domain. (TIF) Figure S10 Reconstruction of a synthetic (computer designed) four state distribution represented by magenta boxes of equal population with N = 1, 2, 4, 8, 16 and 32. Ensembles with 16 or more members fit equally well the synthetic RDCs and best predicted RDCs (20%) left out of the calculations (see Supporting Text S1). The x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. (TIF) 1. Eisenmesser EZ, Millet O, Labeikovsky W, Korzhnev DM, Wolf-Watz M, et al. (2005) Intrinsic dynamics of an enzyme underlies catalysis. Nature 438: 117– 121. 2. Kern D, Zuiderweg ERP (2003) The role of dynamics in allosteric regulation. Curr Opin Struct Biol 13: 748–757. (TIF) Table S1 Agreement between the calculated and exper- imental RDCs for AKe. Scalc refers to the result obtained when S is predicted based on the atomic coordinates of each protein conformation, whereas Sfit refers to the result obtained when S was fit using single value decomposition. The Q values are those of the final ensemble. (DOCX) Figure S12 Reconstruction of synthetic multi-modal inter-domain orientation distributions of AKe represent- ed by magenta boxes of equal population from NH RDCs (see Supporting Text S1). Distributions rebuilt with added random Gaussian error with standard deviations corresponding to 1%, 3% and 6% of error respect the maximum coupling are shown. The best ensemble size was determined by monitoring the agreement with RDCs used to guide the genetic algorithm (Qwork) and RDCs left out of the calculation (free RDCS, Qfree) (Fig. S13). The x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. (TIF) Table S2 Average structural root-mean-squared devia- tion (RMSD) between the calculated ensemble members and reference X-ray structures for AKe. (DOCX) Table S3 NH RDCs measured for E. coli AKe. RDCs used in ensemble refinement are shown in Fig. S15 (average experimental error ,0.3 Hz). (DOCX) (TIF) Figure S16 Alignment of AKe does not induce significant chemical shift perturbations. Shown are chemical shift perturbations to apo AKe induced by the inhibitor Ap5A (red) and by introduction of the enzyme into the anisotropic stretched polyacrylamide gel (black). Chemical shift perturbations were calculated according to: dv = 0.2|D15N|+| D1H |. (TIF) Figure S11 Reconstruction of synthetic unimodal inter- domain orientation distributions of AKe represented by a magenta box from NH RDCs (see Supporting Text S1). Distributions rebuilt with added random Gaussian are shown (standard deviations of 1%, 3% and 6% with respect to the maximum coupling). The best ensemble size was determined by monitoring the agreement with RDCs used to guide the genetic algorithm (Qwork) and RDCs left out of the calculation (free RDCS, Qfree) (Fig. S13). The x and y axis correspond to the hAMPbd and hLID angles (in degrees) shown in Fig. 2a. (TIF) Correlated Inter-Domain Motions in Adenylate Kinase Correlated Inter-Domain Motions in Adenylate Kinase coupling and heteronuclear relaxation measurements. Biochemistry 43: 10678– 10691. 26. Olsson U, Wolf-Watz M (2010) Overlap between folding and functional energy landscapes for adenylate kinase conformational change. Nat Commun 1: 111. 9. Bouvignies G, Bernado´ P, Meier S, Cho K-, Grzesiek S, et al. (2005) Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings. Proc Natl Acad Sci U S A 102: 13885–13890. 27. Mu¨ller CW, Schlauderer GJ, Reinstein J, Schulz GE (1996) Adenylate kinase motions during catalysis: an energetic counterweight balancing substrate binding. Structure 4: 147–156. 10. De Simone A, Richter B, Salvatella X, Vendruscolo M (2009) Toward an accurate determination of free energy landscapes in solution states of proteins. J Am Chem Soc 131: 3810–3811. g 28. Henzler-Wildman KA, Thai V, Lei M, Ott M, Wolf-Watz M, et al. (2007) Intrinsic motions along an enzymatic reaction trajectory. Nature 450: 838–844. g zler-Wildman KA, Thai V, Lei M, Ott M, Wolf-Watz M, et al. (2 29. Hanson JA, Duderstadt K, Watkins LP, Bhattacharyya S, Brokaw J, et al. (2007) Illuminating the mechanistic roles of enzyme conformational dynamics. Proc Natl Acad Sci U S A 104: 18055–18060. J 11. Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278: 1111– 1114. 30. Ade´n J, Wolf-Watz M (2007) NMR identification of transient complexes critical to adenylate kinase catalysis. J Am Chem Soc 129: 14003–14012. 12. Zweckstetter M, Bax A (2000) Prediction of Sterically Induced Alignment in a Dilute Liquid Crystalline Phase: Aid to Protein …. J Am Chem Soc 122: 3791– 3792. 31. Mu¨ller CW, Schulz GE (1992) Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 A resolution. A model for a catalytic transition state. J Mol Biol 224: 159–177. 13. Almond A, Axelsen JB (2002) Physical interpretation of residual dipolar couplings in neutral aligned media. J Am Chem Soc 124: 9986–9987. 32. Schrank TP, Bolen DW, Hilser VJ (2009) Rational modulation of conforma- tional fluctuations in adenylate kinase reveals a local unfolding mechanism for allostery and functional adaptation in proteins. Proc Natl Acad Sci U S A 106: 16984–16989. 14. Berlin K, O’Leary DP, Fushman D (2010) Structural assembly of molecular complexes based on residual dipolar couplings. J Am Chem Soc 132: 8961– 8972. 15. Correlated Inter-Domain Motions in Adenylate Kinase Bruschweiler R, Liao X, Wright PE (1995) Long-range motional restrictions in a multidomain zinc-finger protein from anisotropic tumbling. Science 268: 886– 889. 33. Whitford PC, Gosavi S, Onuchic JN (2008) Conformational transitions in adenylate kinase. Allosteric communication reduces misligation. J Biol Chem 283: 2042–2048. 16. Tjandra N, Garrett DS, Gronenborn AM, Bax A, Clore GM (1997) Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy. Nat Struct Biol 4: 443–449. 34. Whitford PC, Miyashita O, Levy Y, Onuchic JN (2007) Conformational transitions of adenylate kinase: switching by cracking. J Mol Biol 366: 1661– 1671. 35. Kubitzki MB, de Groot BL (2008) The atomistic mechanism of conformational transition in adenylate kinase: a TEE-REX molecular dynamics study. Structure 16: 1175–1182. 17. Ryabov Y, Suh J-Y, Grishaev A, Clore GM, Schwieters CD (2009) Using the experimentally determined components of the overall rotational diffusion tensor to restrain molecular shape and size in NMR structure determination of globular proteins and protein-protein complexes. J Am Chem Soc 131: 9522–9531. 36. Arora K, Brooks CL (2007) Large-scale allosteric conformational transitions of adenylate kinase appear to involve a population-shift mechanism. Proc Natl Acad Sci U S A 104: 18496–18501. 18. Putnam CD, Hammel M, Hura GL, Tainer JA (2007) X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys 40: 191–285. 37. Sinev MA, Sineva EV, V I, E H (1996) Towards a mechanism of AMP-substrate inhibition in adenylate kinase from Escherichia coli. FEBS Lett 397: 273–276. p y 19. Meiler J, Prompers JJ, Peti W, Griesinger C, Bru¨schweiler R (2001) Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins. J Am Chem Soc 123: 6098–6107. 38. Beckstein O, Denning EJ, Perilla JR, Woolf TB (2009) Zipping and unzipping of adenylate kinase: atomistic insights into the ensemble of open/closed transitions. J Mol Biol 394: 160–176. p J 20. Salvatella X, Richter B, Vendruscolo M (2008) Influence of the fluctuations of the alignment tensor on the analysis of the structure and dynamics of proteins using residual dipolar couplings. J Biomol NMR 40: 71–81. 39. Fischer MW, Losonczi JA, Weaver JL, Prestegard JH (1999) Domain orientation and dynamics in multidomain proteins from residual dipolar couplings. Biochemistry 38: 9013–9022. g p p g J 21. References 1. Eisenmesser EZ, Millet O, Labeikovsky W, Korzhnev DM, Wolf-Watz M, et al. (2005) Intrinsic dynamics of an enzyme underlies catalysis. Nature 438: 117– 121. 2. Kern D, Zuiderweg ERP (2003) The role of dynamics in allosteric regulation. Curr Opin Struct Biol 13: 748–757. 3. del Sol A, Tsai C-J, Ma B, Nussinov R (2009) The Origin of Allosteric Functional Modulation: Multiple Pre-existing Pathways. Structure 17: 1042– 1050. 4. Maragakis P, Lindorff-Larsen K, Eastwood MP, Dror R, Klepeis J, et al. (2008) Microsecond Molecular Dynamics Simulation Shows Effect of Slow Loop Dynamics on Backbone Amide Order Parameters of Proteins. J Phys Chem B 112(19):6155–8. July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org 6 PLOS Computational Biology | www.ploscompbiol.org Correlated Inter-Domain Motions in Adenylate Kinase Meier S, Blackledge M, Grzesiek S (2008) Conformational distributions of unfolded polypeptides from novel NMR techniques. J Chem Phys 128: 052204. 40. Wu¨thrich K (2003) NMR studies of structure and function of biological macromolecules (Nobel Lecture). J Biomol NMR 27: 13–39. 22. De Simone A, Montalvao RW, Vendruscolo M (2011) Determination of Conformational Equilibria in Proteins Using Residual Dipolar Couplings. J Chem Theory Comput 7: 4189–4195. 41. Hus J-C, Salmon L, Bouvignies G, Lotze J, Blackledge M, et al. (2008) 16-fold degeneracy of peptide plane orientations from residual dipolar couplings: analytical treatment and implications for protein structure determination. J Am Chem Soc 130: 15927–15937. 23. Nodet G, Salmon L, Ozenne V, Meier S, Jensen MR, et al. (2009) Quantitative description of backbone conformational sampling of unfolded proteins at amino acid resolution from NMR residual dipolar couplings. J Am Chem Soc 131: 17908–17918. 42. Bru¨schweiler R (2011) Protein dynamics: whispering within. Nat Chem 3: 665– 666. 43. Cornilescu G, Marquardt J, Ottiger M, Bax A (1998) Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid …. J Am Chem Soc 120: 6836–6837. 24. 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Imported Inputs and Egyptian Exports: Exploring the Links
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Provided in Cooperation with: Suggested Citation: Parra, María Dolores; Martínez-Zarzoso, Inmaculada (2015) : Imported inputs and Egyptian exports: Exploring the links, Economics: The Open-Access, Open-Assessment E- Journal, ISSN 1864-6042, Kiel Institute for the World Economy (IfW), Kiel, Vol. 9, Iss. 2015-38, pp. 1-31, https://doi.org/10.5018/economics-ejournal.ja.2015-38 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. You are not to copy documents for public or commercial purposes, to exhibit the documents publicly, to make them publicly available on the internet, or to distribute or otherwise use the documents in public. If the documents have been made available under an Open Content Licence (especially Creative Commons Licences), you may exercise further usage rights as specified in the indicated licence. Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ http://creativecommons.or Parra, María Dolores; Martínez-Zarzoso, Inmaculada Article Imported inputs and Egyptian exports: Exploring the links Economics: The Open-Access, Open-Assessment E-Journal Provided in Cooperation with: Kiel Institute for the World Economy – Leibniz Center for Research on Global Economic Challenges Suggested Citation: Parra, María Dolores; Martínez-Zarzoso, Inmaculada (2015) : Imported inputs and Egyptian exports: Exploring the links, Economics: The Open-Access, Open-Assessment E- Journal, ISSN 1864-6042, Kiel Institute for the World Economy (IfW), Kiel, Vol. 9, Iss. 2015-38, pp. 1-31, https://doi.org/10.5018/economics-ejournal.ja.2015-38 This Version is available at: https://hdl.handle.net/10419/122198 Parra, María Dolores; Martínez-Zarzoso, Inmaculada Article Imported inputs and Egyptian exports: Exploring the links Economics: The Open-Access, Open-Assessment E-Journal Provided in Cooperation with: Kiel Institute for the World Economy – Leibniz Center for Research on Global Economic Challenges Suggested Citation: Parra, María Dolores; Martínez-Zarzoso, Inmaculada (2015) : Imported inputs and Egyptian exports: Exploring the links, Economics: The Open-Access, Open-Assessment E- Journal, ISSN 1864-6042, Kiel Institute for the World Economy (IfW), Kiel, Vol. 9, Iss. 2015-38, pp. 1-31, https://doi.org/10.5018/economics-ejournal.ja.2015-38 This Version is available at: https://hdl.handle.net/10419/122198 Article Imported inputs and Egyptian exports: Exploring the links Economics: The Open-Access, Open-Assessment E-Journal Imported Inputs and Egyptian Exports: Exploring the Links María Dolores Parra and Inmaculada Martínez-Zarzoso Vol. 9, 2015-38 | November 12, 2015 | http://dx.doi.org/10.5018/economics-ejournal.ja.2015-38 Vol. 9, 2015-38 | November 12, 2015 | http://dx.doi.org/10.5018/economics-ejournal.ja.2015-38 Received April 15, 2015 Published as Economics Discussion Paper April 27, 2015 Revised October 28, 2015 Accepted October 28, 2015 Published November 12, 2015 © Author(s) 2015. Licensed under the Creative Commons License - Attribution 3.0 Received April 15, 2015 Published as Economics Discussion Paper April 27, 2015 Revised October 28, 2015 Accepted October 28, 2015 Published November 12, 2015 Abstract This paper is the first to explore the links between exporting and importing activities of Egyptian firms using panel data over the period from 2003 to 2007. The main aim is twofold. Firstly, the authors report regression results indicating that firms that both export and import are the most productive, followed by importing-, exporting-only firms and non-traders. Secondly, they estimate the determinants of the extensive and intensive margins of exports and imports using dynamic panel-Probit and panel-Tobit models in combination with the method proposed by Rabe-Hesketh and Skrondal (Avoiding biased versions of Wooldridge’s simple solution to the initial conditions problem, 2013) to tackle the initial conditions problem. The results show that both activities present a high degree of hysteresis, which is higher for imports than for exports pointing to the existence of sunk costs in both activities. Moreover, past productivity does affect the extensive margin of imports, but not of exports and the initial condition status is also only relevant for the import side. Similar outcomes are obtained for the intensive margin of trade. (Published in Special Issue Micro-econometric Analyses of International Firm Activities) EL F14 Keywords Firm level data; Egypt; internationalization; imports; exports; intermediates © Author(s) 2015. Licensed under the Creative Commons License - Attribution 3.0 Citation María Dolores Parra and Inmaculada Martínez-Zarzoso (2015). Imported Inputs and Egyptian Exports: Exploring the Links. Economics: The Open-Access, Open-Assessment E-Journal, 9 (2015-38): 1—31. http:// dx.doi.org/10.5018/economics-ejournal.ja.2015-38 1 There is only a working paper, Kiendrebeogo (2014), investigating the learning by exporting and selecting into exporting hypotheses for the Egyptian case, but importing activities are disregarded. Authors María Dolores Parra, Research Group of Industry and Territory, Department of Economics – CREIP, Universitat Rovira i Virgili, 43204 Reus, Catalonia, Spain, and Institute of International Economics, Universitat Jaume I, Castellón, 12071, Spain, mparra@eco.uji.es Inmaculada Martínez-Zarzoso, Department of Economics, University of Göttingen, 37001 Göttingen, Germany, and Institute of International Economics, Universitat Jaume I, Castellón, 12071, Spain María Dolores Parra, Research Group of Industry and Territory, Department of Economics – CREIP, Universitat Rovira i Virgili, 43204 Reus, Catalonia, Spain, and Institute of International Economics, Universitat Jaume I, Castellón, 12071, Spain, mparra@eco.uji.es Inmaculada Martínez-Zarzoso, Department of Economics, University of Göttingen, 37001 Göttingen, Germany, and Institute of International Economics, Universitat Jaume I, Castellón, 12071, Spain Inmaculada Martínez-Zarzoso, Department of Economics, University of Göttingen, 37001 Göttingen, Germany, and Institute of International Economics, Universitat Jaume I, Castellón, 12071, Spain Citation María Dolores Parra and Inmaculada Martínez-Zarzoso (2015). Imported Inputs and Egyptian Exports: Exploring the Links. Economics: The Open-Access, Open-Assessment E-Journal, 9 (2015-38): 1—31. http:// dx.doi.org/10.5018/economics-ejournal.ja.2015-38 1 Introduction 1 In recent years, there has been a growing interest in the study of the internationalization strategies of small- and medium-size firms in developing countries. According to the related trade literature, a high proportion of trading firms are engaged in both importing and exporting activities. Kasahara and Lapham (2013) show that this is due to the presence of cost complementarities in both activities. Once one of the activities is carried out, the second becomes easier. These cost complementarities have motivated a new strand of research that further investigates the relationship between import and export activities at the firm level, especially those focused on the use of imported intermediates and their role in enhancing exports (Muûls and Pisu 2009; Bas 2012; Aristei et al. 2013; Kasahara and Lapham 2013; Lo Turco and Maggioni 2013). While most of the existent literature on the relationship between productivity and import and export activities has focused on developed countries, the literature concerning developing countries is still scarce. In particular, a relevant question is whether importing intermediates generates productivity gains that add to the gains arising from learning-by-exporting. It is yet to be established to what extent this is also a source of gains for developing countries, which may profit more than others from having access to intermediates from abroad. Therefore, we aim to extend the existing evidence by investigating export and import activities of firms located in Egypt, a developing country that to the best of our knowledge has not yet been investigated.1 Atiyas (2011) summarizes the research that uses firm-level data in MENA (Middle East and North African Countries) countries to analyse productivity and its relation to trade, trade policy and financial constraints. The author emphasizes the fact that researchers have scarcely utilized the recently available firm-level data covering MENA countries provided by the World Bank Enterprise Survey (WBES) to investigate the relationship between trade and productivity. We focus our analysis on Egypt because is one of the most important countries in the MENA (Middle East and North African) region in terms of population and gross domestic product (GDP), and it is a developing country. According to Smeets and Warzynski (2010) and Bas 2 2 www.economics-ejournal.org and Strauss-Kahn (2011), developing countries are able to profit more than developed countries from the benefits of importing intermediate inputs, which they cannot always produce due to the existence of supply side restrictions. 1 Introduction In this paper, we estimate the determinants of the decision to export/import by using a dynamic panel-Probit model applied to data from approximately 500 Egyptian industrial companies. To analyse the extensive margin of trade, we employ a novel technique based on Rabe-Hesketh and Skrondal (2013) that is able to deal with the endogeneity problem of the lagged dependent variable and that controls for initial conditions in dynamic models. We also test whether the same determinants are important in determining the intensive margin of trade; in this case a tobit procedure is employed. The period analysed spans the years from 2003 to 2007, during which the country experienced reductions in tariff barriers and important changes in trade policy. More specifically, the bilateral interim agreement between the EU and Egypt, signed in 2004, will gradually eliminate tariffs on imported products from the EU and eventually increase competition, thus forcing some firms to exit the market. Simultaneously, decreases in trade costs generated by more flexible rules of origin (RoO) for products traded with the EU had a positive effect on Egyptian exports (Bensassi et al. 2011). The main results show that export and import activities have common sunk costs and that those are higher for import than for export activities. Past Total Factor Productivity (TFP) levels explain the decision to import, but not the decision to export and firms with foreign ownership are more prone to export. Our results also show that past experience in both exporting and importing activities are the most important factors determining internationalization strategies and similar outcomes are found for the extensive and intensive margins of trade. This paper is organized as follows: Section 2 describes the theoretical framework and related empirical literature. Section 3 presents the data and some descriptive statistics. Section 4 reports the importer and exporter premia. Section 5 includes the empirical strategy and outlines the main results, and Section 6 concludes. 3 www.economics-ejournal.org 2 Theoretical framework and literature review The empirical investigations focused on explaining the links between productivity and international trade are rich, where they find evidence confirming the self-selection hypothesis (only firms with high productivity levels become exporters), others support the learning-by-exporting hypothesis (firm productivity www.economics-ejournal.org 4 www.economics-ejournal.org increases after they start exporting). Although most investigations focus on the export side, a few recent papers also consider an import perspective.2 Among the studies that focus on the export side, Bernard and Jensen (1999), Delgado et al. (2002), Arnold and Hussinger (2005) and Aw et al. (2000) find support for the self-selection hypothesis for exports, finding that only the most productive firms are able to start exporting, whereas De Loecker (2007), Bustos (2011), Van Biesebroeck (2005), Rizov and Walsh (2009) and Clerides et al. (1998) find evidence of learning-by-exporting. Nevertheless, the results remain mixed and mainly depend on the characteristics of the countries considered in the analysis. A few authors have investigated the self-selection and learning hypotheses from an import perspective and have analysed the role played by intermediate imports in increasing productivity. On the one hand, Halpern et al. (2011), Amiti and Konings (2007) and Kasahara and Rodrigue (2008) find support for a learning-by-importing effect. On the other hand, Wagner (2007) analyses both hypotheses, and only finds evidence to support the self-selection hypothesis. Most of the studies focusing on foreign intermediates find different channels through which imported inputs affect firm productivity. Some authors find that firms that import have access to a wider variety of inputs than firms that only use domestic providers; this in turn leads to firms easily adapting their products to the foreign market. Indeed, Kugler and Verhoogen (2009) show that access to imports increases the availability of different types of inputs. They find that plants which are more productive purchase higher-quality inputs, and that despite import prices being higher than domestic prices for the same input category in the same plant and year, firms still use foreign inputs due mainly to their higher quality. Halpern et al. (2011) find that firms that import all of their inputs have a 12 percent higher productivity in comparison to firms that import only part of them. Access to foreign inputs also means that firms are able to use inputs that are cheaper and of higher quality than domestic inputs, especially in developing countries. Goldberg et al. 2 Theoretical framework and literature review 2 With the introduction of firm heterogeneity in models of international trade by the seminal paper of Melitz (2003), the empirical literature studying the link between trade and productivity has dynamically evolved over time. According to Melitz model there is a fixed cost of exporting and firms can enter in a foreign market by paying it. They then select their level of productivity and if it is too low to be profitable, they are forced to leave the market. This seminal theory has been extended in several directions, one of this works is Kashara and Lapham (2013) that introduces the importance of importing activities in the internationalization process of the firm. These authors extended Melitz (2003) model introducing imported inputs and showed the existence of some productivity gains stemming from importing inputs, which allow importers to start exporting. As a result, a cost complementarity effect emerges between import and export activities. In order to produce final goods, firms can use imported inputs, domestic inputs or a combination of both, and their decision to import/export is linked to the associated import/export fixed costs in which they have to incur. The model is based in an open economy with heterogeneous final goods producers, where the firm makes simultaneously the decision to export their output and the decision to use imported intermediates and firms have to pay a fixed cost to enter into the foreign market in order to import and export. The authors also introduce firm’s productivity, transport costs for importing intermediates and for exporting final goods, and take into account the trade status of the firm in order to capture the observed changes in the firm’s trade status over time. In particular, they consider whether a firm is import- only, export-only, both or only sells in the domestic market. They assume that two- way traders necessarily face higher trade costs, and for this reason only the most productive firms are able to operate as such. The model predict that if there is a common fixed cost for both activities, the firms that are one-way traders are more likely to start exporting and, in due course, become two-way traders. 2 See Silva et al. (2012) for a survey of the learning-by exporting literature and Singh (2010) for a detailed literature review about the effects of international trade on productivity and economic growth at the macro- and micro-levels. 2 Theoretical framework and literature review (2010) show how the combined use of foreign and domestic inputs increases the product scope of Indian firms, and that better access to foreign inputs after trade liberalization is 5 5 www.economics-ejournal.org more important than the price reduction effect produced by the decrease in trade costs. Another important aspect worth mentioning is that the diffusion of modern technologies through the use of foreign intermediate goods appears especially beneficial for developing countries, which benefit the most from these technological spillovers. Meanwhile, the origin of the imported inputs and their impact on productivity have also been analysed in order to understand the technology transfer linked to imported intermediates. In their analysis, Smeets and Warzynski (2010) distinguish between inputs from the OCDE and those from low-income economies, analysing their impact on total factor productivity (TFP). The authors find that both affect productivity in a similar way. However, Bas and Strauss-Kahn (2011), compare imported inputs from developed and developing countries for French firms, and find that foreign intermediates from developed countries increase TFP 20 percent more than inputs from developing countries. They also find that importing more varieties of intermediate inputs increases TFP and also the number of exported varieties of French firms. Other authors have focused their attention on analysing how trade liberalization in intermediate inputs affects productivity. Amiti and Konings (2007) was one of the first studies to estimate the relationship between productivity and the effects of trade liberalization on imported inputs. Using Indonesian data, they analyse the productivity gains that result from reducing tariffs on final goods and on intermediate inputs separately, showing that a ten percent reduction in input tariffs led to a productivity gain of 12 per cent for firms that use imported inputs, and that this gain was twice as large as gains from reducing tariffs on final goods. Bas (2012) studies the impact of input-trade liberalization on Argentinian firms’ export decisions, finding that a reduction in input-tariff on foreign intermediates enhances Argentinian firms' performance in the export market and also increases the percentage of exports. Goldberg et al. (2010) provide evidence indicating that trade liberalization increases productivity not only due to the access to cheap inputs but also due to the opportunity to access new intermediate inputs that allow firms to create new varieties in the domestic market. The literature that directly links both international activities is scarce. Bernard et al. www.economics-ejournal.org 2 Theoretical framework and literature review (2007) were the first authors to consider jointly both activities and they find that two-way traders are more productive than only exporters or importers. Altomonte and Békés (2010) highlight that the previous literature that analyses the 6 6 www.economics-ejournal.org export-productivity link without taking import decisions into account overestimates the export gains. There are a few papers that focus in particular on how imports affect exports. For example, Sjöholm (2003) estimates a static panel model using data for Indonesian manufacturing firms and finds, that the probability to export in the current year are positively affected by the past firm import status. Sjöholm and Takii (2008) estimate instead a dynamic binary model using the approach proposed by Wooldridge (2005). The authors obtain a high degree of hysteresis on the export activity, where past import status does not increase the probability to export. Lo Turco and Maggioni (2013) analyse how imports affect the probability to export for Italian manufacturing firms. They find that importing form low-income countries affects the probability to export and that past export status positively affects the probability to continue with this activity. To our knowledge, only two recent papers analyse the link between both activities in a dynamic framework. Firstly, Muûls and Pisu (2009) test the existence of sunk costs of imports in addition to sunk cost of exports using a dynamic panel probit and taking the coefficient of the lagged dependent variable as a measure of sunk costs. Their results show that exports and imports show a high degree of hysteresis, meaning that past status explain the current status. Also they find that sunk costs decrease when the complementary activity was carried out the past year, meaning that common fixed costs exist, and obtain a higher sunk cost for imports than for exports. Secondly, Aristei et al. (2013) using data for Eastern European and Central Asian firms obtain a high degree of hysteresis for both activities, but higher for exports than for imports and find that past imports affect the probability to export in the current year but not the other way around. The numbers of studies focused on MENA countries are few. Related to the role that imported intermediates could play in technological diffusion, Brach (2010) assesses the role of technological readiness in the MENA region and the implications for Egypt. www.economics-ejournal.org 3 The data comes from a firm-level survey based on a representative sample of manufacturing Egyptian firms classified using ISIC codes 15-37, 45, 50-52, 55, 60-64, and 72 (ISIC Rev.3.1). Formal (registered) companies with 5 or more employees are targeted for interviews and firms with 100% government/state ownership are not eligible to participate in the Enterprise Survey. Business owners and top managers answer the Enterprise Survey from the World Bank. Sometimes the survey respondent calls company accountants and human resource managers into the interview to answer questions concerning the sales and labour sections of the survey, which covers a broad range of business environment topics including access to finance, corruption, infrastructure, crime, competition, and performance measures. Typically, 1200-1800 interviews are conducted in larger economies, 360 interviews in medium-sized economies, and only 150 interviews in small economies. See World Bank (2012) for more details. In our empirical application we have 519 firms for which panel data is available, i.e. they are interviewed in all three years. Hence we have around 50 percent of the original sample. 2 Theoretical framework and literature review The author takes a closer look at the technological progress and innovative activities in the MENA region and within this context investigates the implications for economic development and job creation, as well as the main economic policy recommendations. She finds that one of the major constraints to improving economic performance and sustainable job creation is a general lack of technological capabilities of the MENA countries. Innovation in these countries is mainly linked to the adaptation and modification of existing technologies and the 7 7 www.economics-ejournal.org low level of technological readiness negatively impacts innovation and productivity. Hence, the use of foreign intermediates can be a good way to transfer modern technologies from foreign markets to MENA countries. To the best of our knowledge, only Kiendrebeogo (2014) analyses the Egyptian manufacturing sector and how Egyptian firms perform depending on their export activity using WBES. He finds that exporter firms are larger, more capital-intensive and more productive than domestic-only firms. He examines the self-selection and learning-by-exporting hypotheses, showing that although exporting has a positive impact on firm productivity, supporting the learning by exporting hypothesis of Egyptian firms, the pre-entry differences in productivity do not explain firms’ export decisions. However, the author does not consider importing activities in his analysis. For this reason, in our paper we want to extend this analysis by considering exporting and importing activities, taking advantage of the raw data characteristics. By focusing on the relationship between exporting and importing activities in Egyptian firms, we aim at producing some policy recommendations for this country concerning their participation in regional integration processes and their industrial policies after the Arab Spring revolution. 3 Data and descriptive statistics Data on Egyptian firms are obtained from the World Bank Enterprise Survey dataset.3 The dataset includes 3,129 firms for the years 2004, 2005 and 2007. For some variables, namely sales, exporting and importing status we are able to use 8 www.economics-ejournal.org information for an additional year per questionnaire, since each firm is asked in the questionnaire about the current year of the questionnaire and the previous year. Some firms are only included in one or two years, whereas 554 firms are included in the three questionnaires. Therefore, using the available information for these firms and after data cleaning, we build a panel dataset from 2003 to 2007 keeping 519 firms obtaining around 1,890 observations. Table 1 shows the evolution over time of the exporting and importing status of Egyptian firms in our sample, distinguishing between firms that only sell products in the country (domestic firms), firms that sell in the domestic market and only carry out one international activity (export-only) (import-only) and firms that sell in the domestic market and are involve in both international activities, (two-way traders). The results show that the majority of Egyptian manufacturing firms are focused on the domestic market, results are in line with the empirical literature which highlight that international-trading firms are fairly scarce (Bernard et al 2007). The percentages of export-only and import-only firms remain quite stable over time, around 8 and 11 percent on average, respectively. We observe that only 7 percent of all firms in our sample are involved in both importing and exporting activities in 2003. This number has increased over time and has reached 16 per cent of the number of total firms in 2007. The last rows of Table 1 show the percentage of imported inputs used by import-only firms and two-way traders, showing that on average, more than half of the inputs used in production are imported. In addition, the share has not increased over time and it is relatively stable for both types of firms. The second part of Table 1 displays the relative importance of each industry by status. Firms are classified into nine industrial categories, of which Garments, Non- metal industries and Other industries mainly sell their products in the domestic market, whereas almost half of the two-way traders belong to the Chemical and Machinery and equipment industries. 3 Data and descriptive statistics Despite the fact that Egyptian firms are mainly focused on the domestic market, those that are involved in international activities tend to engage in both import and export activities, rather than in only one of them. There are only a few exceptions in some industries in which one of the international activities is more important than the other. This is the case in the electronics industry, where import of intermediate goods is the only 9 www.economics-ejournal.org www.economics-ejournal.org Table 1: Sample composition by trade status and percentage of imported input Year Import-only Export-only Two-way traders Domestic 2003 13% 7% 7% 73% 2004 12% 7% 13% 68% 2005 10% 6% 15% 69% 2006 9% 10% 10% 71% 2007 11% 9% 16% 64% Average 11% 8% 12% 69% Industries Import-only Export-only Two-way traders Domestic Agro industries 16% 11% 13% 60% Chemicals 22% 9% 26% 43% Electronics 36% 0% 0% 64% Garments 5% 7% 8% 80% Machinery and equipment 22% 11% 20% 47% Metal industries 13% 8% 11% 68% Non-metal industries 9% 7% 8% 76% Other industries 8% 8% 13% 71% Textiles 12% 7% 13% 68% % imported intermediates Import-only Two-way traders 2003 54% 49% 2004 57% 48% 2005 50% 46% 2006 48% 49% 2007 51% 48% Average 52% 48% Note: Authors’ elaboration using data from the World Bank Enterprise Survey. Export-only firms denotes firms that sell in the local market and also export, Import-only firms denotes firms that sell into domestic market and also import, Two-way traders refers to firms that sell into the domestic market and also export and import and Domestic indicate firms that only sell in the local market and are not engaged in international activities. Table 1: Sample composition by trade status and percentage of imported input Note: Authors’ elaboration using data from the World Bank Enterprise Survey. Export-only firms denotes firms that sell in the local market and also export, Import-only firms denotes firms that sell into domestic market and also import, Two-way traders refers to firms that sell into the domestic market and also export and import and Domestic indicate firms that only sell in the local market and are not engaged in international activities. international activity. It seems that firms in this industry import intermediate goods to produce products for the local market; especially the majority are larger factories assembling products for international brands. 3 Data and descriptive statistics Also, the chemical and machinery and equipment industries show a higher share of importers than exporters. This descriptive analysis shows that the nature of the different industries might influence the decision to import/export; indeed, some industries are more likely to participate in international markets. For this reason, we need to take industry effects into account in our analysis. 10 10 www.economics-ejournal.org www.economics-ejournal.org Table 2: Descriptive statistics by trade status Variable Obs Mean Std. Min Max Export-only firms TFP i,t 182 7.12 1.68 0.95 10.35 work i,t 188 251.45 478.84 8.00 2800 foreignowner i,t 191 3.00 15.38 0.00 1.00 px i,t 191 39.92 32.91 0.50 100 pm i,t 191 0.00 0.00 0.00 0.00 capital i,t 180 20229.64 53644.10 50.00 531419 investment i,t 185 129047.50 1601345 0.00 2.18e+07 Import-only firms TFP i,t 258 6.98 1.61 0.95 11.39 work i,t 281 250.75 907.84 8.00 13,695 foreignowner i,t 281 0.06 0.23 0.00 1.00 px i,t 281 0.00 0.00 0.00 0.00 pm i,t 281 50.84 31.19 1.00 100 capital i,t 253 192808.40 1446639.00 0.00 1.57e+07 investment i,t 262 128012.50 1323347.00 0.00 1.52e+07 Two-way traders TFP i,t 297 7.83 1.76 0.98 14.37 work i,t 314 634.40 1206.94 0.00 13,15 foreignowner i,t 316 0.11 0.31 0.00 1.00 px i,t 316 39.02 33.81 0.90 100 pm i,t 316 47.25 29.08 2.00 100 capital i,t 298 129055.70 698418.30 5.00 9,800,000 investment i,t 297 175131.00 1902239 0.00 2.99e+07 Domestic firms TFP i,t 1646 5.44 1.48 1.41 12.93 work i,t 1770 69.11 427.99 0.00 10,500 foreignowner i,t 1783 0.02 0.12 0.00 1.00 px i,t 1783 0.00 0.00 0.00 0.00 pm i,t 1783 0.00 0.00 0.00 0.00 capital i,t 1639 33258.00 476477.50 0.00 1.22e+07 investment i,t 1686 9237.41 165149.10 0.00 6550000 Table 2: Descriptive statistics by trade status Notes: Obs denotes number of observations; Std. Dev denotes standard deviation and Min and Max are the minimum and maximum value of each variable. TFP i,t is total factor productivity, obtained using the Levinsohn-Petrin (2003) procedure. 3 Data and descriptive statistics We explain the choice of this methodology and the estimation in Appendix A.2; work i,t is the average number of workers; foreignowneri,t is a dummy variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise; pxi,t is the share of exports over total sales and pmi,t is the share of imports over total sales; capital i,t is the total fixed tangible assets value of machinery and investment i,t is the net book value of machinery. 11 www.economics-ejournal.org www.economics-ejournal.org Table 2 presents summary statistics of the main variables used in the analysis. The figures show that firms involved in international activities perform better than domestic-only firms in terms of total factor productivity (TFP)4 and size (measured with number of workers: work). Among the three types of international firms, we observe that firms with higher productivity are more often two-way traders than export-only firms or import-only firms, and domestic firms have the lowest average productivity. It is also worth noting that two-way traders are not only bigger in size than import-only and export-only firms but also invest more. We also observe that firms owned by foreigners are more focused on international activities. 4 TFP has been obtained using the Levinsohn-Petrin (2003) methodology. See Appendix A.2. How different are Egyptian traders? Firm-level characteristics of traders vs. non-traders Following studies that analyse how firm trade status affects firm characteristics (Bernard et al. 2007; Muuls and Pisu 2009; Castellani et al. 2010; Seker 2012; Sharma and Mishra 2015), we start this section by computing the exporter and importer premia for Egyptian firms. Exporter/importer premia are conventionally determined estimated by regressing the dependent firm-performance variable indicators, usually expressed as TFP, labour productivity, wages, number of workers or capital, among others, on an exporter/importer dummy and other a number of control variables as explanatory variables using OLS estimations. The estimated coefficients of the dummy trade variables show the exporter/importer premia meaning or simple correlations between the dependent variable and the trade dummy variables used. At this point, a causal interpretation of the results is not possible. Clearly the results cannot be interpreted as causal effects. The main idea aim is to confirm whether an export/import premium for Egyptian international firms is present, which will be in accordance with the related empirical literature. Since some exporters are also importers —as in Bernard et al (2007), Altomonte and Bekés (2010), Muûls and Pisu (2009)—we distinguish between import-only firms, export-only firms and two-way traders to better understand the characteristics of international Egyptian firms compared with domestic-only firms. The estimated equation is: 12 www.economics-ejournal.org 𝑙𝑛𝐹𝑖,𝑡= 𝛼0 + 𝛼1 𝑑𝑖,𝑡 𝑥𝑜+ 𝛼2 𝑑𝑖,𝑡 𝑚𝑜+ 𝛼3𝑑𝑖,𝑡 𝑥𝑚+ β1𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟 𝑖,𝑡+ 𝛽2𝑙𝑛𝑤𝑜𝑟𝑘𝑖,𝑡 +𝛾𝑘+ 𝛿𝑡+ 𝜀𝑖,𝑡 (1) (1) Where ln denotes natural logs; Fi,t denotes a given firm performance indicator5 (TFP, size, sales, capital and investment) used as dependent variable. TFPi,t is obtained using the Levinsohn-Petrin (2003) methodology, worki,t denotes firm size proxied by the average number of workers, salesi,t denotes the total sales of the firm, capitali,t is the total fixed tangible assets deflated using the production price index for manufactures and investmenti,t is the net book value of machinery and equipment, all variables are in natural logs. As explanatory variables we include 𝑑𝑖,𝑡 𝑥o, a dummy variable taking the value of 1 if the firm only exports and zero otherwise. di,t mo takes the value of 1 if the firm only imports and di,t xm takes the value of 1 if the firms are two-way traders, zero otherwise. As control variables, we include the percentage of the firm owned by a foreigner and when the dependent variable is not employment, we include also firm size. 5See Appendix A.1. www.economics-ejournal.org How different are Egyptian traders? Firm-level characteristics of traders vs. non-traders We also include industry dummies and year dummies to take into account any unobserved effects that are industry specific and time invariant and those that are common across industries and time variant. The former could be a proxy of specific comparative advantages and the later controls for the business cycle. Both type of effects also control for potential measurement errors. Table 3 presents the estimated trade-status premia using fixed effects obtained from estimating Equation (1). 13 13 www.economics-ejournal.org Table 3. Exporter and importer premia (1) (2) (3) (4) (6) Dependent Variable lnTFPi,t lnworki,t lnsales i,t lncapital i,t lninvestment i,t Export-only firms 1.107*** 0.896*** 1.329*** 0.213 1.295*** (0.194) (0.142) (0.217) (0.234) (0.269) Import-only firms 0.889*** 0.955*** 1.130*** 0.367* 0.976*** (0.165) (0.119) (0.180) (0.215) (0.217) Two-way traders 1.407*** 1.767*** 1.855*** 0.116 1.608*** (0.199) (0.143) (0.219) (0.198) (0.246) foreignowner i,t 0.322*** 0.448*** 0.787*** 0.376*** (0.0362) (0.0396) (0.0655) (0.0478) lnwork i,t 0.000123 -0.00145 -0.000321 0.00627 -0.0135 (0.00243) (0.00383) (0.00278) (0.00673) (0.0127) Constant 4.785*** 3.143*** 5.387*** 4.406*** 4.572*** (0.135) (0.0408) (0.145) (0.257) (0.189) Observations 1,978 2,547 1,985 1,963 1,968 Number of firms 518 519 518 519 519 Year dummies yes yes yes yes yes Industry dummies yes yes yes yes yes Rho 0.238 0.297 0.298 0.186 0.180 Note: Robust standard errors (clustering at firm level) in parentheses *** p<0.01, ** p<0.05, * p<0.1. Note: Robust standard errors (clustering at firm level) in parentheses *** p<0.01, ** p<0.05, * p<0. We observe that firms involved in international trade, irrespective of their trade pattern, are more productive, are larger in size, sell more, own more capital, and invest more than domestic-only firms. In particular, two-way traders are the best performers in all cases, and only exporters have higher premia than only importers in TFP, sales and investment. These results are in line with those obtained by Bernard et al (2007), Muûls and Pisu (2009), Castellani et al (2010) Seker (2012) and Sharma and Mishra (2015) for other countries. Some recent studies for other developing countries, for example, Seker (2012) using data for 43 developing countries obtain that two-traders are best performers in all measures, followed by only exporters and only importers in comparison to domestic-only firms. Also Sharma and Mishra (2015) using data for Indian manufacturing firms over the period 1994–2006 finds the same pattern. 14 www.economics-ejournal.org www.economics-ejournal.org 5.1.1 Extensive margin of trade In order to estimate the determinants of export and import decisions and analyse how both activities are related, we model the probability of exporting/importing as a function of TFP, size of the firm and ownership structure. In order to account for correlations between exporting and importing activities, we include in the models past import-status in the export equation and past export-status in the import equation. We also add the lagged left hand side variables as explanatory variables. In this dynamic framework we will be able to investigate the existence of state dependence, also termed hysteresis, in export and import activities. In other words, we assume that there is some sort of persistence affecting the decision to export final outputs and import intermediates, and we would like to disentangle the effect of past status from the firm’s initial condition as exporter/importer. The inclusion in the model of the lagged values of the dependent variables has been considered by several authors as a way to introduce a measure of the sunk costs (Bernard and Jensen, 2004; Muûls and Pisu, 2009; and Roberts and Tybout, 1997). The proposed equations for exports and imports are given by, Pr(𝑥𝑖,𝑡= 1) = Pr (𝛽0 + 𝛽1𝑥𝑖,𝑡−1 + 𝛽2 𝑚𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) + 𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5 ln(𝑇𝐹𝑃𝑖,𝑡−1) + 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖,𝑡 >0) (2) (2) Pr(𝑚= 1) = Pr (𝛽0 + 𝛽1𝑚𝑖,𝑡−1 + 𝛽2 𝑥𝑖,𝑡−1 + 𝛽3 + 𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5 ln(𝑇𝐹𝑃𝑖,𝑡−1) + 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖,𝑡 >0) (3) where ln denotes natural logarithms, the subscript i indexes firms; t, indexes time. The dependent variable in Equation (2), Pr(xi,t=1), denotes the probability of exports and is a dummy variable that takes the value of 1 if firm i exports in year t, and 0 otherwise and the dependent variable in Equation (3), Pr(mi,t=1), is the probability of importing, which takes the value of 1 if firm i imports in year t, and 0 otherwise. As explanatory variables we include mi,t-1 is a dummy variable reflecting the import 15 www.economics-ejournal.org www.economics-ejournal.org status of the firm in year t–1 and xi,t-1 is a dummy variable indicating the exporting status of the firm in year t–1, TFPt-1 is total factor productivity of the firm obtained by using the Levinsohn-Petrin (2003) methodology. 6 We also used alternatively the percentage of the firm owned by a foreigner, but since high and a low percentage of foreign ownership have approximately the same effect, we decided to create a 1/0 dummy. 7 Results are obtained using xtprobit command in Stata11. 5.1.1 Extensive margin of trade workit-1 denotes the average number of workers in t–1, and foreigowneri.t is a dummy variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise.6 𝑙𝑛𝑇𝐹𝑃 ̅̅̅̅̅̅̅𝑖 and 𝑤𝑜𝑟𝑘𝑡 ̅̅̅̅̅̅̅̅𝑖 are respectively the average of each variable. Industry (𝛾k) and time dummies (δt) have also been included in the model to proxy for factors that are industry specific and time-invariant and for those that vary over time and are common to all firms. These variables have been commonly included as control variables in models used to estimate the determinants of the decision to export; see for example Greenaway et al (2007) and Muûls and Pisu (2009). The main difficulty of explicitly allowing for lagged effects is that the correlation between the unobserved heterogeneity and the lagged dependent variable in the dynamic binary choice model makes the lagged dependent variable endogenous. Hence, the estimators used before will not be consistent. A familiar alternative approach is based on Wooldridge (2005), which builds on the random effects specification and basically adds the initial condition and the averages over time of the time-variant variables as additional regressors. The solution proposed by Wooldridge (2005) has been improved by Rabe-Hesketh and Skrondal (2013), who exclude the first period when calculating the averages over time of the regressors. Therefore, we follow a similar strategy to Aristei et al (2013) and Muûls and Pisu (2009) but use instead a more reliable estimation technique that will enable us to disentangle the effect of the initial conditions from the effect of the past export/import status of the firm on the decision to export/import. To deal with the so-called “initial condition” problem (basically, we cannot observe the first dependent observation in the data-generating process, hence we cannot treat the stochastic process from its starting point and consequently we cannot treat it as fixed) previous literature used Wooldridge’s auxiliary model. However, as stated by Rabe-Hesketh and Skrondal (2013), Wooldridge’s (2005) method performs poorly for short panels, mainly because if the means are based on all periods, the initial conditions are also used to compute those means, and this induces endogeneity. The authors suggest including the initial-period as explanatory 16 www.economics-ejournal.org variable and calculate the mean only using the remaining periods, which is t+1 until n. variable and calculate the mean only using the remaining periods, which is t+1 until n. Columns (1) and (2) of Table 4 include only the lagged value of the dependent variable. A high degree of hysteresis is obtained for both activities, indicating that past trade status increase the probability to continue with the same activity. We obtain that the sunk costs that firms face to import intermediates are higher than those needed to export. Indeed, liberalization of imports with the EU started in 2004 with the entry into force of the FTA and imports from EU were progressively liberalised during a period of 10 years, whereas exports were already liberalized in 1972 with the bilateral cooperation agreements. Results in Columns (3) and (4) in Table 4 show that the combination of both export and import activities affect the probability of importing/exporting. It can be observed that past export and import participation have a high degree of hysteresis, and firms face higher sunk costs for imports than for exports. The results are similar to those obtained in Muûls and Pisu (2009), in that exporter/importer status in the previous year has a positive effect on the probability of exporting/importing in the current year and the magnitude of the effect is also higher for imports than for exports. Contrary to Aristei et al. (2013) we find that foreign ownership affects positively both the export and import status of the firm, remaining only significant for exports when the imports variable dummy is included in the regression. However, past TFP affects the probability of importing but not of exporting, and the size of the firm affects only the probability to export when the import dummy variable is not included. Both, Aristei et al. (2013) and Muûls and Pisu (2009) find that past TFP influences export and import activities, however in our case, the results show that past TFP only affect the extensive and intensive margin of imports only, but not of exports. There is also a different effect of the variable firm size on the probability of exporting and importing. Firm size has a positive effect only on the decision to start exporting. This could be explained by the fact that larger firms are able to serve the domestic and the foreign market because they have a higher production capacity than smaller firms. 5.1.1 Extensive margin of trade As in Wooldridge (2005), we assume that 𝑢𝑖, (𝜀𝑖), the firm specific effects are determined by, 𝑢𝑖 = 𝛽0 + 𝛽1𝑥|𝑚𝑖0 + 𝛽2𝑙𝑡𝑓𝑝 ̅̅̅̅̅𝑖+ 𝛽3𝑙𝑤𝑜𝑟𝑘𝑡 ̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖,𝑡 (4 (4) where 𝜇𝑖 is an independently and normally distributed error term and the control variables are now the firm-level average of each variable over time. However, as Rabe-Hesketh and Skrondal (2013) suggest, the firm-level average must be obtained excluding the initial period and then adding a dummy in the regression capturing whether the firm exports ( 𝑖𝑒𝑖)/imports ( 𝑖𝑖𝑖) in the first period of the sample. If we now substitute Equation (4) into Equations (2) and (3) we obtain: Pr(𝑥𝑖,𝑡= 1) = Pr (𝛽0 + 𝛽1𝑥𝑖,𝑡−1 + 𝛽2𝑚𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) + 𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5𝑙𝑇𝐹𝑃𝑖+ 𝛽6𝑙𝑇𝐹𝑃 ̅̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡 ̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖,𝑡>0) (5) (5) Pr(𝑚𝑖,𝑡= 1) = Pr (𝛽0 + 𝛽1𝑚𝑖,𝑡−1 + 𝛽2 𝑥𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) + 𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5𝑙𝑇𝐹𝑃𝑖+ 𝛽6𝑙𝑇𝐹𝑃 ̅̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡 ̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖,𝑡>0) (6) (6) In order to test the existence of sunk costs in import and export activity and to measure the importance of these sunk costs, we estimate the parameters of Equations (5) and (6) using a panel-Probit model with random effects7 based on maximum likelihood estimation techniques for the period 2003–2007, and we interpret the estimated coefficients for the dependent lagged variable as a measure of the importance of sunk costs, as the authors cited above. We argue that sunk costs generate hysteresis in the export and import market participation. The results from estimating Equations (5) and (6) are shown in Table 4. 17 www.economics-ejournal.org www.economics-ejournal.org TFPi,t denotes total factor productivity, it is obtained using the Levinsohn-Petrin (2003) procedure; TFPi,t -1 are lagged values of TFPi,t;; worki,t denotes the average number of workers and work i,t–1 are aged value of the variable; xt.i are a dummy variable that takes value 1 if the firm is exporting and 0 otherwise, xt.i–1 are the corresponding lagged value and foreign owneri,t is a dummy variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise. The number of observations differ between column (1) and (2) and between columns (3) and (4) due to the inclusion of lagged imports and initial conditions for which 22 and 19 observations are respectively missing data. However, firm size does not affect the probability of importing indicating that firms import intermediates probably because these are not available in the domestic market, independently of the scale of production 18 www.economics-ejournal.org www.economics-ejournal.org Table 4: Dynamic panel-Probit model controlling for initial conditions (Exports and Imports) (1) (2) (3) (4) P(xit=1) P(mit=1) P(xit=1) P(mit=1) x i, t-1 1.489*** 1.408*** 1.342*** (0.112) (0.136) (0.140) m i, t-1 1.591*** 1.219*** 1.427*** (0.114) (0.135) (0.140) ln work i,t-t 0.130** 0.057 0.071 –0.003 (0.056) (0.055) (0.062) (0.064) foreign owner i,t 0.625*** 0.319* 0.600*** 0.047 (0.175) (0.177) (0.202) (0.218) ln TFP i, t-1 0.041 0.063* 0.034 0.059 (0.036) (0.036) (0.039) (0.041) ln TFP mean i, t-1 0.097 0.094 0.012 0.067 (0.060) (0.061) (0.069) (0.075) ln work mean i,t-t 0.072 0.070 0.096 0.046 (0.073) (0.074) (0.083) (0.088) iei –0.060 0.162 (0.122) (0.157) iii 0.029 0.347** (0.118) (0.170) Year dummies yes yes yes yes Industry dummies yes yes yes yes Number of observations 1889 1867 1882 1863 Notes: The dependent variable P(x=1) is a dummy variable for the exporter status and P(m=1) for the importer status. t–1 denotes lagged values of these variables. iei denotes initial exporter dummy. iii means initial importer dummy. Standard errors are in brackets, where *** p<0.01, **p<0.05, * p<0.1. Industrial and year dummies included. TFPi,t denotes total factor productivity, it is obtained using the Levinsohn-Petrin (2003) procedure; TFPi,t -1 are lagged values of TFPi,t;; worki,t denotes the average number of workers and work i,t–1 are aged value of the variable; xt.i are a dummy variable that takes value 1 if the firm is exporting and 0 otherwise, xt.i–1 are the corresponding lagged value and foreign owneri,t is a dummy variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise. The number of observations differ between column (1) and (2) and between columns (3) and (4) due to the inclusion of lagged imports and initial conditions for which 22 and 19 observations are respectively missing data. Table 4: Dynamic panel-Probit model controlling for initial conditions (Exports and Imports) Notes: The dependent variable P(x=1) is a dummy variable for the exporter status and P(m=1) for the importer status. t–1 denotes lagged values of these variables. iei denotes initial exporter dummy. iii means initial importer dummy. Standard errors are in brackets, where *** p<0.01, **p<0.05, * p<0.1. Industrial and year dummies included. 5.1.2 Intensive margin of trade In this section we analize whether the results are analogous for the impact on the intensive margin of trade. The estimated model is given by Equations (7) and (8), similar to Equations (2) and (3) used for the extensive margin; the difference is that 19 www.economics-ejournal.org the dependent variable is proxied by the percentage of exports over total sales of firm i in year t, and by the percentage of total purchases of materials inputs imported from firm i in year t. We propose a dynamic model following the method proposed by Rabe-Hesketh and Skrondal (2013). 𝑒𝑥𝑝𝑖,𝑡= 𝛽0 + 𝛽1𝑒𝑥𝑝𝑖,𝑡−1 + 𝛽2 𝑖𝑚𝑝𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) + 𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5 ln(𝑡𝑓𝑝𝑖,𝑡−1) + 𝛽6𝑙𝑡𝑓𝑝 ̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡 ̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖 (7) (7) 𝑖𝑚𝑝𝑖,𝑡= 𝛽0 + 𝛽1𝑖𝑚𝑝𝑖,𝑡−1 + 𝛽2 𝑒𝑥𝑝𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) + 𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5 ln(𝑡𝑓𝑝𝑖,𝑡−1) + 𝛽6𝑙𝑡𝑓𝑝 ̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡 ̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖 (8) (8) In this case, the parameters of the model are estimated using a panel-Tobit procedure. The election of this estimation technique is justified by the fact that our dependent variable is continuous and positively distributed, taking censored values from 0 to 100. As stated in Wooldridge (2010), the use of linear models is not recommended in this case where corner solutions are present, and a censored re- gression model is more recommended in this setting. In our sample approximately 80 percent of the observations in the dependent variable take the value of 0. The results are presented in Table 5, and they are similar to those obtained for the extensive margin of trade. Aristei et al. (2013) also used a Tobit model to analyse the relationship between both activities, nevertheless the authors find that only past imports are positively correlated with current exports, but not the other way round. As we can observe in columns (1) and (2) we find a high degree of hysteresis, since the past percentage of exports and imported intermediates explains the current levels of each activity, been more important for imports. When both activities are included in the estimation, sunk costs decrease for both activities, the reduction being more important for imports than for exports (20 percent versus 15 percent), showing the existence of common costs in both activities. 20 www.economics-ejournal.org Conclusions 6 According to the recent literature investigating the relationship between productivity and international trade, firms involved in international activities are larger and more productive than domestic-only firms. The bulk of the literature has mainly focused on the export side, disregarding the importance of importing activity. It has been only recently that more attention has been paid to the import side of internationalization strategies (Muûls and Pisu 2009; Bas 2012; Aristei et al. 2013; Kasahara and Lapham 2013; Lo Turco and Maggioni 2013). These studies show the presence of cost complementarities in both activities, indicating that importing intermediates is crucial to start exporting and to stay in foreign markets. These cost complementarities have motivated a new strand of research that further investigates the relationship between firm’s import and export activities, especially those focused on the use of imported intermediates and their role in enhancing exports. In this paper we contribute to this research by providing new evidence for Egypt. More specifically, using firm-level data for 519 manufacturing companies in Egypt, we first estimate the two-way traders and importer- and exporter-only premia with respect to non-traders. Our findings show that firms involved in international activities have higher productivity, are larger, own more capital and invest more than domestic-only firms. In particular, two-way traders are the best performers in terms of all outcome variables, followed by exporter-, importer-only and non- traders. Secondly, we investigate the relationship between exporting and importing activities by estimating dynamic panel-Probit models for the extensive margin of exports (imports) and panel-Tobit models for the intensive margin of exports (imports). We use the solution proposed by Rabe-Hesketh and Skrondal (2013) that improve Wooldridge (2005) for initial condition problem in dynamic models. The results indicate that both activities are significantly interrelated and that sunk costs are higher for import than for export activities, with both activities showing a high degree of hysteresis. Moreover, past productivity does affect the extensive and intensive margin of imports, but not the export side and the initial condition status is also only relevant for the import side. 8 Results including lagged values of exp, t and imp, t where the dependent variable is exp i, t-1 and imp i,t-1, are available upon request. Acknowledgements: We would like to thank Antonello Maruotti for his comments and useful suggestions and we acknowledge the financial support obtained from the Universitat Jaume I, P1.1B2013-06, PREDOC/2009/55 and E-2012-09. Conclusions 21 www.economics-ejournal.org Table 5: Dynamic panel-Tobit model controlling for initial conditions (Exports and Imports)8 (1) (2) (3) (4) exp i, t imp i, t exp i, t imp i, t exp i, t_1 0.493*** 0.430*** 0.306*** (0.025) (0.031) (0.021) imp i, t-1 0.525*** 0.286*** 0.436*** (0.026) (0.020) (0.032) ln work i,t-t 0.035*** 0.019 0.018 0.002 (0.011) (0.012) (0.011) (0.011) foreign owner i,t 0.179*** 0.080** 0.153*** 0.013 (0.039) (0.039) (0.038) (0.039) ln TFP i, t-1 0.011 0.016** 0.007 0.010 (0.007) (0.007) (0.007) (0.007) ln TFP mean i, t-1 0.011 0.012 -0.005 0.006 (0.011) (0.012) (0.011) (0.012) ln work mean i,t-t 0.016 0.012 0.016 0.004 (0.015) (0.014) iei -0.015 0.022 (0.026) (0.015) (0.028) (0.015) iii 0.018 0.061** (0.026) (0.028) Year dummies yes yes yes yes Industry dummies yes yes yes yes Observations 1889 1867 1882 1863 Table 5: Dynamic panel-Tobit model controlling for initial conditions (Exports and Imports)8 Notes: The dependent variables are the natural logs of the percentage of exports over total sales of a firm and the percentage of material purchases imported. t–1 means lagged values of these variables. Standard errors are in brackets, where *** p<0.01, **p<0.05, * p<0.1. iei denotes initial exporter dummy. iii means initial importer dummy. Industrial and year dummies included. TFP i,t denotes total factor productivity, it is obtained using the Levinsohn-Petrin (2003) procedure; TFP i,t -1 are lagged values of TFP i,t;; work i,t denotes the average number of workers and work i,t-1 are aged value of the variable; xt.i are a dummy variable that takes value 1 if the firm is exporting and 0 otherwise, xt.i–1 are the corresponding lagged value and foreignowner i,t is a dummy variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise. The number of observations differ between column (1) and (2) and between columns (3) and (4) due to the inclusion of lagged imports and initial conditions for which 22 and 19 observations are respectively missing data. 22 www.economics-ejournal.org The main economic policy implication is that governments in developing countries willing to support the international strategy of the firm should be aware of the fact that facilitating importing activities is as much important as facilitating exporting business. 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Journal of applied econometrics, 20(1), 39–54. http://onlinelibrary.wiley.com/doi/10.1002/jae.770/full Wooldridge, J. M. (2010). Econometric analysis of cross section and panel data. MIT press. World Bank (2012). World bank enterprise survey database. World Bank, Washington, D.C. World Bank (2012). World bank enterprise survey database. World Bank, Washington, D.C. 28 www.economics-ejournal.org 28 Appendix A1 Table A1: Variables description Variable Description Dependent Variables xi,t Dummy variable that take value 1 if firm export in year t mi,t Dummy variable that take value 1 if firm import inputs in year t expi,t Percentage of total sales exported in t pmi,t-1 Percentage of purchases of materials inputs imported Firm characteristics work i,t Average number of workers in t foreign owneri, t Percentage of the firm owned by a foreign Arabic owner and by other foreign owner capitali,t Total fixed tangible assets salesi, t Total sales in t. Value in thousands of Egyptian pounds. Not defalted investmenti, t Net book value of machinery and equipment tfpi,t Levinsohn and Petrin (2003) TFP Total Factor Productivity capitaldefi,t Total fixed tangible assets deflated by the Production price index for manufactures materialsde i,t Total purchases of raw material and intermediate goods deflated by the Production price index for manufactures salesdefi, t Total sales in t. Value in thousands of Egyptian pounds. We deflate sales using the Production price index for manufactures using 2005 year as a base years. Table A1: Variables description Total fixed tangible assets deflated by the Production price index for manufactures Total purchases of raw material and intermediate goods deflated by the Production price index for manufactures Total sales in t. Value in thousands of Egyptian pounds. We deflate sales using the Production price index for manufactures using 2005 year as a base years. 29 www.economics-ejournal.org Appendix A2: Total Factor Productivity (TFP) Estimation To calculate TFP estimates of a traditional Cobb-Douglas production are obtained. The Cobb-Douglas production function is given by: 𝑙𝑛𝑠𝑎𝑙𝑒𝑠i,t = 𝛽0 + 𝛽𝑙 ln (𝑤𝑜𝑟𝑘𝑖,𝑡) + βmln (𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙𝑠𝑑𝑒𝑓i,t) + βk ln (𝑐𝑎𝑝𝑖𝑡𝑎𝑙𝑑𝑒𝑓i,t) + ωi,t + ηi,t (9) (9) where all the variables are in natural logarithms, salesi,t is total sales of firm in year t, in thousands of Egyptian pounds. As independent variables we include worki,t defined as the average number of workers, materialsi,t denotes the total purchases of raw material and intermediates goods, capitali,t denotes the total fixed tangible assets of the firm and the error term id discomposed into ωi,t, which indicates productivity socks and an i.i.d. error term given by ηi,t. We deflate firm level sales and input expenditures using the industry level production price index for manufactures using 2005 as a base year, the data comes from the International Financial Statistics (IFS and UN) for manufacturing. When researchers estimate TFP using firm level data they have to deal with different bias associated with input and output firm level data. The existing literature proposes a number of avenues to overcome these problems. The first bias is caused by simultaneity between firm inputs choice and the unobserved productivity level. A second bias emerge when the firm makes its input choice conditional on its survival, which means that there is a correlation between the unobserved productivity and the firm’s capital, conditional to being in the dataset. A third bias appears because industry-level price data are used to deflate output and inputs and to proxy these variables as quantities. The problem is that under imperfect competition in input or output markets, inputs and output prices used as deflators, must be correlated with firm level deviations of inputs or outputs. The literature has not yet provided a formal solution to deal with this bias. Finally, a fourth bias is related to the fact that firms can produce different products with different in production technologies, to deal with this aspect Bernard et al (2009) propose use the number and type of products of the firm and use them to allow for varying technology parameters in the production function. An alternative solution is to estimate different regressions for firms that produce a single product and for multi- product firms. Appendix A2: Total Factor Productivity (TFP) Estimation Also a measurement problem arises because the true value of the 30 www.economics-ejournal.org capital stock is difficult to mesure, in particular due to the fact that the depreciation rate and the initial stock of the firm are unknown. Given that the available methodologies deal with different biases, in what follows we present a number of alternative estimates of the coefficients of the production function used to obtain TFP, as proposed by Van Beveren (2012). Table A.2 shows several estimates that overcome the abovementioned biases. Column 1 in Table A.2 shows the classical OLS estimates that are subject to endogeneity and selection biases. In Column 2 the model is estimated with firm fixed effects, controlling for time-invariant unobserved heterogeneity which is firm- specific. The third alternative, (Column 3), was proposed by Levinsohn and Petrin (2003), who proposed to estimate the production function using inputs as control. Finally, Column 4 shows the coefficients estimated by using Olley and Pakes (1996) method. Olley and Pakes (1996) propose a three steep procedure. In the first steep the unobserved productivity is obtained for each firm using their level of investment, in the second step we obtain the survival probability of the firm and the last steep employs the outcomes of the previous two steps to control for simultaneity and selection biases. Consistent and unbiased estimates of the production function are used to obtain unbiased estimates of TFP, which is computed as the residual of the estimated production function. We decide to use TFP estimated using the Levinsohn and Petrin (2003) as independent variable in the export and import models because this methodology control for two important biases, namely, simultaneity and self- selection and our data availability do not allow use obtain accurate values of the firm investments. Table A.2. Product function estimates OLS FE LP reg OP reg lncapitaldefi.t 0.085*** 0.081*** 0.055*** 0.081*** (0.011 ) (0.012) 0.010 (0.029) lnwork I,t 0.350*** 0.372*** 0.363*** 0.606*** (0.020) (0.025) 0.019 (0.042) lnmaterialsdefi,t 0.603*** 0.611*** 0.608*** 0.315*** (0.012) (0.014) 0.012 (0.043) Number of observations 2429 2429 2429 2480 Note: where OLS denotes Ordinary Least Squares, FE denotes OLS fixed effects, LP denotes, Levinsohn and Petrin, and OP denotes Olley and Pakes. *** p<0.01, ** p<0.05, * p<0.1. Robust standard errors in parentheses. Table A.2. Product function estimates Table A.2. Note: where OLS denotes Ordinary Least Squares, FE denotes OLS fixed effects, LP denotes, Levinsohn and Petrin, and OP denotes Olley and Pakes. *** p<0.01, ** p<0.05, * p<0.1. Robust standard errors in parentheses. © Author(s) 2015. Licensed under the Creative Commons Attribution 3.0. Appendix A2: Total Factor Productivity (TFP) Estimation Product function estimates Note: where OLS denotes Ordinary Least Squares, FE denotes OLS fixed effects, LP denotes, Levinsohn and Petrin, and OP denotes Olley and Pakes. *** p<0.01, ** p<0.05, * p<0.1. Robust standard errors in parentheses. 31 31 www.economics-ejournal.org Please note: You are most sincerely encouraged to participate in the open assessment of this article. You can do so by either recommending the article or by posting your comments. Please go to: http://dx.doi.org/10.5018/economics-ejournal.ja.2015-38 The Editor The Editor The Editor
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To cite this version: Jérôme Darmont, Cécile Favre, Sabine Loudcher, Camille Noûs. Data Lakes for Digital Humanities. 2nd International Digital Tools & Uses Congress (DTUC 2020), Oct 2020, Hammamet, Tunisia. pp.38- 41, ￿10.1145/3423603.3424004￿. ￿hal-02889487￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02889487 https://hal.science/hal-02889487v1 Submitted on 3 Dec 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ABSTRACT Moreover, most source information exploited by humanities and social sciences comes in textual format. Again, such textual documents are difficult to manage without substantial transforma- tions: digitization, encoding and tagging, e.g., via the Text Encoding Initiative (TEI), and even lowercasing, stemming, lemmatization, stopword removal or normalization when it comes to text mining and natural language processing.f Traditional data in Digital Humanities projects bear various for- mats (structured, semi-structured, textual) and need substantial transformations (encoding and tagging, stemming, lemmatization, etc.) to be managed and analyzed. To fully master this process, we propose the use of data lakes as a solution to data siloing and big data variety problems. We describe data lake projects we currently run in close collaboration with researchers in humanities and social sciences and discuss the lessons learned running these projects. Another important methodological issue is the black box effect that occurs when resorting to computer scientists only “as a ser- vice”. How can DH researchers work without mastering the whole process? Furthermore, designing and managing such processes also lead to research issues for computer scientists. CCS CONCEPTS • Information systems →Data management systems; Data- base administration; Information integration; • Applied com- puting; • Digital Humanities; To leverage the above-mentioned issues, we propose the use of data lakes, a concept introduced by Dixon in 2010 as a solution to data siloing and big data variety problems [2]. Even if data exploited by DH are not always big data in terms of volume, they can bear considerable variety, i.e., including structured and semi-structured data, as well as unstructured data such as texts, various types of images, sounds and videos. Traditional data management tends to manage such heterogeneity with different systems, thus separating data into so-called silos. Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. Copyrights for components of this work owned by others than the author(s) must be honored. Abstracting with credit is permitted. To copy otherwise, or republish, to post on servers or to redistribute to lists, requires prior specific permission and/or a fee. Request permissions from permissions@acm.org. ddh20, October 15–17, 2020, Hammamet, Tunisia © 2020 Copyright held by the owner/author(s). Publication rights licensed to the Association for Computing Machinery. KEYWORDS Data Lakes, Digital Humanities, Metadata ACM Reference Format: Jérôme Darmont, Cécile Favre, Sabine Loudcher, and Camille Noûs. 2020. Data Lakes for Digital Humanities. In ddh20: Data and Digital Humanities 2020, October 15–17, 2020, Hammamet, Tunisia. ACM, New York, NY, USA, 4 pages. https://doi.org/ A data lake is a scalable storage and analysis system for data of any type, retained in their native format and used mainly (but not only) by data specialists (statisticians, data scientists or analysts) for knowledge extraction [10]. Data Lakes for Digital Humanities Camille Noûs camille.nous@cogitamus.fr Université de Lyon, Lyon 2, Laboratoire Cogitamus Bron, France Camille Noûs camille.nous@cogitamus.fr Université de Lyon, Lyon 2, Laboratoire Cogitamus Bron, France Jérôme Darmont Cécile Favre Sabine Loudcher jerome.darmont@univ-lyon2.fr cecile.favre@univ-lyon2.fr sabine.loudcher@univ-lyon2.fr Université de Lyon, Lyon 2, ERIC UR 3083 Bron, France © 2020 Copyright held by the owner/author(s). Publication rights licensed to the Association for Computing Machinery. 2.1 HyperThesau The “Hyper thesaurus and data lakes: Mine the city and its archaeo- logical archives” (HyperThesau) project involves a multidisciplinary team consisting of two research laboratories of archaeology and of computer science, a digital library, two archaeological museums and a private company. This project has two main objectives: (3) (3) The data storage layer is core to a data lake. It must have the capacity to store all data in any format. (3) The data storage layer is core to a data lake. It must have the capacity to store all data in any format. (4) The data distillation layer provides a set of tools for data cleaning (eliminating errors such as duplicates and type violations) and encoding formalization (converting various data and character encoding). (1) the design and implementation of an integrated platform to host, search, share and analyze archaeological data;i (5) The data insights layer provides a set of tools for data trans- formation (e.g., into models) and exploratory data analysis (e.g., pattern discovery). Note that transformed data may also be stored into the lake for later reuse. (2) the design of a domain-specific thesaurus taking the whole archaeological data lifecycle into account, from data creation to publication.f Archaeological data may bear many different types, e.g., tex- tual documents, images (photographs, drawings...), sensor data, etc. Moreover, similar documents, e.g., excavation reports, are often created by various software tools that are not compatible with each other. The description of an archaeological object also differs with respect to users, usages and time. Such a variety of archaeological data induces many scientific challenges related to storing heteroge- neous data in a centralized repository, guaranteeing data quality, cleaning and transforming the data to make them interoperable, finding and accessing data efficiently and cross-analyzing the data with respect to their spatial and temporal dimensions. (6) The data application layer provides applications that allow users extracting value from data, e.g., through an interactive query system, reports or dataviz.l (7) The workflow manager layer provides tools to automate the flow of data processes. (8) The communication layer provides tools that allow the other layers to communicate with each other. It must provide syn- chronous and asynchronous communication capability. (9) The data governance layer provides a set of tools to establish and execute plans and programs for data quality control [4]. 1 INTRODUCTION One of the main advantages of data lakes is that data are stored in their initial form, and are thus recognizable by their producers, such as DH researchers. A data lake does not propose a new data model nor new data formats for data archiving. Moreover, when data are transformed for processing, the data lineage is stored as metadata, thus enforcing traceability.fi Traditional data management has long been adopted by many re- searchers involved in Digital Humanities (DH). However, it requires a substantial investment in data modeling, including, at the physical level, technologies such as relational and semi-structured Database Management Systems (DBMSs), various data formats, e.g., XML and JSON for semi-structured data, RDF for linked data, and query languages such as SQL and XQuery. This investment in computer science and the fact that initial data are inevitably transformed are presumably impediments to the adoption of DBMSs and related digital tools for DH. However, a drawback is that unprepared data are difficult to process and require data specialists who can program. Yet, we strongly advocate, with other researchers, for the “industrialization” of data lakes, i.e., providing a software layer that allows non-data scientists such as DH researchers to transform and analyze their own data in autonomy, just as dynamic reports are prepared on top of data warehouses for the use of business (i.e, non technical) users. The remainder of this paper is organized as follows. In Section 2, we describe data lake projects we currently run in close collabora- tion with researchers in social sciences and humanities. In Section 3, we conclude this paper by discussing the lessons learned running these projects. Darmont & al. ddh20, October 15–17, 2020, Hammamet, Tunisia Darmont & al. Darmont & al. 1https://atlas.apache.org 2hhttps://hadoop.apache.org/docs/r1.2.1/hdfs_user_guide.html 3https://hive.apache.org 4https://www.openldap.org 5http://spark.apache.org 6https://lucene.apache.org/solr/ 2 EXAMPLE DH PROJECTS INVOLVING DATA LAKES choose the right tools and plans to ingest data into the data lake with respect to data source properties and the lake’s ca- pacity. During ingestion, metadata provided by data sources, e.g., name of excavation sites or instruments, must be gath- ered as much as possible. 2.1 HyperThesau Each of the above layers is implemented with one or more frame- works of the Apache Hadoop ecosystem, e.g., Atlas1, HDFS2, HIVE3, OpenLdap4, Spark5, etc. This prototype is operational and currently hosts the data of two archaeological research facilities. The meta- data management system instantiates the MEtadata model for Data Lakes (MEDAL), which adopts a graph model [10]. It is implemented with Apache Atlas, which can host not only descriptive metadata, but also several thesauruses. With the help of a search engine, i.e., Solr6, users can find data through descriptive metadata, a thesaurus or the data lineage. To overcome all these challenges, we implement a data lake. Our approach aims to collect all types of archaeological data, save them inside the data lake and propose metadata for better organizing data and for allowing users to easily find data for analysis purposes. Our data lake prototype is architectured in nine layers (Figure 1) [5, 6]. 2.2 Bretez/STRATEGE Bretez [7] is a multidisciplinary project aiming at a visual and sonorous restitution of the XVIIIth-century Paris. It is also an ex- ploratory project constituted of successive, interlinked modules that are (and must be) interoperable and open. The historical urban restitution is achieved with video game engines that bear their own respective characteristics, of course related to gaming. Yet, here, they are used for specific management and traceability needs. Moreover, Bretez’ documentation is a voluminous corpus of hetero- geneous and multimedia data. Within project Bretez, the “Traceability and information manage- ment system for multimedia data” (STRATEGE) aims at designing, storing, querying and analyzing all the project’s data. To master data heterogeneity, manage data quality and volume, warrant data interoperability and an efficient access while keeping data in their Figure 1: HyperThesau data lake’s layered architecture [5] Figure 1: HyperThesau data lake’s layered architecture [5] (1) The data source layer gathers the basic properties of data sources, e.g., volume, format, velocity, connectivity, etc. Based on these properties, data engineers can determine how to import data into the lake. p (2) The data ingestion layer provides a set of tools for perform- ing batch or real-time data integration. Data engineers can Data Lakes for Digital Humanities ddh20, October 15–17, 2020, Hammamet, Tunisia Figure 2: CODAL example screenshot [9] Figure 2: CODAL example screenshot [9] original form so that they remain usable reference for the project’s researchers, we resort to a data lake.i they are quite similar in terms of data: a corpus of various textual documents (e.g., annual reports from companies and organizations, interviews of senior or top executives, press articles; all in French or English) and data from various sources, including the Web, cu- rated and inferred by researchers in management sciences from company legal information and performance indicators such as workforce, annual revenue, stock-exchange price and perceived level of digitization and servicization. STRATEGE is in its first stages: we catalogued all existing data, which included a database, textual documents, sounds, images and a 3D Unity7 (a game engine) model, and more. The database is particularly interesting, for it contains both data and metadata. While retaining it, we also restructured it so as to allow its metadata to interoperate with specific data lake metadata. In short, there are “business” metadata and technical metadata. With such data handy, the objective is to cross-analyze the terms and expressions found in textual resources with structured, qual- itative and quantitative data, in order to discover new insights regarding how companies communicate vs. their actual customer relationship management strategy (for COREL) and how digitiza- tion and servicization impact economic performance (for AURA PMI). The challenges here are to: The remaining tasks include fully designing and integrating the metadata system, on the basis of MEDAL [10]; make the data from the Unity model accessible into the lake; formalize analysis needs; and design tools that must jointly handle textual, visual and audio content, as well as the heterogeneity of data sources. Such software tools must be accessible to all researchers involved in the Bretez project. (1) leverage metadata that allow querying the whole corpus; 7https://unity.com 8http://coactis.org 9https://www.elastic.co/elasticsearch/ 10http://basex.org 11https://neo4j.com ACKNOWLEDGEMENTS (2) proximity analyses such as similarity (what documents are similar or different [8]) and centrality (to identify the docu- ments bearing a specific or common vocabulary, hinting at its importance [3]) analyses; Projects HyperThesau and Bretez/STRATEGE are funded by the Laboratory of Excellence “Intelligence of Urban Worlds” (IMU)12. Project COREL was funded by the University of Lyon 2. Project Projects HyperThesau and Bretez/STRATEGE are funded by the Laboratory of Excellence “Intelligence of Urban Worlds” (IMU)12. Project COREL was funded by the University of Lyon 2. Project Projects HyperThesau and Bretez/STRATEGE are funded by the Laboratory of Excellence “Intelligence of Urban Worlds” (IMU)12. Project COREL was funded by the University of Lyon 2. Project AURA PMI is funded by the Auvergne-Rhône-Alpes Region. (3) custom highlights of the context of terms and, optionally, their synonyms, in textual documents. 2.3 COREL and AURA PMI (2) jointly analyze structured and unstructured data; (3) allow management science researchers to perform analyses by themselves. Both the projects “At the heart of customer relationship” (COREL) and “Digital transformation, servicization and mutations of indus- trial SME business models” (AURA PMI) relate to management sciences and are carried out in collaboration with the Coactis lab- oratory8. Although their respective focus and scope are different, To complete these tasks for the COREL project, we designed a metadata system that prefigured MEDAL [10] and proposed the lightweight COREL Data Lake architecture (CODAL) [9], which is composed of: ddh20, October 15–17, 2020, Hammamet, Tunisia Darmont & al. Darmont & al. (1) a storage layer that notably includes Elasticsearch9 for in- dexing textual contents; (1) a storage layer that notably includes Elasticsearch9 for in- dexing textual contents; In consequence, we take great care of accompanying DH users in their appropriation of our analysis tools, not only by training, but especially by interweaving research methodologies from computer science and other disciplines by design, in close collaboration with partner researchers. (2) a metadata layer leveraging and extending the Metadata Encoding & Transmission Standard (METS) [11], stored in the BaseX XML DBMS10; (3) an analysis layer, i.e., an intuitive Web-based graphical inter- face that allows management science researchers to perform analyses in autonomy, thus enforcing the “industrialization” of CODAL. Moreover, the possibility of having both access to the raw data and the entire possible processing chain is necessary, because black boxes are seldom compatible with a sound methodological approach aiming at producing scientific knowledge. Data lakes precisely allow this much needed transparency. The analysis layer features three kinds of analyses: The analysis layer features three kinds of analyses: (1) data exploration akin to On-Line Analytical Processing (OLAP)[1 All three types of analyses come with various dataviz (Figure 2). All three types of analyses come with various dataviz (Figure 2). [1] E.F Codd, S.B Codd, and C.T Salley. 1993. Providing OLAP (On-line Analytical Processing) to User-Analysts: An IT Mandate. Technical Report. E.F. Codd & Associates. [1] E.F Codd, S.B Codd, and C.T Salley. 1993. Providing OLAP (On-line Analytical Processing) to User-Analysts: An IT Mandate. Technical Report. E.F. Codd & Associates. The AURA PMI Data Lake (AUDAL) is currently being devel- oped, and builds upon CODAL. Its metadata system will notably be a substantial evolution of MEDAL supported by the Neo4J11 graph DBMS. Moreover, the AUDAL analysis layer, which lays on an Application Programming Interface (API), will be much more elaborate and efficient than CODAL’s. [2] James Dixon. 2010. Pentaho, Hadoop, and Data Lakes. (October 2010). https: //jamesdixon.wordpress.com/2010/10/14/pentaho-hadoop-and-data-lakes/ [2] James Dixon. 2010. Pentaho, Hadoop, and Data Lakes. (October 2010). https: //jamesdixon.wordpress.com/2010/10/14/pentaho-hadoop-and-data-lakes/ [3] Ashley Farrugia, Rob Claxton, and Simon Thompson. 2016. Towards Social Network Analytics for Understanding and Managing Enterprise Data Lakes. In Advances in Social Networks Analysis and Mining (ASONAM 2016), San Francisco, CA, USA (IEEE). 1213–1220. ( ) [4] Vijay Khatri and Carol Brown. 2010. Designing data governance. Commununica- tions of the ACM 53 (January 2010), 148–152. [4] Vijay Khatri and Carol Brown. 2010. Designing data governance. Commununica- tions of the ACM 53 (January 2010), 148–152. 12https://imu.universite-lyon.fr 3 CONCLUSION [5] Pengfei Liu, Sabine Loudcher, Jérôme Darmont, and Camille Noûs. 2020. A data lake architecture and implementation for archaeological data management and analytics. Technical Report. Université de Lyon, Lyon 2, ERIC UR 3083, France. In all four data lake projects summarized in Section 2, we use differ- ent versions of the MEDAL metadata system, which is designed to be generic. However, although MEDAL is quite flexible, we do not believe in a single model for data lakes. There are indeed significant differences in data in only four projects, in terms of volume, variety and velocity, which imply different architectures and technologies. Thus, we think that much needed methodological tools for data lakes should be instantiated for each project rather than applied “as is”. [6] Pengfei Liu, Sabine Loudcher, Jérôme Darmont, Emmanuelle Perrin, Jean-Pierre Girard, and Marie-Odile Rousset. 2020. Metadata model for an archeological data lake. Digital Humanities (DH 2020), Ottawa, Canada. (July 2020). https: //dh2020.adho.orgfi g [7] Mylène Pardoën. Last accessed: June 2020. Bretez site officiel. (Last accessed: June 2020). https://sites.google.com/site/louisbretez/home [8] Pascal Pons and Matthieu Latapy. 2006. Computing Communities in Large Networks Using Random Walks. Journal of Graph Algorithms and Applications 10, 2 (2006), 191–218. [9] Pegdwendé-Nicolas Sawadogo, Tokio Kibata, and Jérôme Darmont. 2019. Meta- data Management for Textual Documents in Data Lakes. In 21st International Conference on Enterprise Information Systems (ICEIS 2019), Heraklion, Crete-Greece. INSTICC, 72–83. Vol. 1. Furthermore, the software layer we add to “industrialize” our data lakes might become yet another black box, while there is a strong stake for researchers in humanities and social sciences involved in DH projects not to be dispossessed of data by an analysis layer that would adopt a “click and go” approach. Data are indeed often partly constructed by said researchers themselves as a product of scientific work that takes time, thus giving a significant value to datasets. [10] Pegdwendé-Nicolas Sawadogo, Etienne Scholly, Cecile Favre, Eric Ferey, Sabine Loudcher, and Jérôme Darmont. 2019. Metadata Systems for Data Lakes: Models and Features. In 1st International Workshop on BI and Big Data Applications (BBI- GAP@ADBIS 2019), Bled, Slovenia (Communications in Computer and Information Science), Vol. 1064. Springer, Heidelberg, Germany, 440–451. p g g y [11] The Library of Congress. 2017. METS: An Overview and Tutorial. (March 2017). http://www.loc.gov/standards/mets/METSOverview.v2.html
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Primary sclerosing epithelioid fibrosarcoma of kidney with variant histomorphologic features: report of 2 cases and review of the literature
Diagnostic pathology
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* Correspondence: dertoy@hacettepe.edu.tr 1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey Full list of author information is available at the end of the article Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 DOI 10.1186/s13000-015-0420-z © 2015 Ertoy Baydar et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background supported by MUC4 immunostaining and fluorescence in situ hybridization (FISH). To our knowledge, these are the sixth and seventh genetically confirmed cases of SEF reported in the kidney following very recently described primary renal SEF(s) by Arbajian et al., Argani et al. and Ohlmann, et al. [1, 8, 9]. Some of their morpho- logic features are unique and deserve to be noted for full characterization of this entity in a new environment. Sclerosing epithelioid fibrosarcoma (SEF) is a rare malig- nant mesenchymal tumor of soft tissues composed of cords, nests or sheets of relatively monotonous epithelioid cells within a collagenous background. It has recently been characterized by recurrent FUS-CREB3L1, FUS-CREB3L2 or EWS1-CREB3L1 translocations and immunohistochemi- cal MUC4 expression [1–3]. The tumor occurs over a wide age spectrum at initial presentation without sex predilec- tion. Most reported cases are in the soft tissue of extrem- ities and limb girdles [4–7]. Primary SEF in visceral organs is exceedingly rare, with only a few cases reported. Abstract The authors present two cases of primary sclerosing epithelioid fibrosarcoma (SEF) of the kidney. Both patients had a mass in the upper part of the left kidney without any primary extrarenal neoplastic lesions. Grossly, the tumors were solid masses both measuring 7.5 cm in the greatest diameter. Histologically, one of the lesions exhibited a predominantly lobular growth of round or oval small uniform epithelioid cells in variable cellularity. Circular zones of crowded tumor cells alternating with hypocellular collagenous tissue in a concentric fashion around entrapped native renal tubules were distinctive. The second case was distinctive with significant cytological atypia in the neoplastic cells and prominent reactive proliferations in the trapped renal tubules. Immunohistochemically, vimentin, bcl-2 and MUC4 were diffusely positive in both. They were negative for S-100 protein, CD34, and desmin, whereas CD99 were positive in one lesion. Fluorescence in situ hybridization assay using dual staining probes detected EWSR1-CREB3L1 fusion in each lesion, which is characteristic molecular findings of SEF. One patient presented widespread distant metastases at the time of diagnosis. In the other, no tumor deposits were detected other than primary. Both patients have been alive with 30 and 10 month follow-ups, respectively. These tumors are 6th and 7th cases of primary renal SEF in the literature confirmed by FISH study, which exhibit unique and remarkable histomorphologic features. Keywords: Sclerosing epithelioid fibrosarcoma, Kidney, EWSR1, CREB3L1, Translocation Keywords: Sclerosing epithelioid fibrosarcoma, Kidney, EWSR1, CREB3L1, Translocation Case presentation Two cases of SEF that were found to harbor EWSR1- CREB3L1 fusion were encountered in the diagnostic practice of the authors. The clinical records were retrieved for analysis, and all available pathologic materials were reviewed. Here, we present two cases of SEF with primary renal origin bearing a EWS1-CREB3L1 gene fusion, as Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 2 of 12 Table 1 Features of the antibodies used for the immunohistochemical stains Antigen Clone, dilution, source Pan-Cytokeratin AE1/AE3, 1:200, Leica, Newcastle/UK Epithelial Membrane Antigen E29, 1:300, Biocare, Concord/CA PAX8 PAX8, 1:100, Biocare, Concord/CA WT1 6 F-H2, 1:40, DBS, Pleasenton/CA ER EP1, 1:100, Genemed, San Francisco/CA PR SP2, 1:1000, Thermo, Fremont/CA CD34 QBEND/10, 1:100, DBS, Pleasenton/CA SMA 1A4, 1:1000, NeoMarkers, Fremont/CA Desmin D33, 1:50, Biocare, Concord/CA S100 protein Z0311, 1:6000, DAKO, Glostrup/Denmark GFAP GA-5, 1:50, Thermo, Fremont/CA Bcl2 Bcl-2-100, 1:80, Invitrogen, Paisley/UK CD99 HO36-1.1, 1:100, Thermo, Fremont/CA MUC4 1G8, 1:50, Invitrogen, Paisley/UK HMB45 HMB45, 1:25, DBS, Pleasenton/CA Melan-A A103, 1:100, Thermo, Fremont/CA INI1 25, 1:50, Zeta, Arcadia/CA Fig. 2 Case #1. Solid white tumor filling out the upper half of the kidney Fig. 2 Case #1. Solid white tumor filling out the upper half of the kidney Fluorescence In Situ Hybridization (FISH) Fluorescence In Situ Hybridization (FISH) FISH analysis was performed on representative 4–5 μm thick unstained formalin-fixed, paraffin-embedded tissue sections of the tumor samples of each case. For characte rization of the possible underlying gene rearrangement or fusion gene events, the following FISH probe sets were utilized on both cases: Vysis LSI EWSR1 (22q12) and Vysis LSI FUS (16p11) Dual Color Break Apart Probes Immunohistochemistry For immunohistochemical labeling, a polymer detection system (Leica, DS9800) and the BOND-MAX automated immunostainer was used. The standard antibodies con- sumed, vendors, and dilutions were summarized in Table 1. Fig. 3 Case #1. Alternating hypercellular and a hypocellular areas of neoplastic cells with monomorphic ovoid nuclei and indistinct pale to clear cytoplasm (H&E x 200; inset: H&E x 400) Fig. 3 Case #1. Alternating hypercellular and a hypocellular areas of neoplastic cells with monomorphic ovoid nuclei and indistinct pale to clear cytoplasm (H&E x 200; inset: H&E x 400) Fig. 1 Case #1. Computerized tomography scan showing a large tumor in the left kidney Fig. 3 Case #1. Alternating hypercellular and a hypocellular areas of neoplastic cells with monomorphic ovoid nuclei and indistinct pale to clear cytoplasm (H&E x 200; inset: H&E x 400) Fig. 1 Case #1. Computerized tomography scan showing a large tumor in the left kidney Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 3 of 12 Fig. 4 Case #1. a Lobular/micronodular pattern (H&E x 40). b Concentric hypocellular inner, hypercellular outer zone around entrapped renal tubules (H&E x 100) Fig. 4 Case #1. a Lobular/micronodular pattern (H&E x 40). b Concentric hypocellular inner, hypercellular outer zone around entrapped renal b l (H&E 100) Fig. 4 Case #1. a Lobular/micronodular pattern (H&E x 40). b Concentric hypocellular inner, hypercellular outer zone around entrapped renal tubules (H&E x 100) (Abbott Molecular, Inc., Des Plaines, IL) and EWSR1 and CREB3L1 spanning probe sets using cocktails of BAC clones (RP11-945 M21 and RP11-1126O13, and RP11- 1014A16, RP11-1106 J11 and RP11-481I24 respectively) selected on the basis of their location per the UCSC Human Genome Browser [http://genome.ucsc.edu/cgi- bin/hgGateway] and obtained from BAC/PAC Re- sources Center (Children’s Hospital Oakland Research Institute, Oakland, CA, USA). Subsequently, the slides were incubated for 15 min at 37 °C in protease solution [25 mg of protease in 50 ml of protease solution (Abbott Molecular, Inc., Des Plaines, IL), washed in 1 × PBS at room temperature for 5 min and then dehy- drated in gradient ethanol (75, 85, and 100 %) at room temperature for 1 min each and air-dried. Immunohistochemistry After the cells and probes were co-denatured at 80 °C for 10 min and incubated overnight at 37 °C using the HYBrite™system (Abbott Molecular, Inc., Des Plaines, IL), post-hybridization washing was performed in 2 × SSC/0.1 % NP-40 at 72 °C for 2 min, followed by 2 × SSC/0.1 % NP-40 at room temperature for 1 min. The slides were then counter- stained with DAPI II (Abbott Molecular, Inc., Des Plaines, IL). To confirm correct mapping, optimal signal strength, and lack of cross-hybridization, each probe set was also hybridized to metaphase cell preparations of karyotypically normal peripheral blood lymphocytes before proceeding with analysis of the patient samples. Hybridization studies using the Vysis LSI Dual Color Break Apart Probes for the assessment of rearrangement of the FUS and EWSR1 loci were performed following the manufacturer’s instructions (Abbott Molecular, Inc., Des Plaines, IL). With respect to the custom spanning probes, each BAC clone was directly labeled by nick translation with either Spectrum Green- or Spectrum Orange-dUTP per the manufacturer’s protocol (Abbott Molecular, Inc., Des Plaines, IL). An amount of 3 ug of DNA for each probe or 1.5 ug for each of two probes were combined. All nick translation reagents were then multiplied by the total ug of DNA used in the cocktail. Amounts of 200 ng of each probe were hybridized to the target DNA and blocked with approximately 15 fold excess of a combination of Human Cot-I DNA (Invitrogen, Carlsbad, CA) and human placental DNA. The cutoff level for scoring a specimen as positive for a rearrangement of the FUS or EWSR1 locus or as positive for an EWSR1/CREB3L1 fusion was >15 % of the cells eval- uated. Images were prepared using the Cytovision Image Analysis System (Applied Imaging, Santa Clara, CA). For each probe set, 100–200 interphase nuclei with strong and well-delineated signals were examined. Prior to hybridization, the slides were pretreated at room temperature in 0.2 N HCl for 20 min, washed in water for 3 min, incubated at 80 °C for 25 min in VP 2000 Pretreatment Reagent (Abbott Molecular, Inc., Des Plaines, IL) and then washed again in water for 3 min. Case 1 The patient was a 16-year-old girl who presented at the urology clinics with pain on the left side of the abdo- men radiating to the back. Her past medical history was Page 4 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 5 Case #1. Strong bcl-2 and weak EMA expression by neoplastic cells (Left: Immunohistochemistry, anti-bcl-2 Ab x 200; Right: Immunohistochemistry, anti-EMA Ab x 200) bcl-2 and weak EMA expression by neoplastic cells (Left: Immunohistochemistry, anti-bcl-2 Ab x 200; Right: Immunohistochemistry, Fig. 5 Case #1. Strong bcl-2 and weak EMA expression by neoplastic cells (Left: Immunohistochemistry, anti-bcl-2 Ab x 200; Right: Immunohistochemistry, anti-EMA Ab x 200) noted in many regions where a renal tubule in the center was surrounded by concentric inner hypo and outer hypercellular zones of neoplastic cells (Fig. 4a and b). These lobules were separated from each other by myo- fibroblasts. Tubules entrapped in the tumor were lined by single layered Pax-8 positive cuboidal cells without atypia, some showed shallow papillary hyperplasia and rare mitosis. Mitotic rate was 1/10 hpf in the neoplasm and there were occasional areas of necrosis in the tumor. Hypercellular areas occasionally contained vague nodules of collagen mimicking those seen in hyalinizing spindle cell tumor with giant rosettes (HSCTGR). The surrounding kidney showed no specific pathologic changes. By im- munohistochemistry, neoplastic cells were immunore- active diffusely and strongly for vimentin, bcl-2 and CD99; EMA labelled them in a weak and patchy fashion (Fig. 5). They were negative for pan-cytokeratin, Pax-8, WT-1, CD34, S-100, GFAP, Melan-A, HMB-45, desmin, and estrogen and progesterone receptors. Smooth muscle actin (SMA) stained myofibroblastic cells in-between the neoplastic lobules (Fig. 6). INI-1 was preserved. Then, an insignificant. Computerized axial tomography scans of the chest, abdomen, and pelvis revealed a 70 × 70 × 60 mm left renal mass (Fig. 1), bilateral pulmonary nodules (the largest being 16 mm in diameter), and widespread bone metastases in vertebrae, sacrum and left femoral head. A biopsy from the tumor in the kidney through laparotomy was performed followed by left radical nephrectomy after the diagnosis of malignancy. Tumor occupied the entire upper half of the kidney, extended into the renal sinus, was a solid white unencapsu- lated lesion with sharp borders, measuring 7.5 × 7 × 7 cm in size (Fig. 2). Case 1 Microscopical sections showed diffuse infiltra- tion of the neoplastic tissue in kidney parenchyma separat- ing normal renal elements from each other. The neoplastic cells were small, monotonous and epithelioid with clear to pale eosinophilic cytoplasm, and were arranged in single files, cords, nests or irregular aggregates in collagenous matrix (Fig. 3). Nuclei were generally round to oval, with indistinct nucleoli. Hypercellular areas alternated randomly with hypocellular densely hyalinized or at times myxoid stroma. Additionally, a peculiar lobular organization was Page 5 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 5 of 12 Fig. 6 Case #1. SMA stains myofibroblasts between neoplastic lobules and MUC4 stains neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 100; Right: Immunohistochemistry, anti-MUC4 Ab x 400) Fig. 6 Case #1. SMA stains myofibroblasts between neoplastic lobules and MUC4 stains neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 100; Right: Immunohistochemistry, anti-MUC4 Ab x 400) immunohistochemical stain for MUC4 was performed which showed strong positivity throughout the tumor (Fig. 6). Finally, FISH analysis with the EWSR1 Break Apart probe revealed loss of one copy of the Spectrum Green labeled probe flanking the 3’ (telomeric) side of the EWSR1 gene as well as the presence of a single fused EWSR1/CREB3L1 signal (represented by a juxtaposed or- ange signal and green signal) consistent with the presence of an unbalanced der(22)t(11;22)(p11;q12) (Fig. 7), con- firming the diagnosis of sclerosing epithelioid fibrosar- coma. FISH study was negative for a rearrangement of the FUS gene locus. 6 cm in the largest diameter. Her past medical history was unremarkable except for hypertension, congestive heart fail- ure and arrhythmia. Serum and urine tests were within the normal limits. The patient subsequently underwent open left partial nephrectomy. On gross examination, the specimen had an unencap- sulated, solid - white firm tumor with irregular borders, measuring 7.5 × 5.5 × 4 cm (Fig. 8). Histologic examin- ation revealed that tumor contained abundant entrapped native renal tubules throughout as in the first case, mim- icking a biphasic neoplasm (Fig. 9). These tubules were hyperplastic and proliferating, were of various size and shapes, some being cystic or leaf-like, and lined by single layered cuboidal or occasionally flattened cells, all express- ing nuclear Pax-8. Intraluminal papillary projections were common, but without cytologic atypia. Neoplastic compo- nent had a variable cellularity (Fig. 10). Hypocellular re- gions contained abundant hyalinized collagen and some showed myxoid change. Case 1 Hypercellular areas were divided into anastomosing compartments by sclerotic collagen bands. Neoplastic cells were polygonal epithelioid or plump Patient was given chemotherapy with multiple agents. She is alive with disease after 30 month follow-up. Case 2 The patient was a 57-year-old woman who was investigated for cholelithiasis due to dyspeptic complaints. During ab- dominal ultrasonography, she was found to have a left renal mass incidentally. The CT scan showed that the tumor was located at the upper pole of the kidney and was measured Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 6 of 12 Fig. 7 Case #1. An EWSR1 Break Apart probe set and a custom probe set spanning the EWSR1 (Spectrum Orange) and the CREB3L1 (Spectrum Green) loci demonstrating loss of the Spectrum Green labeled probe signal that flanks the 3’ (telomeric) side of the EWSR1 locus (short arrows, left panel) and a single fusion signal (long arrows, right panel) Fig. 7 Case #1. An EWSR1 Break Apart probe set and a custom probe set spanning the EWSR1 (Spectrum Orange) and the CREB3L1 (Spectrum Green) loci demonstrating loss of the Spectrum Green labeled probe signal that flanks the 3’ (telomeric) side of the EWSR1 locus (short arrows, left panel) and a single fusion signal (long arrows, right panel) spindle type forming short fascicles (Fig. 11). Cytoplasm was clear or eosinophilic. Cellular pleomorphism was prominent with scattered bizarre and hyperchromatic nuclei, intranuclear inclusions were evident in some. Tumor abutted lining urothelium of renal pelvis with- out ulceration and formed large hypocellular sheets of short spindle cells in myxoid matrix encircling pelvic wall. There were rare foci of necrosis. Mitotic rate ranged 5–10/ 10 hpf. Immunohistochemical profile of tumor was similar to the first case including strong and diffuse MUC4, vimen- tin and bcl-2, weak EMA expression (Fig. 12) except nega- tive immunoreactivity for CD99. SMA showed frequently scattered myofibroblasts between the tumor cells. FISH demonstrated an unbalanced der(22)t(11;22)(p11;q12), as in the case #1 (Fig. 13). A FUS gene rearrangement was not identified. showed 1 cm residual mass. No adjuvant treatment was given. Patient is alive without local or distant recurrence 10 months after surgery. Discussion Sclerosing epithelioid fibrosarcoma (SEF) was first de- scribed by Meis-Kindblom et al. in 1995 [6]. The most SEFs are deep seated lesions with a wide age range, but typically occurring in middle aged adults [4, 6]. The most frequent sites of involvement are deep soft tissues of lower extremities or limb girdle followed by trunk, upper extremities, and head and neck region, although primary SEF of unexpected locations such as bone, retro- peritoneum, and pelvis were also described as case reports or small series [10–13]. Primary SEF in visceral organs is exceedingly rare, with only a single case reports in the liver [12], the lower gastrointestinal tract [11], the ovary [14], and the pancreas [10]. Occurrence of primary renal SEF was proven by Argani et al. who have recently reported 2 well-documented cases with rearrangement involving The lesion occupied both renal cortex and medulla, and invaded into peripelvic and perirenal fat tissue. As one of the surgical margins was in continuation with the tumor, the patient underwent left radical nephrectomy which Page 7 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 8 Case #2. Gross photograph of the kidney in patient no. 2 shows a well-defined solid mass that extends to surgical margin of the partial nephrectomy specimen EWSR1 and CREB3L1 genes [8] (Table 2). There are also 3 additional cases in the literature described as primary renal SEFs which were shown to express MUC4 [1, 9]. Two of these had rearranged EWSR1 with undocumented fusion gene partner. EWSR1 and CREB3L1 genes [8] (Table 2). There are also 3 additional cases in the literature described as primary renal SEFs which were shown to express MUC4 [1, 9]. Two of these had rearranged EWSR1 with undocumented fusion gene partner. SEF is a difficult diagnosis in visceral organs because of its rarity and its epithelioid appearance, closely mimicking carcinomas. It is characterized by a proliferation of epitheli- oid cells arranged in nests and cords in a densely hyalinized stroma (Table 3). Neoplastic cells are positive for vimentin, bcl-2, MUC4, weakly and focally for epithelial membrane antigen (EMA), and are negative for broad spectrum cyto- keratins, smooth muscle actin, desmin, CD34, S-100 pro- tein, HMB45 and melan-A [2, 4, 15]. MUC4 itself has been recently reported as highly specific for the diagnosis of SEF [2]. Strong CD99 expression is seen in some cases. Discussion Most common genetic alteration described in pure SEFs is EWSR1-CREB3L1 fusion [1]. Basic histomorphological differentials of SEF will be a variety of tumors with epithe- lioid and sclerotic features, mainly primary or metastatic carcinoma. Epithelioid angiomyolipoma, metanephric stro- mal tumor and synovial sarcoma are the other consider- ations in the differential diagnosis, as well as sclerosing clear cell sarcoma of the kidney (CCSK) being the most challenging. A battery of immunohistochemical stains will help for further characterization in most circumstances, Fig. 8 Case #2. Gross photograph of the kidney in patient no. 2 shows a well-defined solid mass that extends to surgical margin of the partial nephrectomy specimen Fig. 9 Case #2. Proliferating native renal tubules inside the tumor giving an appearance of a biphasic lesion (H&E x 100). Inset shows that hyperplastic tubules maintain nuclear pax-8 (H&E x 40) Fig. 9 Case #2. Proliferating native renal tubules inside the tumor giving an appearance of a biphasic lesion (H&E x 100). Inset shows that hyperplastic tubules maintain nuclear pax-8 (H&E x 40) Page 8 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 10 Case #2. Hyper and hypocellular regions (Left: H&E x 200; Right: H&E x 200) Fig. 10 Case #2. Hyper and hypocellular regions (Left: H&E x 200; Right: H&E x 200) CREB3L1 gene fusion with t(11;22)(p11;q12) chromosome translocation was also found [18]. There have been 5 cases of primary LGFMS described in kidney or renal pelvis so far, one in a 6 year old child, others in adults [19–23]. however SEF and sclerosing type CCSK reveal both similar morphology and immunohistochemical findings Argani et al. suggest that some cases reported as sclerosing clear cell sarcoma of the kidney (CCSK) in the literature might in fact represent SEF [8]. We report 2 distinctive, clinically malignant, renal SEF bearing EWSR1-CREB3L1 fusions through unbalanced translocation with unique histomorphologic features. The common finding in both was the diffuse infiltration of the tumor among renal tubules and glomeruli filling out the space in-between them. Numerous tubules and also glomeruli were seen entrapped throughout the tumors. In case #1, neoplastic cells surrounded these tubules in a concentric onion-skin like pattern giving a lobular or micronodular architecture to the lesion in many areas. Additionally, the neoplastic sleeves around tubules revealed a zonation pattern with inner hypo- cellular and outer hypercellular appearance. Discussion This was a wide-spread occurrence involving also the deeper parts of the tumor, so differed from what is seen in CCSK or metanephric stromal tumor where neoplasm encircles native tubules at the tumor infiltration borders in a SEF and low-grade fibromyxoid sarcoma (LGFMS) are thought to be related members in the fibrosing fibrosar- coma family [4]. Classic variants of LGFMS with or without giant rosettes constitute one end of the spectrum, charac- terized by a protracted clinical course and a low metastatic rate, whereas SEF or cellular variants of LGFMS constitute the other end which appears to be more aggressive. LGFMS may have SEF-like areas, and vice versa occurs in SEF lesions. Both tumors are labelled by immunohistochemical MUC4 staining, 99–100 % in LGFMS and 78 % in SEF, respectively [2, 16]. Furthermore, a genetic link between sclerosing epithelioid fibrosarcoma and low-grade fibro- myxoid sarcoma has been suggested [17]. Although major- ity (>90 %) of LGFMS and hybrid LGFMS/SEF harbor t(7;16)(q33;p11) chromosome translocation resulting in FUS-CREB3L2 gene fusion, rare LGFMS carrying EWSR1- Page 9 of 12 Page 9 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 11 Case #2. Spindle cells forming short fascicles vaguely (H-E x 200). Inset highlights marked pleomorphism among the neoplastic cells (H-E x 400) le cells forming short fascicles vaguely (H-E x 200). Inset highlights marked pleomorphism among the neoplastic cells (H-E x 400) atypia in a gradual transition to small round cells. Immu- nohistochemically, tumor was bcl-2 and CD99+, SMA and S-100 -. SYT-SSX1 and SYT-SSX2 chimeric transcript were not identified. We suspect that at least this published case may represent the entity reported herein. limited extent. No angiodysplasia or juxtaglomerular hyperplasia was found in our case. The second case presented in this study varied from the first one in that the entrapped renal tubules exhib- ited prominent papillary hyperplasia and small gland budding which gave a biphasic appearance to the lesion. They were lined by cuboidal or flattened single layered epithelium devoid of atypia, some were cystic, and some looked fibroadenomatous and leaf-like due to compression by the interstitial neoplastic cells. It is quite likely that we would have diagnosed this lesion as malignant mixed epithelial and stromal tumor of kidney (MEST) only a few years ago when typical genetic alterations or MUC4 staining characteristic of this tumor type were unknown yet. Discussion In fact, we suspect that other examples of renal SEF may have been reported in the lit- erature with an assignment as malignant MEST. Suzuki et al. [24] reported a malignant MEST in a male with a pros- tatic adenocarcinoma under anti-androgen treatment, that was composed of atypical small round cells with a high nuclear cytoplasmic ratio without necrosis and pleo- morphism, and also focal spindle cells without cytologic Another feature of the second case of this study that var- ied from the first one and from 5 cases reported previously was the presence of high grade cytologic atypia with note- worthy pleomorphism and scattered bizarre tumor cells. Most SEFs are cytologically uniform tumors with scanty mitoses; however, it is stated that foci of pleomorphism -particularly in hybrid SEF and LGFMS cases- and high mitotic activity can rarely be encountered [4, 7, 17]. SEF is frequently characterized by aggressive clinical behavior [4, 6]. One or more local recurrences occur in approximately 50 % of cases, with metastatic spread being reported in more than 40 % of cases, most often affecting the pleura, lungs, bone and central nervous system. All of the cases reported previously except one and our first case were already metastatic at the time of presentation. As accurate recognition of SEF is important for appropriate patient management, we believe more cases need to be Page 10 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 12 Case #2. SMA stains interspersed myofibroblasts and MUC4 labels neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 200; Right: Immunohistochemistry, anti-MUC4 Ab x 200) Fig. 12 Case #2. SMA stains interspersed myofibroblasts and MUC4 labels neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 200; Right: Immunohistochemistry anti MUC4 Ab x 200) Fig. 12 Case #2. SMA stains interspersed myofibroblasts and MUC4 labels neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 200; Right: Immunohistochemistry, anti-MUC4 Ab x 200) Fig. 13 Case #2. FISH patterns similar to Case 1. EWSR1 break apart with loss of one copy of the Spectrum Green labeled probe flanking the 3’ (telomeric) side (left panel) and EWSR1/CREB3L1 fusion seen by a juxtaposed orange signal and green signal (right panel) Fig. 13 Case #2. FISH patterns similar to Case 1. Discussion EWSR1 break apart with loss of one copy of the Spectrum Green labeled probe flanking the 3’ (telomeric) side (left panel) and EWSR1/CREB3L1 fusion seen by a juxtaposed orange signal and green signal (right panel) Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 11 of 12 Table 2 Summary of reported cases of primary renal SEF Authors Age (years) Gender Largest Diameter /Laterality Clinical presentation FISH Findings Therapy Metastases Outcome (mo) Arbajian, et al. [1] 41 Female 9 cm/NS NS EWSR1 Del 3′ NS Bone and lung DOD (22) Argani, et al. [8] 17 Male 25 cm/L Left flank, back and abdominal pain, weight loss, dysuria, and decreased appetite EWSR1- CREB3L1 fusion Surgery + RT Rib, vertebrae, epidural spinal cord and liver DOC (1) Argani, et al. [8] 61 Female 5 cm/L Rib pain EWSR1- CERB3L1 fusion Surgery Ribs, bone, lung and lymph nodes AWD (6) Ohlmann, et al. [9] 24 Female 22 cm/R NS No results Surgery + RCT Lungs and vertebrae DOD (82) Ohlmann, et al. [9] 43 Male 4.2 cm/R Incidental EWSR1 split Surgery None ANED (8) Ertoy Baydar, et al. (present case) 16 Female 7.5 cm/L Abdominal pain radiating to back on the left EWSR1- CREB3L1 fusion Surgery + CT Lungs, vertebrae, sacrum and left femoral head AWD (30) Ertoy Baydar, et al. (present case) 57 Female 7.5 cm/L Incidental EWSR1- CREB3L1 fusion Surgery None ANED (10) NS Not specified, L Left kidney, R Right kidney, RT Radiotherapy, CT chemotherapy, RCT Radiochemotherapy, DOD Died of disease, DOC Died of complications related to disease treatment, AWD Alive with disease, ANED Alive with no evidence of disease documented so that its full histomorphologic spectrum is uncovered. are better known to develop in the kidney is a strong possibility as it has been only recently described and is unfamiliar to the pathologists. SEF is a neoplasm with variant morphological features that may overlap with many lesions confusingly. Immunohistochemistry and molecular studies that disclose the characteristic genetic alterations are crucial for accurate recognition. Conclusions Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft tissue tumor that can occur in the kidney as a primary malignancy. Its misdiagnosis with other entities which Table 3 Morphology of SEF Table 3 Morphology of SEF Typical features Macroscopy Large, homogeneously white or white–tan, lobulated, and hard tumors Cell size and shape Small to medium-sized plump to epithelioid cells Cytoplasm Scant clear or eosinophilic cytoplasm Nuclei Oval to slightly elongate angulated nuclei with finely speckled chromatin Cellularity Variable within the neoplasm Atypia Mild Matrix Densely sclerotic, areas of metaplastic bone in some Pattern of cellular arrangement Small clusters, nests and anastomosing cords Hybrid morphology Areas of LGFMS or nodules of collagen reminiscent of HSCTGR in some Immunohistochemistry MUC4 ++, EMA +/−, CD99 +/−, bcl2 +/−, pan-keratin -, S100/HMB45/MelanA - FISH EWSR1 (or rarely FUS) rearrangements Previously unrecognized features in renal SEF Current case 1 A lobular or micronodular architecture due to neoplastic cells surrounding entrapped renal tubules in a concentric fashion Zonation in the neoplastic lobules around tubules with inner hypocellular and outer hypercellular appearance Current case 2 Exuberant epithelial hyperplasia and small gland budding in the entrapped native renal tubules, mimicking MEST High grade cytologic atypia LGFMS Low grade of fibromyxoid sarcoma, HSCTGR Hyalinizing spindle cell tumor with giant rosettes, MEST Mixed epithelial and stromal tumor of kidney A lobular or micronodular architecture due to neoplastic cells surrounding entrapped renal tubules in a concentric fashion Zonation in the neoplastic lobules around tubules with inner hypocellular and outer hypercellular appearance Exuberant epithelial hyperplasia and small gland budding in the entrapped native renal tubules, mimicking MEST High grade cytologic atypia HSCTGR Hyalinizing spindle cell tumor with giant rosettes, MEST Mixed epithelial and stromal tumor of kidney LGFMS Low grade of fibromyxoid sarcoma, HSCTGR Hyalinizing spindle cell tumor with giant rosettes, MEST Mixed epithelial and stromal tumor of kidney Page 12 of 12 Page 12 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Authors’ contribution DEB, KK, OA and BB performed the histological examination. FTA and OA analyzed and interpreted the patients’ data. JAB performed FISH analysis and figures. DEB drafted the manuscript. KK made substantial contribution to analysis. All authors read and approved the final manuscript. 16. Doyle LA, Moller E, Dal Cin P, Fletcher CD, Mertens F, Hornick JL. MUC4 is a highly sensitive and specific marker for low-grade fibromyxoid sarcoma. Am J Surg Pathol. 2011;35(5):733–41. doi:10.1097/PAS.0b013e318210c268. The authors declare that they have no competing interest. The authors declare that they have no competing interest. 15. Goldblum JR, Folpe AL, Weiss SW. Borderline and Malignant Fibroblastic/ Myofibroblastic Tumors. In: Goldblum JR, Folpe AL, Weiss SW, editors. Enzinger and Weiss's soft tissue tumors. 6th ed. Philadelphia, PA: Saunders/ Elsevier; 2014. p. 288–340. Acknowledgment 17. Guillou L, Benhattar J, Gengler C, Gallagher G, Ranchere-Vince D, Collin F, et al. Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group. Am J Surg Pathol. 2007;31(9):1387–402. doi:10.1097/PAS.0b013e3180321959. The authors thank Jonathan I. Epstein, M.D. and Pedram Argani, M.D. (The Johns Hopkins Medical Institutions, Baltimore, MD) who reviewed first case and provided a significant diagnostic input. The authors also would like to thank Dr. Irina Vaysertreyger, Dr. Dali Huang, and Dr. Yimin Sun for their expert technical assistance. 18. Lau PP, Lui PC, Lau GT, Yau DT, Cheung ET, Chan JK. EWSR1-CREB3L1 gene fusion: a novel alternative molecular aberration of low-grade fibromyxoid sarcoma. Am J Surg Pathol. 2013;37(5):734–8. doi:10.1097/ PAS.0b013e31827560f8. Consent 9. Ohlmann CH, Brecht IB, Junker K, van der Zee JA, Nistor A, Bohle RM, et al. Sclerosing epithelioid fibrosarcoma of the kidney: clinicopathologic and molecular study of a rare neoplasm at a novel location. Ann Diagn Pathol. 2015. doi:10.1016/j.anndiagpath.2015.04.005. Written informed consent was obtained from the parent (case #1) and the patient (case #2) for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in- Chief of this journal. 10. Bai S, Jhala N, Adsay NV, Wei S. Sclerosing epithelioid fibrosarcoma of the pancreas. Ann Diagn Pathol. 2013;17(2):214–6. doi:10.1016/ j.anndiagpath.2011.06.006. 10. Bai S, Jhala N, Adsay NV, Wei S. Sclerosing epithelioid fibrosarcoma of the pancreas. Ann Diagn Pathol. 2013;17(2):214–6. doi:10.1016/ j.anndiagpath.2011.06.006. 11. Frattini JC, Sosa JA, Carmack S, Robert ME. Sclerosing epithelioid fibrosarcoma of the cecum: a radiation-associated tumor in a previously unreported site. Arch Pathol Lab Med. 2007;131(12):1825–8. doi:10.1043/ 1543-2165(2007)131[1825:SEFOTC]2.0.CO;2. Author details 1 f 1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. 2Department of Pathology, Ondokuz Mayis University Hospital, Samsun, Turkey. 3Departments of Pathology/Microbiology, Pediatrics and Orthopaedic Surgery, Nebraska Medical Center, Omaha, NE, USA. 4Department of Urology, Hacettepe University Hospital, Ankara, Turkey. 1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. 2Department of Pathology, Ondokuz Mayis University Hospital, Samsun, 3 1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. 2Department of Pathology, Ondokuz Mayis University Hospital, Samsun, Turkey. 3Departments of Pathology/Microbiology, Pediatrics and Orthopaedic Surgery, Nebraska Medical Center, Omaha, NE, USA. 4Department of Urology, Hacettepe University Hospital, Ankara, Turkey. Turkey. 3Departments of Pathology/Microbiology, Pediatrics and Orthopaedic Surgery, Nebraska Medical Center, Omaha, NE, USA. 4Department of Urology, Hacettepe University Hospital, Ankara, Turkey. 19. Alevizopoulos A, Mygdalis V, Tyritzis S, Stravodimos K, Constantinides CA. Low-grade fibromyxoid sarcoma of the renal pelvis: first report. Case Rep Nephrol Urol. 2012;2(2):87–91. doi:10.1159/000341191. 20. Arancio M, Ranzoni S, Delsignore A, Maffei N, Landi G, Mina A, et al. Fibromyxoid sarcoma of the kidney. A case report. Urologia. 2010;77(4):263–6. Received: 1 August 2015 Accepted: 1 October 2015 20. Arancio M, Ranzoni S, Delsignore A, Maffei N, Landi G, Mina A, et al. Fibromyxoid sarcoma of the kidney. A case report. Urologia. 2010;77(4):263–6. 21. del Valle GN, Santos Largo J, Martinez-Sagarra Oceja J, Rodriguez TV. Fibromyxoid sarcoma of kidney. Actas Urol Esp. 2009;33(9):1036–9. Received: 1 August 2015 Accepted: 1 October 2015 21. del Valle GN, Santos Largo J, Martinez-Sagarra Oceja J, Rodriguez TV. Fibromyxoid sarcoma of kidney. Actas Urol Esp. 2009;33(9):1036–9. 21. del Valle GN, Santos Largo J, Martinez-Sagarra Oceja J, Rodriguez TV. Fibromyxoid sarcoma of kidney. Actas Urol Esp. 2009;33(9):1036–9. Abbreviations l SEF: Sclerosing epithelioid fibrosarcoma; FISH: Fluorescence in situ hybridization; HSCTGR: Hyalinizing spindle cell tumor with giant rosettes; CT: Computerized tomography; CCSK: Clear cell sarcoma of the kidney; LGFMS: Low-grade fibromyxoid sarcoma; MEST: Mixed epithelial and stromal tumor of kidney. 12. Tomimaru Y, Nagano H, Marubashi S, Kobayashi S, Eguchi H, Takeda Y, et al. Sclerosing epithelioid fibrosarcoma of the liver infiltrating the inferior vena cava. World J Gastroenterol. 2009;15(33):4204–8. 12. Tomimaru Y, Nagano H, Marubashi S, Kobayashi S, Eguchi H, Takeda Y, et al. Sclerosing epithelioid fibrosarcoma of the liver infiltrating the inferior vena cava. World J Gastroenterol. 2009;15(33):4204–8. 13. Wojcik JB, Bellizzi AM, Dal Cin P, Bredella MA, Fletcher CD, Hornicek FJ, et al. Primary sclerosing epithelioid fibrosarcoma of bone: analysis of a series. Am J Surg Pathol. 2014;38(11):1538–44. doi:10.1097/PAS.0000000000000265. 13. Wojcik JB, Bellizzi AM, Dal Cin P, Bredella MA, Fletcher CD, Hornicek FJ, et al. Primary sclerosing epithelioid fibrosarcoma of bone: analysis of a series. Am J Surg Pathol. 2014;38(11):1538–44. doi:10.1097/PAS.0000000000000265. References 22. Rubinstein JC, Visa A, Zhang L, Antonescu CR, Christison-Lagay ER, Morotti R. Primary Low-Grade Fibromyxoid Sarcoma of the Kidney in a Child with the Alternative EWSR1-CREB3L1 Gene Fusion. Pediatr Dev Pathol. 2014;17(4):321–6. doi:10.2350/14-05-1487-CR.1. 1. Arbajian E, Puls F, Magnusson L, Thway K, Fisher C, Sumathi VP, et al. Recurrent EWSR1-CREB3L1 gene fusions in sclerosing epithelioid fibrosarcoma. Am J Surg Pathol. 2014;38(6):801–8. doi:10.1097/PAS.0000000000000158. 1. Arbajian E, Puls F, Magnusson L, Thway K, Fisher C, Sumathi VP, et al. Recurrent EWSR1-CREB3L1 gene fusions in sclerosing epithelioid fibrosarcoma. Am J Surg Pathol. 2014;38(6):801–8. doi:10.1097/PAS.0000000000000158. 2. Doyle LA, Wang WL, Dal Cin P, Lopez-Terrada D, Mertens F, Lazar AJ, et al. MUC4 is a sensitive and extremely useful marker for sclerosing epithelioid fibrosarcoma: association with FUS gene rearrangement. Am J Surg Pathol. 2012;36(10):1444–51. doi:10.1097/PAS.0b013e3182562bf8. 23. Silverman JF, Nathan G, Olson PR, Prichard J, Cohen JK. Fine-needle aspiration cytology of low-grade fibromyxoid sarcoma of the renal capsule (capsuloma). Diagn Cytopathol. 2000;23(4):279–83. 24. Suzuki T, Hiragata S, Hosaka K, Oyama T, Kuroda N, Hes O, et al. Malignant mixed epithelial and stromal tumor of the kidney: report of the first male case. Int J Urol. 2013;20(4):448–50. doi:10.1111/j.1442-2042.2012.03155.x. 3. Prieto-Granada C, Zhang L, Chen HW, Sung YS, Agaram NP, Jungbluth AA, et al. A genetic dichotomy between pure sclerosing epithelioid fibrosarcoma (SEF) and hybrid SEF/low-grade fibromyxoid sarcoma: A pathologic and molecular study of 18 cases. Genes Chromosomes Cancer. 2014. doi:10.1002/gcc.22215. 4. Antonescu CR, Rosenblum MK, Pereira P, Nascimento AG, Woodruff JM. Sclerosing epithelioid fibrosarcoma: a study of 16 cases and confirmation of a clinicopathologically distinct tumor. Am J Surg Pathol. 2001;25(6):699–709. 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 Competing interest g 14. Watanabe K, Suzuki T. Epithelioid fibrosarcoma of the ovary. Virchows Arch. 2004;445(4):410–3. doi:10.1007/s00428-004-1068-3. Submit your next manuscript to BioMed Central and take full advantage of: 5. Chow LT, Lui YH, Kumta SM, Allen PW. Primary sclerosing epithelioid fibrosarcoma of the sacrum: a case report and review of the literature. J Clin Pathol. 2004;57(1):90–4. 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 • Convenient online submission 6. Meis-Kindblom JM, Kindblom LG, Enzinger FM. Sclerosing epithelioid fibrosarcoma. A variant of fibrosarcoma simulating carcinoma. Am J Surg Pathol. 1995;19(9):979–93. • Thorough peer review 7. Ossendorf C, Studer GM, Bode B, Fuchs B. Sclerosing epithelioid fibrosarcoma: case presentation and a systematic review. Clin Orthop Relat Res. 2008;466(6):1485–91. doi:10.1007/s11999-008-0205-8. 8. Argani P, Lewin JR, Edmonds P, Netto GJ, Prieto-Granada C, Zhang L, et al. Primary Renal Sclerosing Epithelioid Fibrosarcoma: Report of 2 Cases With EWSR1-CREB3L1 Gene Fusion. Am J Surg Pathol. 2014. doi:10.1097/PAS.0000000000000338.
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Phenalenyl-based ligand for transition metal chemistry: Application in Henry reaction
Journal of chemical sciences/Proceedings of the Indian Academy of Sciences. Chemical sciences
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J. Chem. Sci. Vol. 123, No. 2, March 2011, pp. 139–144. c⃝Indian Academy of Sciences. J. Chem. Sci. Vol. 123, No. 2, March 2011, pp. 139–144. c⃝Indian Academy of Sciences. ARUP MUKHERJEEa, TAMAL K SENa, SWADHIN K MANDALa,∗, DANIEL KRATZERTb, DIETMAR STALKEb, ALEXANDER DÖRINGb and CAROLA SCHULZKEc aDepartment of Chemical Sciences, Indian Institute of Science Education and Research (I Mohanpur 741252, India bGeorg-August-Universität Göttingen, Tammannstr. 4, 37077 Göttingen, Germany cSchool of Chemistry, Trinity College Dublin, Dublin 2, Ireland e-mail: swadhin.mandal@iiserkol.ac.in aDepartment of Chemical Sciences, Indian Institute of Science Education and Research Mohanpur 741252, India bGeorg-August-Universität Göttingen, Tammannstr. 4, 37077 Göttingen, Germany cSchool of Chemistry, Trinity College Dublin, Dublin 2, Ireland e-mail: swadhin.mandal@iiserkol.ac.in Abstract. We report the synthesis and characterization of the first transition metal complex of a phenalenyl- based ligand. The reaction of Cu(OAc)2.H2O with 9-N-methylamino-1-N′-methylimino-phenalene (LH) in 1:1 stoichiometric ratio results in the formation of a mononuclear copper complex [LCu(OAc)] (1). The molecular structure of 1 was established by X-ray crystallography. The electrochemistry of 1 indicates the formation of an anionic radical by one electron reduction into the non-bonding molecular orbital of the phenalenyl system. The complex 1 efficiently catalyses the C–C bond forming Henry reaction. Keywords. Cu-complex; phenalenyl; radical; X-ray crystallography; Henry reaction. ∗For correspondence X = X' = O, R = lone pair of electrons, R' = H or Me; X= O, X' = N, R = alkyl or aryl, R' = H; X = X' = N, R = alkyl, R' = H or Me 2.1 Materials and methods It was filtered and kept at 25◦C. Red-brown crystals appeared in one day (332 mg, yield 95%). M.pt. 175◦C (decomposes to black mass). IR (KBr, 600– 4000 cm−1): 3032(w), 2925(w), 2896(w), 2792(w), 2353(w), 1637(s), 1587(s), 1512(w), 1477(w), 1430(w), 1395(m), 1346(w), 1279(s), 1254(w), 1192(m), 1164(s), 1018(m), 927(m), 908(w), 830(s), 812(s), 776(m), 746(w), 720(w), 690(w). Elemental analysis: Anal. Calcd. for CuC17H16N2O2: C, 59.47%; H, 4.66%; N, 8.16%; Found: C, 58.44%; H, 4.42%; N, 7.83%. 9-N-methylamino-1-N′-methylimino-phenalene (222 mg, 1.0 mmol) and Cu(OAc)2.H2O (199 mg, 1.0 mmol) were mixed in dichloromethane (DCM)/ethanol mixture (30 mL, 1:1) and stirred at 25◦C overnight. The starting materials slowly dissolved and resulted in a brown-red colour. Finally, the reaction mixture was refluxed for 30 min. It was filtered and kept at 25◦C. Red-brown crystals appeared in one day (332 mg, yield 95%). M.pt. 175◦C (decomposes to black mass). IR (KBr, 600– 4000 cm−1): 3032(w), 2925(w), 2896(w), 2792(w), 2353(w), 1637(s), 1587(s), 1512(w), 1477(w), 1430(w), 1395(m), 1346(w), 1279(s), 1254(w), 1192(m), 1164(s), 1018(m), 927(m), 908(w), 830(s), 812(s), 776(m), 746(w), 720(w), 690(w). Elemental analysis: Anal. Calcd. for CuC17H16N2O2: C, 59.47%; H, 4.66%; N, 8.16%; Found: C, 58.44%; H, 4.42%; N, 7.83%. 1. Introduction systems has not yet been explored for designing tran- sition metal catalysts. Herein we report the synthesis and characterization of a copper complex bearing a symmetrical phenalenyl-based N,N-ligand (LH). The reaction of Cu(OAc)2.H2O with 9-N-methylamino-1- N′-methylimino-phenalene (LH) in 1:1 stoichiometric ratio results in the formation of a mononuclear copper complex [LCu(OAc)] (1). The molecular structure of 1 was established by X-ray crystallography. The elec- trochemistry of 1 reveals the presence of a reversible The chemistry of phenalenyl-based ligand systems has not yet been explored except on the development of neutral boron containing free radical conductors.1,2 Phenalenyl is well-known odd alternant hydrocarbon with high symmetry (D3h) which has the ability to form three redox species: cation, radical, and anion.3 The formation of this redox triad involves the use of the nonbonding molecular orbital (MO) of the phenalenyl moiety and hence does not affect the stability of the resulting species greatly. The neutral radical state is sta- bilized by the extended electronic delocalization and this way a number of phenalenyl-based organic conduc- tors (chart 1) have been reported exhibiting the high- est room temperature conductivity among any neutral radical solids.1,2 Such characteristic features have been widely utilized for exploring new conjugated electronic systems, such as multifunctional electronic and mag- netic materials.2 The chemistry so far developed in this system is limited to the spiro-conjugated boron system.1,2 The chemistry of phenalenyl-based ligand X' X R' B X X' X = X' = O, R = lone pair of electrons, R' = H or Me; X= O, X' = N, R = alkyl or aryl, R' = H; X = X' = N, R = alkyl, R' = H or Me R' R R R R + Chart 1. Phenalenyl-based neutral radical conductors. X' X R' B X X' R' R R R R + R' Chart 1. Phenalenyl-based neutral radical conductors. Chart 1. Phenalenyl-based neutral radical conductors. 139 Arup Mukherjee et al. 140 one electron reduction process. The complex 1 exhibits high catalytic activity in C–C bond forming Henry reaction.4–15 gen atoms were refined isotropically on calculated posi- tions using a riding model with their Uiso values con- strained to 15 times the Ueq of their pivot atoms for terminal sp3 carbon atoms and 1.2 times for all other carbon atoms. 2.1 Materials and methods All manipulations were carried out under dry nitro- gen atmosphere using standard Schlenk line tech- nique. Solvents were dried using conventional proce- dure. 9-N-methylamino-1-N ′-methylimino-phenalene was prepared according to the literature procedure.1a All manipulations were carried out under dry nitro- gen atmosphere using standard Schlenk line tech- nique. Solvents were dried using conventional proce- dure. 9-N-methylamino-1-N ′-methylimino-phenalene was prepared according to the literature procedure.1a Copper acetate monohydrate was purchased from Merck, India and was used as received. Aldehydes were purchased from Aldrich and were used with- out further purifications. Triethylamine (Et3N) was dried over KOH and nitromethane (CH3NO2) was dried over CaCl2. Elemental analyses were performed at the Institut für Anorganische Chemie, Universität Göttingen. The melting point was measured on a Buchi B-545 melting point apparatus. Infrared spectra were recorded on a Perkin Elmer FT-IR spectrometer. 1H and 13C NMR spectra were recorded on a Bruker 500 MHz spectrometer. Electrochemical measure- ments were performed with an AUTOLAB PGSTAT12 potentiostat/galvanostat using a platinum disc electrode with a reaction surface of 1 mm2 as working elec- trode. A platinum rod electrode (together with internal referencing versus ferrocene/ferrocenium) was used as reference electrode and a platinum knob electrode as auxiliary electrode. The scan rate of the cyclic voltam- metric experiment was 100 mV/s. The sample was dissolved in acetonitrile (0.1 mmol/L) together with the electrolyte tetrabutylammonium hexafluorophos- phate (0.1 mol/L). The electrochemical experiments were conducted inside a glovebox under argon atmo- sphere at 25◦C. The crystallographic data for 1 was col- lected from shock-cooled crystals at 100 K mounted on a glass fibre. The crystals were cooled and protected from air with a low temperature mounting device16 in inert oil. The data of 1 was collected on a INCOATEC Mo Microsource with Quazar mirror optics equipped with a low temperature device using monochromatic MoKα radiation (λ = 0.71073 Å). The data was inte- grated with SAINT,17 and an empirical absorption cor- rection with SADABS18 was applied. The structure was solved by direct methods (SHELXS-97)19 and refined by full-matrix least-squares methods against F2 (SHELXL-97).18 All non-hydrogen atoms were refined with anisotropic displacement parameters. The hydro- 9-N-methylamino-1-N′-methylimino-phenalene (222 mg, 1.0 mmol) and Cu(OAc)2.H2O (199 mg, 1.0 mmol) were mixed in dichloromethane (DCM)/ethanol mixture (30 mL, 1:1) and stirred at 25◦C overnight. The starting materials slowly dissolved and resulted in a brown-red colour. Finally, the reaction mixture was refluxed for 30 min. 2.3 General procedure for the catalytic Henry reaction Complex 1 (0.066 mmol) was dissolved in ethanol (2 mL) and aldehyde (0.66 mmol) was added followed by the addition of nitromethane (3.3 mmol) and triethy- lamine (0.066 mmol). The mixture was allowed to stir at 25◦C for 15 h. The volatiles were removed in vacuo, and the residue was purified by flash chromatography to afford the nitroaldol adduct. 2.3a 1-(2-nitrophenyl)-2-nitroethanol: 1H NMR (500 MHz, CDCl3, ppm)20: δ 3.33 (br, 1H, OH), 4.55 (dd, 1H, J = 9 Hz and 13.8 Hz, CHHNO2), 4.87 (dd, 1H, J = 2.5 Hz and 13.8 Hz, CHHNO2), 6.05 (d, 1H, J = 9 Hz, CHOH), 7.48 (t, 1H, J = 7.5 Hz, ArH), 7.68 (t, 1H, J = 7.5 Hz, ArH), 7.95 (d, 1H, J = 7.8 Hz, ArH), 8.08 (d, 1H, J = 7.8 Hz, ArH). 13C NMR (125 MHz, CDCl3, ppm): δ 66.6, 80.0, 125.2, 128.6, 129.6, 134.2, 134.6, 147.0. 2.3a 1-(2-nitrophenyl)-2-nitroethanol: 1H NMR (500 MHz, CDCl3, ppm)20: δ 3.33 (br, 1H, OH), 4.55 (dd, 1H, J = 9 Hz and 13.8 Hz, CHHNO2), 4.87 (dd, 1H, J = 2.5 Hz and 13.8 Hz, CHHNO2), 6.05 (d, 1H, J = 9 Hz, CHOH), 7.48 (t, 1H, J = 7.5 Hz, ArH), 7.68 (t, 1H, J = 7.5 Hz, ArH), 7.95 (d, 1H, J = 7.8 Hz, ArH), 8.08 (d, 1H, J = 7.8 Hz, ArH). 13C NMR (125 MHz, CDCl3, ppm): δ 66.6, 80.0, 125.2, 128.6, 129.6, 134.2, 134.6, 147.0. 2.3b 1-(4-nitrophenyl)-2-nitroethanol: 1H NMR (500 MHz, CDCl3, ppm)20: δ 3.3 (br, 1H, OH), 4.61 (m, 2H, CH2NO2), 5.6 (m, 1H, CHOH), 7.63 (d, 2H, J = 8.6 Hz, ArH), 8.27 (d, 2H, J = 8.6 Hz, ArH). Phenalenyl-based ligand for transition metal chemistry: application in Henry reaction 141 Figure 1. Perspective ORTEP view of the molecular struc- ture of 1. Thermal ellipsoids are drawn with 50% probability. Hydrogen atoms have been omitted for the sake of clarity. 13C (125 MHz, CDCl3, ppm): δ 69.9, 80.6, 124.1, 126.9, 145.0, 148.1. 3.1 Synthesis of complex 1 The title compound was synthesized by the treatment of the ligand 9-N-methylamino-1-N ′-methylimino- phenalene (LH) with Cu(OAc)2.H2O in ethanol and dichloromethane mixture (1:1) (scheme 1). The reac- tion mixture was refluxed for 30 min at the end to ensure complete conversion of the starting materials into the title compound. The compound 1 was characterized by IR spectroscopy, elemental analysis and single crystal X-ray crystallography. The reddish-brown single crys- tals of 1 were grown from the reaction mixture at 25◦C under slow evaporation of the solvents. 3.2 X-ray crystal structures of 1 The structure of 1 was established by X-ray diffrac- tion study. The molecular structure of 1 is shown in figure 1, and the selected bond lengths and angles are presented in table 1. The X-ray structure reveals a distorted square-planar geometry around the copper (II) centre with two nitrogen atoms of the phenalenyl ligand and two oxygen atoms of the acetate group coordinated to the metal centre. The two Cu–N bond lengths are not equal revealing an inherent asym- metry in the structure. However, the two bond angles N(1)–Cu(1)–O(1) and N(2)–Cu(1)–O(2) are equal (162.7◦). The Cu–N bond lengths [Cu(1)–N(2) = 1.907 Å and Cu(1)–N(1) = 1.912 Å] in complex 1 fall in the range reported for other Cu(II) square- planar complexes. For example, the Cu–N bond lengths Figure 1. Perspective ORTEP view of the molecular struc- ture of 1. Thermal ellipsoids are drawn with 50% probability. Hydrogen atoms have been omitted for the sake of clarity. (∼1.92 Å) in propylene-bis(biuret)-copper complex, Na2[Cu(pr(bi)2)]·3Me2SO21 and in LCuCl [where L = 2,4-bis(2,6-diisopropylphenylimido)pentane]22 are similar to the Cu–N bond length observed in 1. HN N +Cu(OAc)2 DCM/Ethanol 1 .H2O LH O Cu O N N + Scheme 1. Synthesis of complex 1. (∼1.92 Å) in propylene-bis(biuret)-copper complex, Na2[Cu(pr(bi)2)]·3Me2SO21 and in LCuCl [where L = 2,4-bis(2,6-diisopropylphenylimido)pentane]22 are similar to the Cu–N bond length observed in 1. The electrochemistry of 1 is presented in figure 2 where it may be seen that complex 1 shows a well- behaved reversible single reduction corresponding to the generation of an anionic radical (scheme 2). Similar reduction process is well-known in related phenalenyl-based spiro-conjugated boron systems.1,2 This reduction wave appears at a more negative value (−1.75 V) than the spiro-conjugated boron compounds (∼−1.0 V) bearing the N,N-phenalenyl ligands.1a This HN N +Cu(OAc)2 DCM/Ethanol 1 .H2O LH O Cu O N N + Scheme 1. Synthesis of complex 1. ( ) p py ( ) pp p Na2[Cu(pr(bi)2)]·3Me2SO21 and in LCuCl [where L = 2,4-bis(2,6-diisopropylphenylimido)pentane]22 are similar to the Cu–N bond length observed in 1. The electrochemistry of 1 is presented in figure 2 where it may be seen that complex 1 shows a well- behaved reversible single reduction corresponding to the generation of an anionic radical (scheme 2). 3.2 X-ray crystal structures of 1 Similar reduction process is well-known in related phenalenyl-based spiro-conjugated boron systems.1,2 This reduction wave appears at a more negative value (−1.75 V) than the spiro-conjugated boron compounds (∼−1.0 V) bearing the N,N-phenalenyl ligands.1a This HN N +Cu(OAc)2 DCM/Ethanol 1 .H2O LH O Cu O N N + Scheme 1. Synthesis of complex 1. The electrochemistry of 1 is presented in figure 2 where it may be seen that complex 1 shows a well- behaved reversible single reduction corresponding to the generation of an anionic radical (scheme 2). Similar reduction process is well-known in related phenalenyl-based spiro-conjugated boron systems.1,2 This reduction wave appears at a more negative value (−1.75 V) than the spiro-conjugated boron compounds (∼−1.0 V) bearing the N,N-phenalenyl ligands.1a This LH LH Scheme 1. Synthesis of complex 1. 142 Arup Mukherjee et al. Table 1. Selected bond lengths [Å] and angles [◦] for complex 1. Bond Distance Bond Angles Cu(1)–N(2) 1.907(6) N(2)–Cu(1)–N(1) 95.2(3) Cu(1)–N(1) 1.912(6) N(2)–Cu(1)–O(2) 162.7(2) Cu(1)–O(2) 2.051(5) N(1)–Cu(1)–O(2) 101.1(2) Cu(1)–O(1) 2.079(5) N(2)–Cu(1)–O(1) 100.8(2) N(1)–C(11) 1.31(10) N(1)–Cu(1)–O(1) 162.7(2) N(1)–C(15) 1.475(10) O(2)–Cu(1)–O(1) 63.9(2) N(2)–C(1) 1.333(10) C(11)–N(1)–C(15) 119.3(6) N(2)–C(14) 1.481(10) C(11)–N(1)–Cu(1) 125.4(5) O(1)–C(16) 1.271(10) C(15)–N(1)–Cu(1) 115.1(5) O(2)–C(16) 1.272(10) C(1)–N(2)–Cu(1) 127.1(5) C(1)–C(2) 1.449(11) C(14)–N(2)–Cu(1) 114.6(5) C(1)–C(12) 1.461(11 C(16)–O(1)–Cu(1) 88.1(5) Table 1. Selected bond lengths [Å] and angles [◦] for complex 1. indicates that the reduction is comparatively more dif- ficult in this system to that of boron-based systems.1a This may be attributed to the lower oxidation state of the copper (+2) in neutral complex 1 as compared to that of cationic spiro-conjugated boron (in +3 oxidation state) compounds. important intermediate such as 1,2-amino alcohol or α- hydroxy carboxylic acids. As a result, there have been considerable efforts towards the development of syn- thetic methods using Henry reaction.4–8 Recent stud- ies have revealed that copper can actively catalyse the Henry reaction.9–15 The new copper complex 1 was tested for its effi- ciency as catalyst in Henry reaction. The catalytic reac- tion was carried out at 25◦C for 15 h resulting in a moderate yield (∼60%) of the corresponding nitroaldol adduct. Addition of Et3N (10 mol%) greatly enhances the conversion rate (> 90%) of the catalytic reaction (table 2). We carried out the catalytic Henry reaction in a range of solvents such as methanol, ethanol, iso- propanol, THF, toluene, dichloromethane, and chloro- form to find out the best solvent. 3.2 X-ray crystal structures of 1 Ethanol was found to be the best solvent leading to more than 90% isolated yield (table 3). 3.3 Application to Henry reaction The Henry reaction or the nitroaldol reaction is an important organic transformation leading to the new C– C bonds. It allows access to a range of synthetically -2.0 -1.9 -1.8 -1.7 -1.6 -1.5 -2.0 -1.5 -1.0 -0.5 0.0 Current(µA) Potential (Volts vs.SCE ) E1/2 = -1.75 V Figure 2. Cyclic voltammetry of complex 1 generating an anionic radical by one electron reduction process. -2.0 -1.9 -1.8 -1.7 -1.6 -1.5 -2.0 -1.5 -1.0 -0.5 0.0 Current(µA) Potential (Volts vs.SCE ) E1/2 = -1.75 V Figure 2. Cyclic voltammetry of complex 1 generating an anionic radical by one electron reduction process. form to find out the best solvent. Ethanol was found to be the best solvent leading to more than 90% isolated yield (table 3). After the most productive reaction condition was established, we carried out the Henry reaction on another nitro aldehyde (2-nitro benzaldehyde) using the +e -e O Cu O N N O Cu O N N + Scheme 2. Generation of an anionic radical by one elec- tron reduction of 1. -2.0 -1.9 -1.8 -1.7 -1.6 -1.5 -2.0 -1.5 -1.0 -0.5 0.0 Current(µA) Potential (Volts vs.SCE ) E1/2 = -1.75 V After the most productive reaction condition was established, we carried out the Henry reaction on another nitro aldehyde (2-nitro benzaldehyde) using the Current(µA) +e -e O Cu O N N O Cu O N N + Scheme 2. Generation of an anionic radical by one elec- tron reduction of 1. Potential (Volts vs.SCE ) Scheme 2. Generation of an anionic radical by one elec- tron reduction of 1. Figure 2. Cyclic voltammetry of complex 1 generating an anionic radical by one electron reduction process. 143 Phenalenyl-based ligand for transition metal chemistry: application in Henry reaction Table 2. Standardization of catalytic Henry reactiona using complex 1 as catalyst. Table 2. Standardization of catalytic Henry reactiona using complex 1 as catalyst. H O O2N CH3NO2 Catalyst (10 mol%) OH O2N NO2 Ethanol, 25 ˚C Entry Catalyst Time (h) Yield (%)b 1 Complex 1 22 65 2 Complex 1 + Et3N (10 mol%) 15 92 aTh i i d i h 0 66 l f C H NO CHO d 3 3 l f aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of CH3NO2 in 2 mL EtOH at 25◦C. 3 2 bIsolated yield after flash chromatographic purification. Table 3. 4. Conclusion 2. 2. (a) Mandal S K, Samanta S, Itkis M E, Jensen D W, Reed R W, Oakley R T, Tham F S, Donnadieu B and Haddon R C 2006 J. Am. Chem. Soc. 128 1982; (b) Pal S K, Itkis M E, Tham F S, Reed R W, Oakley R T and Haddon R C 2005 Science 309 281; (c) Itkis M E, Chi X, Cordes A W and Haddon R C 2002 Science 296 1443 In summary, we have synthesized and characterized the first transition metal complex of a phenalenyl-based lig- and to be utilized as catalyst for carrying out Henry reaction. This result establishes that the phenalenyl- based ligands can be used for the development of transition metal chemistry to design catalyst for homo- geneous organic transformation. The complex was syn- thesized by direct mixing of the reagents in high yield. The electrochemistry of this complex clearly demonstrates that the empty non-bonding orbital of the phenalenyl ligand can accept an electron to generate the anionic radical. This result supports that this system has potential to prepare the mixed organic (free electron in phenalenyl part) inorganic [free electron in copper(II)] hybrid magnetic system. Also it could find application for preparation mixed organic–inorganic tunnel junc- tion by external spin injection. Complex 1 exhibits high catalytic efficiency in the C–C bond forming Henry reactions. This study establishes that the phenalenyl- based ligand system can not only be used as building block for the preparation of molecular conductors but also they can be used as useful ligands for making effi- cient catalysts. Currently, we have been working on different metals to synthesize active catalysts bearing phenalenyl-based ligands for useful organic catalytic transformations. 3. Haddon R C 1975 Nature 256 394 4. Palomo C, Oiarbide M and Laso A 2007 Eur. J. Org. Chem. 2561 5. Bandini M, Piccinelli F, Tommasi S, Umani-Ronchi A and Ventrici C 2007 Chem. Commun. 616 6. Trost B M, Yeh V S C, Ito H and Bremeyer N 2002 Org. Lett. 4 2621 7. Kudoh T, Ishikawa T, Shimizu Y and Saito S 2003 Org. Lett. 5 3875 8. Paintner F F, Allmendinger L, Bauschke G and Klemann P 2005 Org. Lett. 7 1423 9. Christensen C, Juhl K and Jørgensen K A 2001 Chem. Commun. 2222 10. Christensen C, Juhl K, Hazell R G and Jørgensen K A 2002 J. Org. Chem. 67 4875 11. References complex 1 as a catalyst. The results of the catalytic reac- tions are presented in table 4 exhibiting high isolated yield. 1. (a) Mandal S K, Itkis M E, Chi X, Samanta S, Lidsky D, Reed R W, Oakley R T, Tham F S and Haddon R C 2005 J. Am. Chem. Soc. 127 8185; (b) Chi X, Itkis M E, Patrick B O, Barclay T M, Reed R W, Oakley R T, Cordes A W and Haddon R C 1999 J. Am. Chem. Soc. 121 10395 4. Conclusion Evans D A, Seidel D, Rueping M, Lam H W, Shaw J T and Downey C W 2003 J. Am. Chem. Soc. 125 12692 y 12. Ginotra S K and Singh V K 2001 Org. Biomol. Chem. 5 3932 13. Arai T, Watanabe M and Yanagisawa A 2007 Org. Lett. 9 3595 14. Bandini M, Benaglia M, Sinisi R, Tommasi S and Umani-Ronchi A 2007 Org. Lett. 9 2151 15. Mei L, Ming T H, Rong L Q, Jie S, Zhong Y S and Liang L X 2009 J. Chem. Sci. 121 435 16. Kottke T and Stalke D 1993 J. Appl. Crystallogr. 26 615 17. Bruker SAINT v7.34A. 2005 Bruker AXS Inc. Madison 280 (WI, USA) 3.3 Application to Henry reaction Effect of solvent on Henry reaction using 1 as catalyst.a H O O2N CH3NO2 Catalyst (10 mol%) OH O2N NO2 Solvent, 25 ˚C Entry Catalyst Solvent Time (h) Yield (%)b 1 1+ Et3N Methanol 15 88 2 1+ Et3N Ethanol 15 92 3 1+ Et3N Isopropanol 15 86 4 1+ Et3N THF 15 75 5 1+ Et3N Toluene 15 65 6 1+ Et3N DCM 15 72 7 1+ Et3N CHC13 15 68 aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of CH3NO2 in 2 mL solvent for 15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 3. Effect of solvent on Henry reaction using 1 as catalyst.a aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of CH3NO2 in 2 mL solvent for 15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 4. Henry reaction using 1 as catalyst on different alde- hyde substrates.a CH3NO2 Catalyst (10 mol%) Ethanol, 25 ˚C OH ArCHO Ar NO2 Entry Catalyst Aldehyde Yield (%)b 1 Complex 1 4-NO2C6H4CHO 92 2 Complex 1 2-NO2C6H4CHO 90 aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of CH3NO2 in 2 mL EtOH for 15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 4. Henry reaction using 1 as catalyst on different alde- hyde substrates.a CH3NO2 Catalyst (10 mol%) Ethanol, 25 ˚C OH ArCHO Ar NO2 Entry Catalyst Aldehyde Yield (%)b 1 Complex 1 4-NO2C6H4CHO 92 2 Complex 1 2-NO2C6H4CHO 90 aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of CH3NO2 in 2 mL EtOH for 15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 4. Henry reaction using 1 as catalyst on different alde- hyde substrates.a Table 4. Henry reaction using 1 as catalyst on different alde- hyde substrates.a 144 Arup Mukherjee et al. Acknowledgements 18. Sheldrick G M 2008 SADABS 2008/1 Göttingen Financial support from the Department of Science and Technology, New Delhi, India through SERC Fast- Track Grant SR/FT/CS-020/2008 and IISER, Kolkata is acknowledged. AM and TKS are thankful to IISER, Kolkata and Council of Scientific and Industrial Research (CSIR), New Delhi for research fellowships. 19. Sheldrick G M 2008 Acta Crystallogr. Sect. A. 64 112 20. Lai G, Wang S and Wang Z 2008 Tetrahedron: Asymme- try 19 1813 21. Barbier J P, Biyyadh A E, Kappenstein C, Mabiala N and Hugel R P 1985 Inorg. Chem. 24 3615 22. Holland P L and Tolman W B 1999 J. Am. Chem. Soc. 121 7270
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Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter
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PLOS ONE PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Duy PN, Lan DT, Pham Thu H, Thi Thu HP, Nguyen Thanh H, Pham NP, et al. (2021) Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter. PLoS ONE 16(9): e0255470. https://doi.org/10.1371/journal. pone.0255470 Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter Phuong Nguyen Duy1☯, Dai Tran Lan1,2☯, Hang Pham Thu1, Huong Phung Thi Thu1, Ha Nguyen Thanh1, Ngoc Phuong Pham1¤, Florence Auguy3, Huong Bui Thi Thu4, Tran Bao Manh5, Sebastien Cunnac3, Xuan Hoi PhamID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Molecular Pathology, Institute of Agricultural Genetics, Vietnam Academy of Agricultural Sciences, Hanoi, Vietnam, 2 Faculty of Natural Sciences, Department of Applied Biology and Agriculture, Quynhon University, Quynhon, Vietnam, 3 PHIM Plant Health Institute, Univ Montpellier, IRD, CIRAD, INRAE, Institut Agro, Montpellier, France, 4 Vietnam National University of Agriculture, Hanoi, Vietnam, 5 ThaiBinh Seed Corporation, Thaibinh, Vietnam ☯These authors contributed equally to this work. ¤ Current address: Faculty of Biology, Hanoi University of Sciences, Hanoi, Vietnam * xuanhoi.pham@gmail.com ☯These authors contributed equally to this work. q y ¤ Current address: Faculty of Biology, Hanoi University of Sciences, Hanoi, Vietnam * xuanhoi.pham@gmail.com Abstract TBR225 is one of the most popular commercial rice varieties in Northern Vietnam. However, this variety is highly susceptible to bacterial leaf blight (BLB), a disease caused by Xantho- monas oryzae pv. oryzae (Xoo) which can lead to important yield losses. OsSWEET14 belongs to the SWEET gene family that encodes sugar transporters. Together with other Clade III members, it behaves as a susceptibility (S) gene whose induction by Asian Xoo Transcription-Activator-Like Effectors (TALEs) is absolutely necessary for disease. In this study, we sought to introduce BLB resistance in the TBR225 elite variety. First, two Vietnam- ese Xoo strains were shown to up-regulate OsSWEET14 upon TBR225 infection. To inves- tigate if this induction is connected with disease susceptibility, nine TBR225 mutant lines with mutations in the AvrXa7, PthXo3 or TalF TALEs DNA target sequences of the OsS- WEET14 promoter were obtained using the CRISPR/Cas9 editing system. Genotyping analysis of T0 and T1 individuals showed that mutations were stably inherited. None of the examined agronomic traits of three transgene-free T2 edited lines were significantly different from those of wild-type TBR225. Importantly, one of these T2 lines, harboring the largest homozygous 6-bp deletion, displayed decreased OsSWEET14 expression as well as a sig- nificantly reduced susceptibility to a Vietnamese Xoo strains and complete resistance to another one. Our findings indicate that CRISPR/Cas9 editing conferred an improved BLB resistance to a Vietnamese commercial elite rice variety. Introduction Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major bacterial disease that causes 10%-20% annual reduction in rice production worldwide [1]. The use of improved rice varieties resistant to Xoo is probably the most efficient, economical and environ- mentally-friendly way to control BLB. The virulence of Xoo depends on the transcriptional activation of specific host disease-sus- ceptibility (S) genes by a subgroup of bacterial type III effectors, called transcription activator- like effectors (TALEs) [2]. Upon translocation into the plant cell, TALEs bind to specific host nuclear gene promoter sequences termed Effector-Binding Elements (EBEs) and induce target gene expression to the benefit of the pathogen. The central repetitive domain of TALEs is responsible for DNA target sequence binding. DNA binding involves recognition principles that have been largely deciphered and applied to the computational prediction of TALEs target DNA sequences [3,4]. This and earlier work has fostered the identification of TALEs transcrip- tional targets in the rice genome and ultimately, of rice BLB S genes [2]. All Xoo strains recurrently target S genes belonging to the SWEET gene family and coding for transmembrane sugar exporter proteins [3]. The over accumulation of SWEETs due to TALE induction is presumed to provide an additional ration of apoplastic carbohydrates for full bacterial pathogen multiplication and disease expression [5]. Although all five rice clade III SWEET genes can function as S genes for bacterial blight, only three, namely OsSWEET11, OsSWEET13 and OsSWEET14, are known to be targeted by several unrelated TALEs in nature [6–11]. OsSWEET11 is activated by PthXo1 [6], OsSWEET13 is targeted by different variants of PthXo2 [11,12], while OsSWEET14 is a target of multiple TAL effectors, including AvrXa7, PthXo3, TalC and TalF [7–9]. Previous studies established that rice resistance to Xoo resulting from "TALE-unresponsive" alleles can be conferred by natural DNA polymorphisms or targeted editing of EBEs located in OsSWEET genes promoters of rice germplasm accessions or engineered rice varieties, respec- tively [6,13–16]. For example, early resistance engineering work has used TALENs to individu- ally alter the AvrXa7, TalC or TalF EBEs in the OsSWEET14 promoter and successfully obtained resistance to some Asian Xoo strains [13,15]. Editor: R. M. Sundaram, ICAR-Indian Institute of Rice Research, INDIA Editor: R. M. Sundaram, ICAR-Indian Institute of Rice Research, INDIA Received: October 22, 2020 Accepted: July 17, 2021 Published: September 9, 2021 Published: September 9, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0255470 Copyright: © 2021 Duy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. 1 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Funding: This work was supported by the National Technology Innovation Program of Vietnam (Grant No. M.36.DN/18) funded by the Vietnam Ministry of Science and Technology (https://most.gov.vn/ vn/pages/Trangchu.aspx) and ThaiBinh Seed Corporation (https://thaibinhseed.com.vn/trang- chu.aspx?lang=en-US). - All equipments, labs and nethouses for this work were supported by Institute of Agricultural Genetics, Vietnam Academy of Agricultural Sciences - Financial support came from the Vietnam Ministry of Science and Technology (https://most.gov.vn/vn/pages/ Trangchu.aspx) and ThaiBinh Seed Corporation (https://thaibinhseed.com.vn/trang-chu.aspx?lang= en-US). - The funders had no role in study design, data collection and analysis, decision to publish the manuscript. However, Tran Manh Bao who’s employed by ThaiBinh Seed Corporation has contributed to the reviewing and editing the manuscript. His name was added as an author of the manuscript. - The funder (ThaiBinh Seed Corporation) supported the research materials (rice cultivar TBR225) for this study. - The funders provided support in the form of salaries for authors (Pham Xuan Hoi, Nguyen Duy Phuong, Pham Thu Hang and Nguyen Thanh Ha and Tran Manh Bao), but did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 Introduction However, strains collected in Asian countries such as China, Japan, Phillippines, Taiwan, Thailand, India, Nepal or South Korea can express combinations of up to three major TALEs redundantly targeting clade III OsS- WEET genes with either PthXo3 or AvrXa7 being occasionally associated with PthXo2 [11,17]. Broad BLB resistance engineering thus required multiplex OsSWEET promoters EBE editing using the CRISPR/Cas9 system [11,12]. Competing interests: Authors XP, PD, HPT, HNT, and TB are employee of ThaiBinh Seed Corporation. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein- 9 nuclease (CRISPR/Cas9) system is a simple and efficient gene-editing tool developed in the past few years [18,19]. Moreover, the targeted mutations generated by CRISPR/Cas9 can be stably transmitted to the next generation. Thus, CRISPR/Cas9 has become a routine tool in plant laboratories around the world to create various mutants for many applications, including the genetic improvement of crops [20]. BLB is a major rice disease which occurs in many rice cultivating areas of Vietnam [21,22]. Most Vietnamese commercial rice varieties, including TBR225, are susceptible to BLB, result- ing in annual yield loss of about 15–30% on average [23]. A few studies have identified rice resistance genes effective against Vietnamese Xoo lineages [22,23]. However, no information is currently available on the nature of Vietnamese Xoo TALEs and their corresponding S genes. Despite the large number of mapped rice BLB resistance genes [24,25], there is a need for alter- native breeding approaches that enable the rapid introduction of broad BLB resistance in elite varieties in order to cope with swift pathogen populations adaptive shifts in the fields [11,26]. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 2 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Here, we report on the identification of OsSWEET14 as a transcriptional target of Vietnam- ese Xoo. CRISPR/Cas9-mediated mutagenesis of the OsSWEET14 promoter in TBR225, a major elite variety in rice production areas of North Vietnam, is shown to confer BLB resis- tance without detectable yield penalty. The current study found this quintessential S gene to be associated with the virulence of Vietnamese Xoo strains. Gene expression analysis Gene expression analyses were carried out as described previously [29] by RT-PCR method. The rice leaves were infiltrated with the indicated bacterial strains and used for total RNA extraction 48 h post inoculation using the TRIzol reagent (Invitrogen, USA). One microgram of RNA was used for each RT-PCR with oligo (dT) primer followed by PCR with OsS- WEET14-specific primers (forward 50-ACTTGCAAGCAAGAACAGTAGT-30 and reverse 50-AT GTTGCCTAGGAGACCAAAGG-30). An Eppendorf Mastercycler ep Gradient S was used for 35 PCR cycles. The OsEF1α gene was used as a constitutive control [15] using specific primers (forward 50-GAAGTCTCATCCTACCTGAAGAAG-30 and reverse 50-GTCAAGAGCCTCAAGCA AGG-30). gRNA design The OsSWEET14 promoter (GenBank, accession number: AP014967.1) was amplified by PCR with forward primer 5’-TTGCGGCTCATCAGTTTCTC-3’ and reverse primer 5’-CTAGGAGAC CAAAGGCGAAG-3’ from genomic DNA of TBR225 rice plants and ligated in pGEM-T Easy vector (Promega) for sequencing. The gRNA target sequence (Fig 1A) for editing the TBR225 OsSWEET14 promoter was designed based on the sequence of the cloned TBR225 OsS- WEET14 promoter using a combination of two bioinformatics tools CRISPR-P v2.0 [30] and CCTop [31]. A gRNA sequence with high on-target and low off-target scores in both predic- tion tools was chosen for vector construction. Plant and pathogen materials Rice cultivar TBR225 (Oryza sativa L. ssp. indica) were obtained from ThaiBinh Seed Cor. [27]. All edited and wild-type (WT) TBR225 plants were grown in a net-house under the fol- lowing average conditions: 30˚C for 14 h (light) and 25˚C for 10 h (dark) with 80% humidity. The Xoo VXO_11 and VXO_15 strains used in this study were isolated from diseased leaves collected in Hanoi-Vietnam in 2013 and 2016, respectively. Bacteria were cultured as described in Zhou et al. (2015) [28]. Introduction This is an important step for the future design and implementation of broad-spectrum BLB-resistance in elite rice varieties using genome editing in Vietnam. Vector construction The Cas9 rice expression vector (pUbi-Cas9) [32] and the sgRNA expression vector (pENTR- sgRNA) under the control of the OsU6 promoter [33] were used to construct the pCas9/OsS- WEET14-gRNA expression vector. The complementary oligonucleotides with appropriate 4-bp overhangs were synthesized by Macrogen (Korea). After heat denaturation, the comple- mentary oligonucleotides (50-gtgtGGTGCTAAGCTCATCAAGCC-3’ and 50-aaacGGCTT GATGAGCTTAGCACC-3’) were first annealed to each other, phosphorylated, and ligated into the BsaI-digested vector pENTR-sgRNA. The integrity of the inserted fragment was verified by sequencing. Subsequently, the sgRNA cassette was cloned into pUbi-Cas9 using the Gateway 3 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 1. CRISPR/Cas9-induced OsSWEET14 promoter modification in TBR225 rice. (A) A region of the OsSWEET14 promoter containing four EBEs (TalC, PthXo3, AvrXa7 and TalF) and putative TATA box from TBR225. The target site (complementary to the guide RNA) is shown in the box, immediately following the protospacer adjacent motif (PAM). (B) T-DNA region of the CRISPR/Cas9-mediated genome editing construct carrying OsSWEET14-sgRNA (indicated by the black box). The expression of Cas9 is driven by the maize ubiquitin promoter (P-Ubi); the expression of the OsSWEET14-sgRNA is driven by the rice OsU6 promoter (P-OsU6a); the expression of HPT is driven by two CaMV35S promoters (P-2×35S); T-35S, T-Nos and TTTTTT: Gene terminators; LB and RB: Left and right border, respectively. (C) Alignment of the OsSWEET14 promoter fragment in the nine T0 transgenic TBR225 rice plants edited in the AvrXa7, PthXo3 and TalF EBEs. The lines on top of the wild-type sequence represent the binding sites of AvrXa7, PthXo3 and TalF. The arrow indicates the expected cutting site of the Cas9 complex used in this study. The labels on the left indicate the name of examined mutant lines; (a1) and (a2) distinguish alleles in the same line. The numbers on the right indicate the type of mutation and the number of nucleotides involved; (+) and (-) indicate insertion and deletion, respectively. Fig 1. CRISPR/Cas9-induced OsSWEET14 promoter modification in TBR225 rice. (A) A region of the OsSWEET14 promoter containing four EBEs (TalC, PthXo3, AvrXa7 and TalF) and putative TATA box from TBR225. The target site (complementary to the guide RNA) is shown in the box, immediately following the protospacer adjacent motif (PAM). (B) T-DNA region of the CRISPR/Cas9-mediated genome editing construct carrying OsSWEET14-sgRNA (indicated by the black box). Vector construction The expression of Cas9 is driven by the maize ubiquitin promoter (P-Ubi); the expression of the OsSWEET14-sgRNA is driven by the rice OsU6 promoter (P-OsU6a); the expression of HPT is driven by two CaMV35S promoters (P-2×35S); T-35S, T-Nos and TTTTTT: Gene terminators; LB and RB: Left and right border, respectively. (C) Alignment of the OsSWEET14 promoter fragment in the nine T0 transgenic TBR225 rice plants edited in the AvrXa7, PthXo3 and TalF EBEs. The lines on top of the wild-type sequence represent the binding sites of AvrXa7, PthXo3 and TalF. The arrow indicates the expected cutting site of the Cas9 complex used in this study. The labels on the left indicate the name of examined mutant lines; (a1) and (a2) distinguish alleles in the same line. The numbers on the right indicate the type of mutation and the number of nucleotides involved; (+) and (-) indicate insertion and deletion, respectively. https://doi.org/10.1371/journal.pone.0255470.g001 https://doi.org/10.1371/journal.pone.0255470.g001 LR clonase (Life Technologies) (Fig 1B). The resulting construct was confirmed by Sanger sequencing of the insertion junctions. LR clonase (Life Technologies) (Fig 1B). The resulting construct was confirmed by Sanger sequencing of the insertion junctions. Agrobacterium-mediated rice transformation The pCas9-OsSWEET14-gRNA was electroporated into Agrobacterium tumefaciens EHA105 and the resulting strain was used to transform rice using the method described by Hiei et al. (1994) [34]. The presence of the transgene in the genome of T0 hygromycin-resistant plants or segregating T1 individuals was evaluated by PCR using 50-ATGGCCCCAAAGAAGAAG-30 and 50- GCCTCGGCTGTCTCGCCA-30 primers specific for Cas9. T1 individuals were analyzed by PCR using Cas9, OsSWEET14-gRNA (50- GGATCATGAACCAACG-30 and 50- GAATTCGATAT CAAGCTT-30) and HPT (5’-AAACTGTGATGGACGACACCGT-3’ and 50- GTGGCGATCCTGC AAGCTCC -30) specific diagnostic primer pairs together with a positive control pair (50-TTG CGGCTCATCAGTTTCTC-30 and 50- TGGATCAGATCAAAGGCAAC -30) specific to the OsS- WEET14 promoter. Bacterial blight inoculation Rice cultivation and disease assays were done according to the methods of Blanvillain-Bau- fume´ et al. (2017) [15]. Bacteria were cultured in PSA media (10 g/liter peptone, 10 g/liter sucrose, 1 g/liter glutamic acid, 15 g/liter Bacto Agar) at 28˚C for two days [35] and inoculated at an optical density (OD600) of 0.5 (infiltrations) or 0.4 (leaf clipping) in water. For lesion length measurements, at least three inoculated leaves per plant and three plants for each line were measured 14 days after inoculation (DAI), and scored as follows: high resistance (lesion length < 8 cm), moderate resistance (lesion length 8–12 cm) and susceptibility (lesion PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 4 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 length > 12 cm). For gene expression analyses, 4-cm leaf sections infiltrated with bacterial sus- pensions were collected at 48 h after inoculation for RNA extraction. Experiments included samples from three pooled biological replicate leaves. The plants inoculated with distilled water only were used as negative controls. Analysis of OsSWEET14 edited allele sequences To determine the nature of the mutation at the target site, all transgenic T0 or T1 plants were analyzed by PCR using genomic DNA (50 ng) as a template and OsSWEET14 specific primers (50-TTGCGGCTCATCAGTTTCTC-30 and 50- TGGATCAGATCAAAGGCAAC -30). The PCR products were directly sequenced using the Sanger method. The sequencing chromatograms were decoded using the Degenerate Sequence Decoding method [36] in order to identify the mutations. Evaluation of major agronomic traits under net-house conditions WT and selected mutant plants were planted under net-house conditions in a randomized pot design experiment. At maturity, five plants of each line were investigated for the following agronomic traits: growth duration, plant height, number of tillers per plant, number of grains per panicle, number of filled grains per panicle and yield (seed mass) per plant. The experi- ment was repeated three times, so a total of fifteen plants were evaluated for each line. Analysis of potential off-target editing Off-target sequences were predicted with the CCTop tool [31] against the OsSWEET14 pro- moter sgRNA and the rice Nipponbare genome with default parameters. A total of 18 potential off-target sequences were identified. Three of them were located in coding regions (S2 Table). These regions were amplified by PCR using the specific primers listed in S2 Table and analyzed by sequencing. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 Vietnamese Xoo strains induce OsSWEET14 during infection of the TBR225 rice variety OsSWEET14/Os11N3 was previously identified as a susceptibility gene for Xoo strains relying on either of the AvrXa7, PthXo3, TalF (formerly Tal5) or TalC TALEs for infection of the rice cultivars Nipponbare and Kitaake [11]. Because Xoo strains tend to frequently target this gene, we first sequenced a region of the OsSWEET14 promoter from rice cultivar TBR225 to exam- ine if it also carries documented target EBEs. Based on the Nipponbare genome sequence in database (AP014967.1), the region encompassing 1343 bp sequence upstream and 52 bp sequence downstream of the predicted transcription start site of OsSWEET14 gene from TRB225 rice cultivar was PCR amplified and sequenced (S1 Fig). The promoter region includ- ing the putative TATA box (TATAAA) and the AvrXa7, PthXo3, TalF/Tal5 and TalC EBEs (Fig 1A), located 319 bp to 216 bp upstream of the ATG initiation codon, showed 100% iden- tity to the Nipponbare sequence. This therefore implied that in principle, the TBR225 OsS- WEET14 promoter can be recognized by characterized major Xoo TALEs. As shown in Fig 2A, we also challenged TBR225 plants with two Vietnamese Xoo strains VXO_11 and VXO_15, both originating from the Hanoi province, using leaf clipping assays. We consistently obtained typical extended disease lesions 14 days after inoculation (25.5 cm and 26.6 cm average lesions length for VXO_11 and VXO_15, respectively in the experiment of S2 Fig), indicating that the TBR225 variety is susceptible to BLB. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 5 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 2. OsSWEET14 is likely a susceptibility gene for Vietnamese Xoo strains in rice cultivar TBR225. (A) Representative images of the disease lesions obtained 14 days after leaf clipping inoculation of TBR225 rice leaves with Vietnamese Xoo strains VXO_11 and VXO_15 or with water (CT). The chevrons above the leaves indicate the maximum visible extent of lesions away from the inoculation point on the left (B). OsSWEET14 expression pattern obtained by RT-PCR two day post-infiltration of TBR225 rice leaves with Vietnamese Xoo strains. CT Plants were inoculated with water only. The experiment was repeated three times. https://doi org/10 1371/journal pone 0255470 g002 Fig 2. OsSWEET14 is likely a susceptibility gene for Vietnamese Xoo strains in rice cultivar TBR225. Vietnamese Xoo strains induce OsSWEET14 during infection of the TBR225 rice variety (A) Representative images of the disease lesions obtained 14 days after leaf clipping inoculation of TBR225 rice leaves with Vietnamese Xoo strains VXO_11 and VXO_15 or with water (CT). The chevrons above the leaves indicate the maximum visible extent of lesions away from the inoculation point on the left (B). OsSWEET14 expression pattern obtained by RT-PCR two day post-infiltration of TBR225 rice leaves with Vietnamese Xoo strains. CT Plants were inoculated with water only. The experiment was repeated three times. https://doi.org/10.1371/journal.pone.0255470.g002 https://doi.org/10.1371/journal.pone.0255470.g002 To test if OsSWEET14 is a potential direct virulence target of Vietnamese Xoo strains, we infiltrated TBR225 rice leaves with the two Vietnamese Xoo strains. Forty-eight hours post infiltration, TBR225 plants inoculated with VXO strains displayed a strong induction of OsS- WEET14 relative to water controls (Fig 2B). These results suggest that OsSWEET14 is a tran- scriptional target of VXO strains and that it may act as a susceptibility gene in TBR225. CRISPR/Cas9 design for OsSWEET14 promoter editing Our main objective was to engineer resistance to BLB caused by Vietnamese Xoo strains. To this end, we subsequently sought to specifically modify the OsSWEET14 promoter in TBR225 rice with CRISPR/Cas9-mediated editing. Previous work revealed that while African Xoo strains rely on TalC and occasionally, TalF, all Asian Xoo strains use either PthXo3- or Avr- Xa7-like TALEs to activate OsSWEET14 [11]. Because the talC gene is currently exclusively found in African strains, we reasoned that it is unlikely that Vietnamese strains carry a talC copy. Thus, to maximize our chances to perturb all remaining documented EBEs, we selected a 20-bp nucleotide target site overlapping the PthXo3, AvrXa7 and TalF EBEs and having a predicted cut site located near the 3’-end of the AvrXa7 EBE (Fig 1A). The recombinant binary plasmid pCas9/OsSWEET14-gRNA for CRISPR/Cas9 mediated editing of OsSWEET14 was transformed into the rice variety TBR225 via Agrobacterium-mediated transformation (S1 Table). A total of nine TBR225 transformants were selected from 10 independent PCR-vali- dated transgenic T0 TBR225 plants to further investigate CRISPR/Cas9-targeted mutagenesis of the OsSWEET14 promoter. In order to decipher the nature of the editing events in OsS- WEET14, the promoter sequencing data of transgenic lines were analyzed using the Degener- ate Sequence Decoding software [36]. All 9 T0 transgenic plants harbored at least an editing event (Fig 1C): two were heterozygous mutant/wild type, two had homozygous mutations, and five had bi-allelic mutations. Regarding the type of mutations, 66.7% were nucleotide dele- tions, 11.1% of the mutations were nucleotide insertions and no substitution was detected (Table 1). Inheritance of CRISPR/Cas9-induced mutations in the T1 generation To assess the inheritance of the CRISPR/Cas9-induced OsSWEET14 mutations in the next generation, all T0 mutant transgenic plants (Fig 1C) were allowed to self-pollinate, and T1 transgenic plants were randomly selected in the progeny of T0 plants for sequencing and PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 6 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 analysis of their edited site (Table 2). All T1 individuals derived from T0 plants previously gen- otyped as homozygous possessed the same allele as their parent, suggesting stable inheritance Table 1. Frequencies of mutant genotypes and target mutation types in T0 transgenic plants. Mutant genotype ratiosa (%) Mutation type ratiosb (%) Heterozygote Homozygote Bi-allelic Deletion Insertion Substitution 22.2 (2/9) 22.2 (2/9) 55.6 (5/9) 66.7 (12/18) 11.1 (2/18) 0 (0/18) a (Number of on-target mutant genotype/total number of on-target mutant genotypes) x 100%. b (Number of allele mutation type/number of all allele mutation types) x 100%. https://doi.org/10.1371/journal.pone.0255470.t001 Table 1. Frequencies of mutant genotypes and target mutation types in T0 transgenic plants. Mutant genotype ratiosa (%) Mutation type ratiosb (%) Heterozygote Homozygote Bi-allelic Deletion Insertion Substitution 22.2 (2/9) 22.2 (2/9) 55.6 (5/9) 66.7 (12/18) 11.1 (2/18) 0 (0/18) a (Number of on-target mutant genotype/total number of on-target mutant genotypes) x 100%. b (Number of allele mutation type/number of all allele mutation types) x 100%. https //doi org/10 1371/journal pone 0255470 t001 Table 1. Frequencies of mutant genotypes and target mutation types in T0 transgenic plants. a (Number of on-target mutant genotype/total number of on-target mutant genotypes) x 100%. b (Number of allele mutation type/number of all allele mutation types) x 100%. analysis of their edited site (Table 2). All T1 individuals derived from T0 plants previously gen- otyped as homozygous possessed the same allele as their parent, suggesting stable inheritance of the mutations to the next generation. Similarly, the T1 progeny of each of both bi-allelic and heterozygous mutation T0 lines showed a segregation ratio which is consistent with Mendelian segregation (χ2 < χ2 0.05, 2 = 5.99), indicating that the CRISPR/Cas9-induced mutations in T0 plants were transmitted as expected to the next generation. Interestingly, no new mutant allele was detected in the T1 generation of both heterozygous mutants L-21 and L-27, even though most of them still carried the transgene. Inheritance of CRISPR/Cas9-induced mutations in the T1 generation Overall, consistent with previous similar studies, our results indicate that the CRISPR/Cas9-mediated mutations generated here are stably transmit- ted to the next generation in a Medelian fashion. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides. “w”, wild type. yp Number of homozygous mutant plants without T-DNA. and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides. , wild type. https://doi.org/10.1371/journal.pone.0255470.t002 w , wild type. Number of homozygous mutant plants without T-DNA. yp Number of homozygous mutant plants without T-DNA. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides. “w”, wild type. ectively, insertion and deletion, of the indicated number of nucleotides. n and deletion, of the indicated number of nucleotides. pe. homozygous mutant plants without T-DNA. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides. “ ” ild t Xoo To characterize the BLB-resistance phenotype of the generated rice mutants, three T-DNA- free, homozygous TBR225 edited lines, namely, L-5.7(-6), L-31.12(-3) and L-15.4(+1) with OsSWEET14 promoter alleles corresponding respectively to L-5-a1 (6bp deletion), L-31 (3bp deletion) and L-15 (1bp insertion) in Fig 1C, were established. Selected T1 individuals were propagated to obtain T2 seeds which were used to perform BLB susceptibility assays. Edited T2 and WT TBR225 plants were inoculated by leaf-clipping with the VXO_11 and VXO_15 strains at the eight-week stage. The inoculated leaves of wild type TBR225 plants and of edited lines L-15.4(+1) and L-31.12(-3) developed long water-soaked lesions typical of BLB, ranging from 18.3 cm to 29.0 cm in length. In contrast, the edited line L-5.7(-6), harboring a longer 6-bp deletion at the target site, displayed high (1.2 cm average lesion length) and moderate (7.3 cm average lesion length) resistance to VXO_11 and VXO_15 strains, respectively (Fig 3). Means comparisons with a Tukey’s HSD test further indicated that irrespective of the inoculated strain, the mean lesion lengths measured on the L-15.4 (+1), L-31.12(-3) or wild type lines were not significantly different. In contrast, the mean lesion lengths recorded on the L-5.7(-6) mutant line were significantly different from those obtained on the wild type and the two other edited lines challenged with either of the Viet- namese strains (Fig 3B). Furthermore, our off-target editing analysis on line L-5.7(-6) did not reveal unintended modifications of other annotated rice loci (S2 Table and S5 Fig), indi- cating that the 6-bp deletion in the OsSWEET14 promoter is probably responsible for this phenotype. Consistent with disease assays and as shown in Fig 3C, whereas a semiquantitative RT-PCR signal for OsSWEET14 expression was detected on the parental variety and the L-15.4(+1) and L-31.12(-3) edited lines following VXO_11 and VXO_15 infiltration, this amplicon was unde- tectable in the resistant L-5.7(-6) line. In conclusion, this data shows that the 6-bp deletion in the AvrXa7/PthXo3 EBE reduces dramatically OsSWEET14 expression following VXO strains inoculation and confers resis- tance to these strains. In contrast, shorter modifications on the 3’-end of this EBE are insuffi- cient to perturb OsSWEET14 expression after inoculation and do not confer detectable protection against the corresponding strains. Xoo Finally, while these results strongly support the view that OsSWEET14 functions as a unique susceptibility gene in the interaction between strain VXO_11 and the TBR225 rice variety, the resistance to strain VXO_15 is not as dramatic and may suggest that other mechanisms partially counteract the effects of the AvrXa7/PthXo3 EBE 6-bp deletion in edited TBR225 plants. Selection of transgene-free mutant TBR225 rice lines To identify T-DNA free T1 rice plants containing a mutation in EBEs of the OsSWEET14 pro- moter, PCR analysis was carried out using primers specific to Cas9, sgRNA and HPT sequences (Table 2). A T1 individual was considered devoid of the transgene if the control amplification of the OsSWEET14 promoter was successful and if none of the PCR reactions with independent primer pairs designed on the T-DNA produced a detectable diagnostic band. The results of this PCR screen show that the T-DNA could be segregated out in the prog- eny of most T0 lines, with 88.9% of the T0 lines generating T-DNA-free progeny. In total, 44 of 221 analyzed edited T1 plants did not generate a specific amplicon from the T-DNA construct and 15 of them were homozygous mutant harboring the desired OsSWEET14 modifications. Our results demonstrate that transgene-free, homozygous mutant individuals could be obtained in the segregating progeny of selfed T0 individuals. Table 2. Transmission of CRISPR/Cas9 editing events to the T1 generation. T0 plant Genotype Allele(s) No. of T1 plants tested Mutation inheritance in the T1 generation No. of T-DNA-free plants Alleles segregation χ2 (1:2:1) L-4 Bi-allelic -5/-3 32 10 (-5), 18 (-5/-3), 4 (-3) 2,750 5 (2) L-5 Bi-allelic -6/+1 44 9 (-6), 22 (-6/+1), 13 (+1) 0,727 10 (2) L-7 Bi-allelic -4/-3 38 14 (-4), 17 (-4/-3), 7 (-3) 3,000 11 (4) L-15 Homozygote +1 5 5 (+1) - 1 (1) L-21 Heterozygote -3 26 3 (-3), 13 (-3/wt), 10 (wt) 3,769 7 (1) L-27 Bi-allelic -5/-4 7 1 (-5), 3(-5/-4), 3 (-4) 1,286 0 L-29 Heterozygote -5 33 6 (-5), 19 (-5/wt), 8 (wt) 1,000 2 (0) L-31 Homozygote -3 15 15 (-3) - 5 (5) L-54 Bi-allelic -3/-2 21 3 (-3), 12 (-3/-2), 6(-2) 1,286 3 (0) Table 2. Transmission of CRISPR/Cas9 editing events to the T1 generation. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 7 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 TBR225 OsSWEET14 promoter edited lines agronomic performances are undistinguishable from the parental variety To determine if mutations in the OsSWEET14 promoter affect agronomic traits of TRB225 rice plants, three independent homozygous mutant lines were analyzed by measuring their growth duration, plant height, number of tillers per plant, number of grains per panicle, num- ber of filled grains per panicle, yield per plant and amylose content under net-house conditions (see picture of S3 Fig). ANOVA tests and Student’s t tests showed that the mutant lines dis- played no significant difference to TBR225, in terms of the examined agronomic traits, under our net-house conditions (Table 3). These results suggest that the tested CRISPR/Cas9-in- duced mutations in the OsSWEET14 promoter did not negatively impact the main agronomic traits of TBR225. 8 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 3. BLB resistance assays for homozygous mutant rice lines L-5.7(-6), L-15.4(+1) and L-31.12(-3). (A) Leaves were photographed 14 days post-leaf clipping inoculation of Xoo strains VXO_11 and VXO_15; arrow heads indicate the end of the lesion. (B) Mean lesion lengths (bars) and standard deviations (error bars). Values were measured 14 days post-leaf clipping inoculation of two Xoo strains VXO_11 and VXO_15 and were computed from at least three leaves from each of three plants. Asterisks indicate significant differences relative to wild type plants (Tukey’s HSD test; P < 0.05). The number in the parentheses following the line name indicates the type of mutation and the number of nucleotides involved. The letters above strain labels indicate susceptibility score (R—high resistance; M–moderate resistance; S—susceptibility). The experiment was repeated three times. (C) OsSWEET14 expression pattern obtained by RT-PCR two day post-infiltration of genome edited homozygous mutant rice lines L-31.12(-3), L-15.4(+1) and L-5.7(-6) and parental TBR225 rice leaves with Vietnamese Xoo strains. This experiment was repeated two times with similar results. S ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 3. BLB resistance assays for homozygous mutant rice lines L-5.7(-6), L-15.4(+1) and L-31.12(-3). (A) Leaves were photographed 14 days post-leaf clipping inoculation of Xoo strains VXO_11 and VXO_15; arrow heads indicate the end of the lesion. (B) Mean lesion lengths (bars) and standard deviations (error bars). Values were measured 14 days post-leaf clipping inoculation of two Xoo strains VXO_11 and VXO_15 and were computed from at least three leaves from each of three plants. TBR225 OsSWEET14 promoter edited lines agronomic performances are undistinguishable from the parental variety Asterisks indicate significant differences relative to wild type plants (Tukey’s HSD test; P < 0.05). The number in the parentheses following the line name indicates the type of mutation and the number of nucleotides involved. The letters above strain labels indicate susceptibility score (R—high resistance; M–moderate resistance; S—susceptibility). The experiment was repeated three times. (C) OsSWEET14 expression pattern obtained by RT-PCR two day post-infiltration of genome edited homozygous mutant rice lines L-31.12(-3), L-15.4(+1) and L-5.7(-6) and parental TBR225 rice leaves with Vietnamese Xoo strains. This experiment was repeated two times with similar results. https://doi.org/10.1371/journal.pone.0255470.g003 PLOS ONE PLOS ONE Table 3. Agronomic traits evaluation of homozygous T2 mutant lines. Lines Growth duration (day) Plant height (cm) No. of tillers per plant No. of grains per panicle No. of filled grains per panicle Amylose content (%) WT 108.4 ± 1.1a 86.6 ± 3.2a 5 ± 0.7a 144.4 ± 4.9a 125 ± 4.5a 13.2 ± 0.38a L-5.7(-6) 108 ± 1.2a 86.4 ± 4.3a 5.2 ± 0.4a 144.2 ± 4.4a 123.4 ± 5.5a 13.7 ± 0.35a L-15.4(+1) 107.8 ± 0.8a 86.4 ± 5.0a 4.8 ± 0.4a 147.8 ± 5.1a 121.8 ± 3.0a 13.5 ± 0.41a L-31.12 (-3) 108 ± 1.2a 88.4 ± 4.3a 5.4 ± 0.5a 144.6 ± 5.3a 124.2 ± 7.4a 13.8 ± 0.21a Five plants per line were measured. Experiments were repeated three time. Means followed by the same letter do not differ significantly (P < 0.05). https://doi.org/10.1371/journal.pone.0255470.t003 Table 3. Agronomic traits evaluation of homozygous T2 mutant lines. Five plants per line were measured. Experiments were repeated three time. Means followed by the same letter do not differ significantly (P < 0.05). https://doi.org/10.1371/journal.pone.0255470.t003 improvement of agriculturally relevant elite rice cultivars for pertinent traits using the CRISPR/Cas9 technology (see for example [38–42]) is gradually increasing but is still limited. improvement of agriculturally relevant elite rice cultivars for pertinent traits using the CRISPR/Cas9 technology (see for example [38–42]) is gradually increasing but is still limited. TBR225 [27], a major commercial rice variety cultivated in large areas of Northern Viet- nam, has the advantages of early maturity, high and stable yield, as well as cooking quality. However, it is very susceptible to BLB. Here, the CRISPR/Cas9-mediated editing method was applied in order to rapidly improve the BLB resistance of TBR225 by modifying the AvrXa7, PthXo3 and TalF EBEs on the promoter of OsSWEET14. Of the three generated homozygous mutant lines tested for resistance, the one carrying the largest deletion at the target site (6 bp) showed a significantly improved resistance to infection with two Xoo strains VXO_11 and VXO_15. Therefore, using the major commercial rice variety TBR225 as an example, we illus- trate the advantages of CRISPR/Cas9 tool for rice breeding. In the present study, the frequency of individuals with CRISPR/Cas9-induced mutations in T0 transgenic plants was 90%, which is similar to previous observation [33]. We obtained only two heterozygous mutant/wild type lines versus seven homozygous or bi-allelic mutant lines. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 Discussion Recently, the CRISPR/Cas9 system has emerged as a powerful tool for gene editing in many organisms including plants. Because of its specificity and efficiency, this system has been widely used to improve important agronomic traits of major crops such as rape, tomato, soy- bean, rice, wheat and maize [37]. Excluding easy-to-transform reference accessions such as Nipponbare and Kitaake that are widely used in the laboratory, the number of reports on the PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 9 / 16 Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 PLOS ONE [11] who studied 30 combinations of EBE mutations in the OsSWEET11, OsSWEET13 and OsSWEET14 promoters of the IR64 or Ciher- ang-Sub1 varieties and detected only a single line with abnormal agronomic traits. Some individual Xoo strains have evolved a set of distinct TALE effectors that collectively target several members of the clade III SWEET family. The presence of these redundant TALEs thereby trumps single “loss-of-tale-responsiveness” resistance alleles [11,12,17,47]. For example, Kitaake lines carrying TALEN-induced mutation in the SWEET14 promoter [13,15] exhibit resistance to strains which depend exclusively on matching AvrXa7/PthXo3 for clade III SWEET family induction. Likewise, the natural xa13 allele [48] or CRISPR/Cas9-induced mutation in the SWEET11 promoter [11] exhibit resistance to strains such as PXO99 which depend exclusively on PthXo1, for virulence. However, the BLB resistance of the Kitaake lines harboring mutations in both AvrXa7/PthXo3 (OsSWEET14) and PthXo1 (OsSWEET11) EBEs was defeated by Xoo strains expressing simultaneously the AvrXa7/PthXo3 and PthXo2B TALEs [11]. Recently, the stacking of EBE-edited alleles in several OsSWEET promoters have overcome this limitation and was shown to achieve a broad spectrum of resistance to strains from most BLB-prone countries in Asia [11,12]. All of the three T2 lines tested for BLB resistance were affected for the AvrXa7/PthXo3 EBE and conserved an otherwise wild type TalF EBE (Fig 1C). The homozygous mutant TBR225 line L-5.7(-6) carrying a 6-bp deletion in the AvrXa7/PthXo3 EBE exhibited a significantly enhanced resistance to two Vietnamese Xoo strains compared to WT TBR225. The L-15.4(+1) and L-31.12(-3) lines that harbored more subtle alterations in the 3’-end of this EBE (a 1-bp insertion and a 3-bp deletion, respectively) in contrast remained susceptible to VXO strains. Our OsSWEET14 expression analysis after Vietnamese Xoo strains inoculation (Fig 1C) sug- gests that these editing events did not alter the EBE sequence sufficiently to compromise pro- moter recognition by an AvrXa7/PthXo3-like Vietnamese TALE. With less than 2 cm average lesion length, the resistance of line L-5.7(-6) (6-bp deletion) to the VXO_11 strain is rather extreme (versus average lesion length of 20.1 cm on wild type plants). Moreover, in this line, OsSWEET14 expression following bacterial inoculation is strongly reduced relative the paren- tal line and the two other edited lines, which suggest that in this case, recognition by an AvrXa7/PthXo3-like Vietnamese TALE is abrogated. PLOS ONE This high frequency of mutated alleles is another proof that the CRISPR/Cas9 system is indeed an efficient tool for gene editing in plant. We also observed the stable transmission of edited alleles to subsequent generations. This is a common phenomenon that has been repeatedly documented for rice plants carrying CRISPR/Cas9-induced mutations [38,40,43,44]. In this study, we obtained only two types of induced mutations in T0 plants: insertion (11.1%) and deletion (66.7%), but no substitution were observed. In some earlier studies, new mutations were continuously obtained in the T1 offspring of heterozygous T0 mutants because the Cas9 complex remains active on edited targets until the seed or PAM regions cease to be functional [35,37,43]. In contrast, here, all the T1 plants generated from both heterozygous lines L-21 and L-29, regardless of whether they had a CRISPR/Cas9 T-DNA transgene integrated in their genome, did not show any new mutation. We could also readily obtain transgene-free plants from most of the T1 segregation populations without any laborious crossing or backcrossing steps, which illustrates an advantage of the CRISPR/Cas9 technology compared to conven- tional breeding. Clade III SWEET family proteins are involved in a number of biological processes such as seed and pollen development or pathogen susceptibility [45]. Their inactivation has previously been shown to cause pleiotropic and/or detrimental effects. For example, both ossweet11 single and ossweet11-ossweet15 double Kitaake rice mutants showed defects in endosperm develop- ment and filling [46]. In addition, RNA-mediated silencing of either Os11N3/OsSWEET14 [7] or Os8N3/OsSWEET11 [6] in BLB resistant Kitaake lines causes negative effects on seed pro- duction. In contrast, here, we show that T-DNA-free TBR225 plants harboring homozygous mutations generated with the CRISPR/Cas9 system in the AvrXa7/PthXo3 EBE of the OsS- WEET14 promoter exhibited enhanced Xoo resistance but did not show any significant PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 10 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 difference in all examined agronomic traits compared to wild-type plants under net-house growth conditions. It is conceivable that limited modifications in promoter regions do not affect the normal expression of SWEET genes in contrast to KO or silenced lines. Our findings are consistent with the previous work of Oliva et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 PLOS ONE Consistent with OsSWEET14 expression analysis and as shown in S4 Fig, the Talvez [49] target prediction scores for AvrXa7 and PthXo3 on the OsSWEET14 promoter L-5-a1 allele sequence of line L-5.7(-6) are markedly lower than on the wild type promoter sequence. This is not the case however for the edited alleles carried by lines L-15.4(+1) and L-31.12(-3) (respectively L-15 and L-31 in S4 Fig) whose Talvez scores are identical or slightly lower than those of the wild type promoter sequence. The magnitude of the effect of the 6-bp deletion allele on susceptibility to VXO_11 is com- parable to the dramatic effect of previously characterized alterations of the same EBEs in the Kitaake background against the PXO86 strain that possesses a single TALE, AvrXa7, targeting OsSWEET14 for clade III OsSWEET gene induction [15]. By analogy, this suggests that OsS- WEET14 is also the only clade III OsSWEETs target of VXO_11 in the TBR225 background but, in order to confirm this hypothesis an examination of other clade III OsSWEET genes expression patterns in response to this strain would be required. The situation with the VXO_15 strain is not as straightforward to interpret and will require further investigations. Although the 6-bp deletion in the AvrXa7/PthXo3 EBE did provide an increased resistance to the edited plants, the VXO_15 strain caused intermediate disease severity (7.3 cm average lesion length on Fig 3). This incomplete resistance is unlikely to result from the partial but still PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 11 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 productive recognition of subsequences of the altered EBE by a VXO_15 AvrXa7/PthXo3-like TALE because OsSWEET14 expression is similarly decreased in response to either this strain or VXO_11 (Fig 3C). Alternatively, contrary to all Asian Xoo examined so far, but similar to African Xoo [11,15], VXO_15 may have the intrinsic potential to cause disease in the absence of clade III OsSWEET gene induction, a phenomenon that seems to be dependent on the edited rice variety genetic background [42]. More likely, analogous to other Asian strains, VXO_15 may encode alternative TALEs, such as PthXo2B or PthXo1 that compensate the loss of OsSWEET14 induction by targeting other clade III OsSWEET genes. S1 Fig. Nucleotide sequence of the OsSEET14 promoter in TBR225. (TIF) S2 Fig. Virulence of Vietnamese Xoo strains VXO_11 and VXO_15 on TBR225 rice. Grey points correspond to individual lesion length measurements while the black points indicate the calculated average value. The line range represents standard deviation. (TIF) PLOS ONE In this regard, deci- phering clade III OsSWEET genes expression patterns in combination with long read genome sequencing will ultimately help describe TALEs variability in Vietnamese Xoo strains and its functional impact on OsSWEET genes induction. In conclusion, we showed that editing specific EBEs of Xoo TALEs via CRISPR/Cas9 tool is an efficient method for improving BLB resistance of elite rice varieties such as TBR225 without detectable yield penalties. This also uncovered the potential diversity of TALEs in Vietnamese Xoo population, which will thus require future investigations to address the TALE repertoires of Vietnamese Xoo strains in order to generate broad-spectrum BLB-resistant rice varieties in Vietnam. S3 Fig. Picture of an individual plant from the homozygous mutant rice lines L-5.7(-6). (TIF) S4 Fig. Talvez scoring of AvrXa7, PthXo3 and TalF target EBES in the edited OsSWEET14 promoter allele sequences. Score values are represented both by the length of the horizontal bar and a fill color scale. Higher Talvez prediction scores reflect a better match between a pre- dicted EBE and the sequence of RVD of the query TALE. (TIF) S5 Fig. Amplicon sequencing of predicted off-target sites for the OsSWEET14 promoter- sgRNA in annotated exons of the TBR225 edited line L-5.7(-6). Potential unintended target sequences including the PAM are highlighted in boxes. They are all identical to the expected wild type Nipponbare sequences. (TIF) S1 Table. Key figures on the TBR225 transformation procedure for OsSWEET14 promoter editing. (DOCX) S1 Table. Key figures on the TBR225 transformation procedure for OsSWEET14 promoter editing. (DOCX) S2 Table. Output of the CCTop tool used with the OsSWEET14 promoter sgRNA for off- target prediction on the rice Nipponbare genome. (DOCX) S1 Raw images. Original photograph used in Fig 2 for the RT-PCR gels panel. (DOCX) 12 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470 September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Author Contributions Conceptualization: Phuong Nguyen Duy, Sebastien Cunnac, Xuan Hoi Pham. Conceptualization: Phuong Nguyen Duy, Sebastien Cunnac, Xuan Hoi Pham. Formal analysis: Phuong Nguyen Duy, Dai Tran Lan, Sebastien Cunnac, Xuan Hoi Pham. Funding acquisition: Phuong Nguyen Duy, Tran Bao Manh, Xuan Hoi Pham. Investigation: Phuong Nguyen Duy, Dai Tran Lan, Hang Pham Thu, Huong Phung Thi Thu, Ha Nguyen Thanh, Ngoc Phuong Pham, Florence Auguy, Sebastien Cunnac. Methodology: Phuong Nguyen Duy, Dai Tran Lan, Hang Pham Thu, Ha Nguyen Thanh, Sebastien Cunnac, Xuan Hoi Pham. Project administration: Xuan Hoi Pham. Project administration: Xuan Hoi Pham. Resources: Hang Pham Thu, Ha Nguyen Thanh, Florence Auguy. Resources: Hang Pham Thu, Ha Nguyen Thanh, Florence Auguy. Supervision: Sebastien Cunnac, Xuan Hoi Pham. Validation: Sebastien Cunnac, Xuan Hoi Pham. Writing – original draft: Phuong Nguyen Duy, Dai Tran Lan. Writing – review & editing: Huong Bui Thi Thu, Tran Bao Manh, Sebastien Cunnac, Xuan Hoi Pham. Acknowledgments We are grateful to Msc. Pham Thi Van, Dr. Cao Le Quyen and Dr. Nguyen Van Cuu from the Institute of Agricultural Genetics for rice transformation experiments, Msc. Nguyen Thi Thu Ha from the Institute of Agricultural Genetics for managing the Xoo strains collection and Msc. 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Functional and genome sequence-driven characterization of tal effector gene repertoires reveals novel variants with altered specificities in closely related malian Xanthomonas oryzae pv. oryzae strains. Front Microbiol. 2018; 9:1657. https://doi.org/10.3389/fmicb.2018.01657 PMID: 30127769. 48. Chu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, et al. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev. 2006; 20(10):1250–1255. https://doi.org/ 10.1101/gad.1416306 PMID: 16648463. 49. Pe´rez-Quintero AL, Rodriguez-R LM, Dereeper A, Lo´pez C, Koebnik R, Szurek B, et al. An improved method for TAL effectors DNA-binding sites prediction reveals functional convergence in TAL reper- toires of Xanthomonas oryzae strains. PLoS One. 2013; 8(7):68464. https://doi.org/10.1371/journal. pone.0068464 PMID: 23869221 16 / 16
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Relationship Between Epicardial Fat volume on Cardiac CT and Atherosclerosis Severity in Three-Vessel Coronary Artery Disease: A Single-Center Cross-Sectional Study
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Sun et al. BMC Cardiovascular Disorders (2022) 22:76 https://doi.org/10.1186/s12872-022-02527-7 Sun et al. BMC Cardiovascular Disorders (2022) 22:76 https://doi.org/10.1186/s12872-022-02527-7 Open Access Relationship between epicardial fat volume on cardiac CT and atherosclerosis severity in three‑vessel coronary artery disease: a single‑center cross‑sectional study Yu Sun1,2,3, Xiao‑gang Li2,3, Kai Xu4, Jie Hou1,2,3, Hong‑rui You2, Rong‑rong Zhang2, Miao Qi1,2,3, Li‑bo Zhang2,3, Li‑sheng Xu1, Stephen E. Greenwald5 and Ben‑qiang Yang2,3* Abstract Background:  The ideal treatment strategy for stable three-vessel coronary artery disease (CAD) patients are difficult to determine and for patients undergoing conservative treatment, imaging evidence of coronary atherosclerotic severity progression remains limited. Epicardial fat volume (EFV) on coronary CT angiography (CCTA) has been con‑ sidered to be associated with coronary atherosclerosis. Therefore, this study aims to evaluate the relationship between EFV level and coronary atherosclerosis severity in three-vessel CAD. Methods:  This retrospective study enrolled 252 consecutive patients with three-vessel CAD and 252 normal control group participants who underwent CCTA between January 2018 and December 2019. A semi-automatic method was developed for EFV quantification on CCTA images, standardized by body surface area. Coronary atherosclerosis severity was evaluated and scored by the number of coronary arteries with ≥ 50% stenosis on coronary angiography. Patients were subdivided into groups on the basis of lesion severity: mild (score = 3 vessels, n = 85), moderate (3.5 vessels ≤ score < 4 vessels, n = 82), and severe (4 vessels ≤ score ≤ 7 vessels, n = 85). The independent sample t-test, analysis of variance, and logistic regression analysis were used to evaluate the associations between EFV level and severity of coronary atherosclerosis. Results:  Compared with normal controls, three-vessel CAD patients had significantly higher EFV level (65 ± 22 mL/ m2 vs. 48 ± 19 mL/m2; P < 0.001). In patients with three-vessel CAD, there was a progressive decline in EFV level as the score of coronary atherosclerosis severity increased, especially in those patients with a body mass index (BMI) ≥ 25 kg/ m2 (75 ± 21 mL/m2 vs. 72 ± 22 mL/m2 vs. 62 ± 17 mL/m2; P < 0.05). Multivariable regression analysis showed that both BMI (OR 3.40, 95% CI 2.00–5.78, P < 0.001) and the score of coronary atherosclerosis severity (OR 0.49, 95% CI 0.26–0.93, P < 0.05) were independently related to the change of EFV level. Conclusion:  Three-vessel CAD patients do have higher EFV level than the normal controls. While, there may be an inverse relationship between EFV level and the severity of coronary atherosclerosis in patients with three-vessel CAD. *Correspondence: bqyang888@sina.com 2 Department of Radiology, General Hospital of Northern Theater Command, 83 Wenhua RoadLiaoning Province, Shenyang 110016, People’s Republic of China Full list of author information is available at the end of the article Background three-vessel CAD were initially included. Patients with previous percutaneous coronary intervention (n = 147) or coronary artery bypass grafting (n = 297), pericardial effusion (n = 38), coronary anomalies (n = 187), malig- nant chest tumor (n = 12), or systemic autoimmune disease (n = 3) were excluded. A further 418 patients without invasive coronary angiography (ICA) data were excluded. Patients with ICA-confirmed one- (n = 39) and two-vessel (n = 215) CAD were also excluded. Finally, 252 consecutive patients with ICA-confirmed three-vessel CAD were enrolled (Fig. 1). The time interval between CCTA imaging and ICA operating of all patients was (37 ± 15) days. Three-vessel CAD was defined as ≥ 50% luminal diameter stenosis present in three main epicar- dial coronary arteries on the ICA data, including the left anterior descending artery, left circumflex, and right coronary artery, with or without the left main artery involvement. Patients suffering from three-vessel coronary artery dis- ease (CAD) often have long-standing and complex coro- nary atherosclerosis and are at an increased risk of adverse cardiovascular events [1]. Many randomized clinical tri- als results have revealed no significant differences in sur- vival benefit between the two most widely used treatment, namely percutaneous coronary intervention and coronary artery bypass grafting, for the therapy of multivessel CAD patients [2–4]. Recently, another multicenter randomized trial reported that, in stable CAD, there was no evidence that an initial invasive strategy reduced the risk of adverse cardiovascular events in comparison to medical therapy alone [5]. Therefore, it is difficult to make the best choice of treatment strategy for stable three-vessel CAD patients. For patients who have undergone initial conservative treat- ment, evaluation of coronary atherosclerotic severity and assessment of its progression are in great demand, as an important aid in deciding further treatment, such as adjust- ment of drug dose or the ideal timing of invasive strategies. For the normal control group, 252 age-, gender- and body mass index (BMI)-matched subjects were selected. They underwent CCTA because of chest pain from Janu- ary 2018 to December 2019 and all had negative coronary atherosclerosis and no known cardiovascular disease. Epicardial adipose tissue is a type of visceral fat that is located between the surface of the myocardium and the visceral pericardial layer. Background By secreting a large variety of bio- active molecules, it can modulate vascular inflammation via paracrine signaling mechanisms which, in turn con- tribute to the development of coronary atherosclerosis and the destabilization of existing atherosclerotic lesions [6]. Many clinical studies have reported that the epicardial fat volume (EFV) using coronary CT angiography (CCTA) is associated with the presence and severity of coronary ath- erosclerosis and with the characteristics of atherosclerotic plaques [7–9]. Measuring the volume of epicardial fat has been considered as a potential imaging biomarker of dis- ease progression and response to treatment [10]. However, clinical evidence of the relationship between EFV level and coronary atherosclerosis severity in three-vessel CAD patients remains limited, except for that arising from sub- group analysis in previously reported literature [11]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑ mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 2 of 10 Keywords:  Epicardial adipose tissue, Epicardial fat volume, Computed tomography angiography, Three-vessel coronary artery disease CCTA data acquisition CCTA was performed using a 256-slice CT scanner (Bril- liance CT, Philips Medical Systems, Cleveland, USA). Detailed scanning parameters were listed as follows: tube voltage, 120 kVp; detector collimation, 128 × 0.625 mm; pitch, 0.16; rotation time, 0.27 s; slice thickness, 0.9 mm; section increment, 0.45 mm. Tube current was set using the ECG-based tube current modulation technique. About 60–70  mL of iodine contrast agent was injected at a rate of 4.5–5.5  mL/s via a high-pressure injector, followed by a 20–30 mL flush of saline at the same rate. Threrfore, in the present study, we sought to investigate the relationship between EFV level assessed with CCTA and coronary atherosclerosis severity in three-vessel CAD. Cardiovascular risk factors and clinical data Demographic characteristics and clinical data (summa- rized in Table 1) were collected for all patients, includ- ing BMI, smoking history, hypertension, dyslipidemia, diabetes mellitus and family history of CAD. In addition, measures of left ventricular (LV) function assessed with transthoracic echocardiography were obtained from the hospital records. These included LV end-diastolic diame- ter, LV ejection fraction (LVEF), and the ratio of diastolic mitral inflow velocity E peak and A peak (E/A). Coronary collateral circulation status and SYNTAX score assessed with ICA data were also collected. Study population In this single-center retrospective cross-sectional study, between 1 January 2018 and 31 December 2019, 1608 consecutive patients who underwent CCTA scan- ning in our hospital and had CCTA-based diagnosis of Sun et al. BMC Cardiovascular Disorders Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Page 3 of 10 Fig. 1  Study flowchart. CT computed tomography, CAD coronary artery disease, PCI percutaneous coronary intervention, CABD coronary artery bypass grafting, ICA invasive coronary angiography the heart. Epicardial fat was defined as all voxels with attenuation values between −  190 HU and −  30HU. Thus all voxels having attenuation values within this range and lying within the pericardium were thresh- olded accordingly and used for EFV level quantifica- tion (Fig. 2). The value of EFV was normalized to body surface area (BSA) where the BSA was calculated with the Mosteller equation [13]. Epicardial fat attenuation index (FAI) was defined as the average attenuation of adipose tissue within the pre-specified attenuation window of − 190 HU to − 30 HU. A prospective ECG-gated CCTA was triggered using a bolus tracking technique with a trigger threshold of 100 Hounsfield units (HU) in the descending aorta. The mean estimated effective radiation dose of the CCTA scan was (4.6 ± 1.2) mSv. EFV level and epicardial FAI measurement EFV level quantification was quantified on axial CCTA images at 75% of the R-R interval. A semi-automatic method was developed for segmenting the pericar- dium and measuring the amount of EFV. Briefly, the pericardial contour was automatically delineated by a U-net framework, details of which are described in a previous report from our group [12]. The segmen- tation results were further checked and modified by two experienced cardiac imaging physicians (J.H. and M.Q.) blinded to both the study plan and the clini- cal data. Modifications, including manual small scale enlargment or shrinking of the imaging annotation range, were made to ensure that the segmentation results of the pericardial boundary matched exactly with its anatomical structure. The upper boundary of the pericardium was taken as the bifurcation of the pulmonary trunk and the lower limit was the apex of For reproducibility assessment, images from 30 patients with three-vessel CAD were randomly selected. The pericardial contour was automatically delineated using the U-net framework mentioned above. Two radiologists (J.H. and M.Q.) reviewed and modified the automatic pericardial segmentation to ensure a good match with the pericardial anatomy. After four weeks, one of the radiologists (J.H.) reviewed and modified the pericardial segmentation output again. The value of EFV and epicardial FAI derived from the semi-automatic segmentation results of the pericardium was employed for reproducibility assess- ment using intraclass correlation coefficient analysis. Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 4 of 10 Table 1  Basic characteristics and clinical data of study population CAD coronary artery disease, EFV epicardial fat volume, BSA body surface area, FAI fat attenuation index, BMI body mass index, LV left ventricular, EF ejection fraction, CCC​ coronary collateral circulation *Patients were divided into two subgroups according to median EFV level (118 mL) # P values refer to the comparison between three-vessel CAD patients and normal controls. Statistical analysis Coronary atherosclerosis severity was quantified by the number of coronary arteries having ≥ 50% stenosis on ICA [11, 14]. Stenoses in the left anterior descending, left circumflex, and right coronary artery were each scored as a single vessel lesion and stenoses in the left main coronary artery were more strongly weighted by being scored as two vessel lesions. Stenoses in the remaining secondary branches of the coronary arter- ies (such as the diagonal artery, obtuse marginal artery, posterolateral artery, and posterior descending artery) with ≥ 50% stenosis were each scored as 0.5 of a lesion. The three-vessel CAD patients were subdivided into three groups according to coronary atherosclerosis severity: mild (score = 3 vessels, n = 85), moderate (3.5 vessels ≤ score < 4 vessels, n = 82), and severe (4 ves- sels ≤ score ≤ 7 vessels, n = 85). Patients with more than three vessel lesions were divided into two subgroups based on the median score (4 lesions) of coronary ath- erosclerosis severity. Continuous variables were expressed as mean ± standard deviation or medians and interquartile range. Categori- cal variables were expressed as number and percentage. The Kolmogorov–Smirnov test was employed to test for the normal distribution of continuous data. Com- parisons of continuous variables were conducted using the independent t-test and one-way analysis of variance (ANOVA), with the least significant difference (LSD) method for multiple comparisons. Comparisons of cat- egorical variables were performed with the Chi-square test. The relation of EFV level to mean epicardial FAI, LV function, and BMI were assessed with Pearson’s cor- relation analysis. Multivariable logistic regression analy- sis was used for co-variables that had a significant effect in the univariable logistic regression analysis, aiming to identify the independent factors associated with a change of EFV level. Interobserver and intraobserver agree- ment for EFV level and epicardial FAI quantification were assessed by evaluating the intraclass correlation Coronary atherosclerosis severity was quantified by the number of coronary arteries having ≥ 50% stenosis on ICA [11, 14]. Stenoses in the left anterior descending, left circumflex, and right coronary artery were each scored as a single vessel lesion and stenoses in the left main coronary artery were more strongly weighted by being scored as two vessel lesions. EFV level and epicardial FAI measurement The remaining P values refer to comparisons between the low and high EFV level subgroups of the CAD patients Variables Normal control (n = 252) Three-vessel CAD patients* P-value All patients (n = 252) Low EFV level (< 118 mL, n = 125) High EFV level (≥ 118 mL, n = 127) EFV (mL) 89 ± 32 120 ± 42 86 ± 21 154 ± 29 < 0.001# EFV/BSA (mL/m2) 48 ± 19 65 ± 22 49 ± 12 82 ± 16 < 0.001# Epicardial FAI (HU) − 84 ± 9 − 81 ± 5 − 78 ± 4 − 84 ± 4 < 0.001# Cardiovascular risk factors  Age (years) 61 ± 8 61 ± 8 61 ± 9 62 ± 7 0.163  Male sex, n (%) 193 (77) 193 (77) 93 (74) 100 (79) 0.416  BMI (kg/m2) 25 ± 3 25 ± 3 24 ± 3 26 ± 3 < 0.001  Smoking, n (%) 37 (19) 125 (50) 60 (48) 65 (51) 0.614  Hypertension, n (%) 0 (0) 179 (71) 85 (68) 94 (74) 0.293  Dyslipidemia, n (%) 0 (0) 45 (19) 21 (17) 24 (19) 0.664  Diabetes, n (%) 0 (0) 104 (41) 48 (38) 56 (44) 0.359  CAD family history, n (%) 0 (0) 29 (12) 17 (14) 12 (9) 0.302 LV functional parameters  LV diameter (mm) 48 ± 3.8 48 ± 4.3 48 ± 4.5 49 ± 4.0 0.023  LVEF (%) 61 ± 5.0 60 ± 6.0 60 ± 6.4 60 ± 5.6 0.911  E/A 1.6 ± 0.3 1.3 ± 0.4 1.2 ± 0.4 1.3 ± 0.4 0.056 Invasive coronary angiography  CCC status, n (%) NA 78 (31) 40 (32) 38 (30) 0.721  SYNTAX score NA 29 (23, 36) 30 (23, 36) 29 (23, 35) 0.793 Table 1  Basic characteristics and clinical data of study population d into two subgroups according to median EFV level (118 mL) isease, EFV epicardial fat volume, BSA body surface area, FAI fat attenuation index, BMI body mass index, LV left ventricular, EF ejection fra al circulation Statistical analysis Stenoses in the remaining secondary branches of the coronary arter- ies (such as the diagonal artery, obtuse marginal artery, posterolateral artery, and posterior descending artery) with ≥ 50% stenosis were each scored as 0.5 of a lesion.h The three-vessel CAD patients were subdivided into three groups according to coronary atherosclerosis severity: mild (score = 3 vessels, n = 85), moderate (3.5 vessels ≤ score < 4 vessels, n = 82), and severe (4 ves- sels ≤ score ≤ 7 vessels, n = 85). Patients with more than three vessel lesions were divided into two subgroups based on the median score (4 lesions) of coronary ath- erosclerosis severity. Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 5 of 10 Fig. 2  Semi-automatic quantification of EFV level from CCTA images. Axial CCTA images at 75% of the R-R interval were processed for pericardial segmentation using the U-Net framework. The initial results were checked and modified by two radiologists and the epicardial fat was identified as those voxels lying between upper and lower threshold values of − 30 and − 190 HU. EFV epicardial fat volume, CCTA coronary computed tomography angiography Fig. 2  Semi-automatic quantification of EFV level from CCTA images. Axial CCTA images at 75% of the R-R interval were processed for pericardial segmentation using the U-Net framework. The initial results were checked and modified by two radiologists and the epicardial fat was identified as those voxels lying between upper and lower threshold values of − 30 and − 190 HU. EFV epicardial fat volume, CCTA coronary computed tomography angiography significantly higher EFV in overweight patients than in those with normal body weight (EFV: 134  ±  41 vs 105  ±  39  mL, t = 5.786, P < 0.001; EFV/BSA: 70  ±  21 vs 60  ±  21  mL/m2, t = 3.603, P < 0.001). Hypertensive patients had a significantly higher EFV (123  ±  42 vs 110 ± 42 mL, t = 2.229, P = 0.027; EFV/BSA: 67 ± 21 vs 60 ± 22 mL/m2, t = 2.322, P = 0.021). Study population characteristics A total of 252 consecutive three-vessel CAD patients and 252 control group participants were included in this retrospective study. Detailed baseline demographic and clinical data are listed in Table 1. Compared with lesion- free controls, three-vessel CAD patients had significantly higher EFV level (EFV: 120 ± 42 vs 89 ± 32 mL, EFV/ BSA: 65 ± 22 vs 48 ± 19 mL/m2; P < 0.001) and higher mean epicardial FAI (− 81 ± 5 vs − 84 ± 9 HU, P < 0.001). According to the median EFV level (118 ­cm3), three- vessel CAD patients were categorized into low (< 118 mL, n = 125) and high EFV level subgroups (≥ 118  mL, n = 127). Compared with the low EFV subgroup, patients with high EFV had significantly higher BMI (26 ± 3 vs 24 ± 3 kg/m2, P < 0.001) and larger LV diameter (49 ± 4.0 vs 48 ± 4.5 mm, P = 0.023) (Table 1). Pearson correlation analysis showed that, for all CAD subjects, EFV was positively and significantly correlated with BMI (r = 0.407, P < 0.001) and negatively, with mean epicardial FAI (r = − 0.709, P < 0.001). Statistical analysis In comparison to patients who had normal LV diastolic function (E/A > 1, n = 203), those with LV diastolic dysfunction (E/A < 1, n = 49) had a lower EF and lower EFV relative to BSA, although only the latter was statistically significant (EFV: 110 ± 43 vs 122 ± 42 mL, t = − 1.868, P = 0.063; EFV/ BSA: 60 ± 22 vs 67 ± 21 mL/m2, t = − 1.988, P = 0.048). However, there were no significant differences of EFV level between the two subgroups in the variables related to gender, smoking, dyslipidemia, diabetes mellitus, or family history of CAD (P > 0.05). coefficient (ICC). Statistical analysis was performed with SPSS software (SPSS statistics, version 26.0, IBM Corp.). Two-sided testing was used and P < 0.05 was considered to be statistically significant. Relation of EFV, FAI, and LV function to the severity of coronary atherosclerosis Relation of EFV level to risk factors and LV function Subgroup analysis of EFV level, separated into two groups by median BMI (25  kg/m2), revealed a Relation of EFV level to risk factors and LV function Subgroup analysis of EFV level, separated into two groups by median BMI (25  kg/m2), revealed a Table  2 and Fig. 3a, b show that increasing steno- sis severity was significantly associated with reduced Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. Relation of EFV level to risk factors and LV function BMC Cardiovascular Disorders Page 6 of 10 Table 2  Comparison of EFV level, FAI, and LV function in three- vessel CAD EFV epicardial fat volume, FAI fat attenuation index, LV left ventricular, CAD coronary artery disease, BSA body surface area, EF ejection fraction, BMI body mass index *Patients were subdivided into groups according to their coronary atherosclerosis severity: mild (score = 3 vessels), moderate (3.5 ≤ score < 4 vessels), and severe (4 ≤ score ≤ 7 vessels) # Patients were categorized by median BMI (25 kg/m2) Variables Score of coronary atherosclerosis severity* P-value Mild Moderate Severe All patients (n = 252) 85 (34) 82 (32) 85 (34)  EFV (mL) 126 ± 45 124 ± 45 110 ± 34 0.045  EFV/BSA (mL/m2) 69 ± 23 67 ± 23 60 ± 17 0.036  Epicardial FAI (HU) − 81 ± 5.5 − 77 ± 4.5 − 73 ± 4.3 < 0.001  LV diameter (mm) 48 ± 4.3 48 ± 4.5 48 ± 4.0 0.967  LVEF (%) 60 ± 6.3 60 ± 5.6 60 ± 6.2 0.952  E/A 1.3 ± 0.4 1.3 ± 0.3 1.3 ± 0.4 0.931 Patients stratified by ­BMI#  BMI < 25 kg/m2 (n = 124) 41 (33) 37 (30) 46 (37)   EFV (mL) 107 ± 43 107 ± 42 103 ± 31 0.86   EFV/BSA (mL/m2) 62 ± 24 62 ± 24 58 ± 17 0.857   Epicardial FAI (HU) − 79 ± 6.6 − 77 ± 4.7 − 72 ± 3.9 < 0.001   LV diameter (mm) 48 ± 4.3 47 ± 4.3 48 ± 4.0 0.448   LVEF (%) 60 ± 5.8 61 ± 6.1 60 ± 6.2 0.788   E/A 1.2 ± 0.5 1.3 ± 0.3 1.2 ± 0.3 0.945  BMI ≥ 25 kg/m2 (n = 128) 44 (34) 45 (35) 39 (31)   EFV (mL) 144 ± 40 139 ± 43 118 ± 35 0.01   EFV/BSA (mL/m2) 75 ± 21 72 ± 22 62 ± 17 0.013   Epicardial FAI (HU) − 82 ± 4.0 − 78 ± 4.4 − 74 ± 4.5 < 0.001   LV diameter (mm) 49 ± 4.2 49 ± 4.4 49 ± 4.2 0.802   LVEF (%) 60 ± 6.7 60 ± 5.2 59 ± 6.2 0.725   E/A 1.3 ± 0.4 1.3 ± 0.3 1.3 ± 0.4 0.825 level from the mild, moderate to severe coronary ath- erosclerosis subgroups in the higher BMI group (EFV: 144 ± 40 vs 139 ± 43 vs 118 ± 35 mL, P = 0.01; EFV/ BSA: 75 ± 21 vs 72 ± 22 vs 62 ± 17 mL/m2, P = 0.013) (Table  2; Fig. Reproducibility assessmentfi EFV epicardial fat volume, FAI fat attenuation index, LV left ventricular, CAD coronary artery disease, BSA body surface area, EF ejection fraction, BMI body mass index Intraclass correlation coefficient analysis revealed excel- lent interobserver and intraobserver agreement for EFV level and epicardial FAI measurement. Interobserver analysis showed 0.97, 0.95 agreement for EFV level and epicardial FAI, respectively. Intraobserver analysis showed 0.99, 0.97 agreement for EFV level and epicardial FAI, respectively. # Patients were categorized by median BMI (25 kg/m2) EFV. There was a statistically significant inverse rela- tionship between EFV and severity of coronary ath- erosclerosis (126  ±  45 vs 124  ±  45 vs 110  ±  34  mL, F = 3.818, P = 0.023). Post hoc tests using the LSD method showed that the differences between the mild and severe coronary atherosclerosis subgroups were statistically significant (t = 16.212, P = 0.012), as well as for the comparison between the moderate and severe subgroups (t = 14.301, P = 0.028). When normalized to BSA, the relationship was maintained and remained significant although the effect was weaker (69 ± 23 vs 67 ± 23 vs 60 ± 17 mL/m2, P = 0.036). EFV. There was a statistically significant inverse rela- tionship between EFV and severity of coronary ath- erosclerosis (126  ±  45 vs 124  ±  45 vs 110  ±  34  mL, F = 3.818, P = 0.023). Post hoc tests using the LSD method showed that the differences between the mild and severe coronary atherosclerosis subgroups were statistically significant (t = 16.212, P = 0.012), as well as for the comparison between the moderate and severe subgroups (t = 14.301, P = 0.028). When normalized to BSA, the relationship was maintained and remained significant although the effect was weaker (69 ± 23 vs 67 ± 23 vs 60 ± 17 mL/m2, P = 0.036). Analysis of factors related to the change of EFV level Analysis of factors related to the change of EFV level Univariate and multivariable logistic regression analysis showed that BMI (OR 3.40, 95% CI 2.00–5.78, P < 0.001) and coronary atherosclerosis severity (OR 0.49, 95% CI 0.26–0.93, P = 0.03) were independently associated with the change of EFV level in all patients (Table 3). Relation of EFV level to risk factors and LV function 3c, d). Post hoc tests using the LSD method showed that, in comparison to the severe ath- erosclerosis subgroup, both the mild (EFV: t = 25.678, P = 0.004; EFV/BSA: t = 12.670, P = 0.005) and the moderate atherosclerosis subgroups (EFV: t = 20.480, P = 0.020; EFV/BSA: t = 10.164, P = 0.023) had signifi- cantly higher EFVs. However, no significant relation- ship between EFV level and atheroslcerotic severity was seen in patients with BMIs below the median value Additionally, there was a significant positive associa- tion between the severity of coronary atherosclerosis and the mean attenuation due to epicardial fat (FAI) (− 81 ± 5.5 vs − 77 ± 4.5 vs − 73 ± 4.3 HU, P < 0.001) for the mild, moderate and severe atherosclerosis lev- els, respectively. However, there were no significant differences between the mild, moderate, and severe atherosclerosis subgroups in the variables related to LV function, namely LV diameter, LVEF, or E/A (P > 0.05). Discussionh The results show that three-vessel CAD patients had significantly higher EFV levels than those of the normal controls. Furthermore, stratifying the severity of athero- sclerosis into mild, moderate and severe levels showed that there was an inverse relationship between EFV level assessed with CCTA and coronary atherosclerosis sever- ity in three-vessel CAD. Many clinical studies have demonstrated that EFV level on CCTA is related to the presence and severity of coronary atherosclerosis. For instance, in the Framing- ham Heart Study, measurements in more than 3000 indi- viduals showed that increased EFV was associated with the presence of CAD [8]. EFV level was also related to When grouped according to the median BMI (25 kg/ m2), there was, similarly, a progressive decline in EFV Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 7 of 10 Fig. 3  Relation of EFV level to coronary atherosclerosis severity in three-vessel CAD. Three-vessel CAD patients with more severe coronary atherosclerosis have significantly lower EFV level (a, b), especially in those patients with higher BMI (≥ 25 kg/m2) (c, d). EFV, epicardial fat volume; CAD, coronary artery disease; BMI, body mass index. Fig. 3  Relation of EFV level to coronary atherosclerosis severity in three-vessel CAD. Three-vessel CAD patients with more severe coronary atherosclerosis have significantly lower EFV level (a, b), especially in those patients with higher BMI (≥ 25 kg/m2) (c, d). EFV, epicardial fat volume CAD, coronary artery disease; BMI, body mass index. atherosclerosis, chronic hypoxia and an unfavorable hemodynamic environment would affect the regula- tory component of the epicardial fat proteasome, which would accelerate the apoptosis of epicardial adipocytes. This hypothesis is supported by evidence that several genes involved in cellular function are downregulated in the epicardial adipocytes of advanced CAD [15]. Fur- thermore, recent evidence has revealed a bidirectional signaling between epicardial adipose tissue and vascular inflammatory cells [16, 17]. Epicardial adipocytes could, therefore, modulate structural changes and inflamma- tion in the vascular wall via paracrine mechanisms and vascular wall-derived inflammatory factors could block the differentiation of epicardial adipocytes. In three- vessel CAD patients, the remodelling of adipocytes due to vessel inflammation was more obvious than that in patients with mild or moderate CAD. Therefore, the end result could be large proportion of small and immature epicardial perivascular pre-adipocytes around vessels, thus leading to a reduction of EFV. Discussionh These observa- tions are also consistent with previous studies which have reported increased epicardial FAI in patients with advanced CAD or acute coronary syndrome [20, 21]. Additionally, we observed a significantly higher EFV in hypertensive subjects and those in the higher BMI group, again in line with previous studies [22, 23]. We also found that in patients with LV diastolic dysfunction there was a tendency towards lower EFV, although this was not sta- tistically significant. A possible explanation is that epicar- dial fat is associated with both visible epicardial coronary atherosclerosis and coronary microvascular dysfunction, both of which would lead to impaired myocardial perfu- sion and contractile function [24].h There are several limitations to the study. First, it was a retrospective single-center study and the sample size was relatively small. Second, even though we adopted a commonly used method for classifying the severity of coronary atherosclerosis [11, 14], it did not account for the stability of the atherosclerotic plaque. Third, it has been reported that statin therapy is associated with a reduction in epicardial fat accumulation, especially when given in high doses (atorvastatin 80 mg/day) [25]. However, in this study, all the CAD patients were given moderate doses (atorvastatin 10–20 mg/day), this being based on expert recommendation for Chinese subjects [26]. Whether a significant reduction of EFV in three- vessel CAD patients with severe coronary atherosclerosis is associated with moderate statin therapy needs further clinical studies to confirm. to changes in the composition of epicardial adipocytes which could increase the degree to which they attenu- ate incident x-rays. If this were to happen, fewer voxels would fall within the thresholded range and, this would lead to an apparent reduction in EFV. This effect is likely to be seen in CAD patients who have had long-standing and complex coronary atherosclerosis, which is associ- ated with irreversible changes in epicardial perivascular adipocyte composition, such as inflammatory infiltra- tion, microvascular remodeling and extracellular fibro- sis [18, 19]. Furthermore, according to the bidirectional interaction hypothesis mentioned above [16, 17], epicar- dial fat preadipocytes tend to be of smaller size and con- tain less intracellular lipid than that of mature epicardial adipocytes. The loss of balance between lipid and water content in epicardial perivascular preadipocytes would result in a rise of epicardial FAI. Discussionh The second explana- tion for reduced EFV in those with severe CAD is related coronary atherosclerosis severity, coronary artery cal- cium score, and the characteristics of unstable athero- sclerotic plaques [8–10]. Here, we found that three-vessel CAD patients had significantly higher EFVs than those of the normal controls, which is consistent with the previ- ous studies [8–10]. Interestingly, we observed a progressive decline in EFV level with increasing coronary atherosclerotic severity in all three-vessel CAD patients, although it was statisti- cally significant only those with BMI ≥ 25 kg/m2. Gorter et  al. [11] also reported that patients with more severe coronary atherosclerosis tended to have lower EFVs although their study sample contained only 128 subjects. We believe there are two plausible explanations for this inverse association between disease severity and EFV. One is that fewer numbers of epicardial adipocytes and an increased number of small and immature epicardial perivascular preadipocytes in patients with severe coro- nary atherosclerosis would imply a reduced absolute EFV level. In the three-vessel CAD patients with chronic coronary vascular inflammation and severe coronary Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 8 of 10 Page 8 of 10 Table 3  Factors associated with EFV level in three-vessel CAD EFV epicardial fat volume, CAD coronary artery disease, OR odds ratio, BMI body mass index, LV left ventricular, EF ejection fraction, CCC​ coronary collateral circulation * Patients were divided into lower (< 25 kg/m2) and higher (≥ 25 kg/m2) BMI groups # Patients were divided into groups according to their coronary atherosclerosis severity: mild (score = 3 vessels), moderate (3.5 ≤ score < 4 vessels), and severe (4 ≤ score ≤ 7 vessels) Variables Univariable analysis Multivariable analysis (univariable P < 0.05) OR (95% CI) P value OR (95% CI) P value Age 1.02 (0.99; 1.06) 0.163 Gender 0.70 (0.38; 1.19) 0.168 Smoking 1.21 (0.74; 1.98) 0.452 Hypertension 1.45 (0.81; 2.60) 0.211 Dyslipidemia 1.40 (0.75; 2.63) 0.294 Diabetes 1.26 (0.76; 2.07) 0.366 BMI* 3.60 (2.14; 6.05) < 0.001 3.40 (2.00; 5.78) < 0.001 CAD family history 0.66 (0.30; 1.45) 0.304 LV diameter 1.07 (1.00; 1.14) 0.025 1.06 (0.99; 1.13) 0.104 LVEF 1.00 (0.96; 1.05) 0.91 E/A 1.94 (0.98; 3.84) 0.058 Score of coronary atherosclerosis ­severity# 0.49 (0.27; 0.90) 0.022 0.49 (0.26; 0.93) 0.03 CCC status 0.91 (0.53; 1.55) 0.72 SYNTAX score 0.99 (0.97; 1.02) 0.57 had significantly higher epicardial FAI. Discussionh The change of epicar- dial fat composition in three-vessel CAD patients with more severe coronary atherosclerosis leads to an increase of epicardial FAI, which, in turn, leads to a significant reduction of EFV level when measuring CCTA images using threshold-based methods. This hypothesis is con- sistent with the results reported here in which three- vessel CAD patients with severe coronary atherosclerosis Conclusions Patients with three-vessel CAD have significantly higher EFV level than age-, gender- and BMI-matched normal controls. Furthermore, our study results show that there is an potential inverse relationship between EFV level and coronary atherosclerosis severity in three-vessel CAD. Therefore, the value of clinical applications of the current study may be that a fall in EFV level on CCTA images during follow-up of patients with three-vessel CAD is a potential imaging biomarker of disease progres- sion, which will contribute to the clinical management of three-vessel CAD. However, future larger-scale studies are required for the validation of our findings. Abbreviations BMI: Body mass index; BSA: Body surface area; CAD: Coronary artery disease; CCTA​: Coronary computed tomography angiography; EF: Ejection fraction; EFV: Epicardial fat volume; FAI: Fat attenuation index; HU: Hounsfield units; ICA: Invasive coronary angiography; LV: Left ventricular. Abbreviations Abbreviations BMI: Body mass index; BSA: Body surface area; CAD: Coronary artery disease; CCTA​: Coronary computed tomography angiography; EF: Ejection fraction; EFV: Epicardial fat volume; FAI: Fat attenuation index; HU: Hounsfield units; ICA: Invasive coronary angiography; LV: Left ventricular. Declarations 11. Gorter PM, Vos AMD, Graaf YVD, Stella PR, Doevendans PA, Meijs MFL, et al. Relation of epicardial and pericoronary fat to coronary athero‑ sclerosis and coronary artery calcium in patients undergoing coronary angiography. Am J Cardiol. 2008;102:380–5. Acknowledgements Not applicable. Acknowledgements Not applicable. Page 9 of 10 Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Availability of data and materials 10. Mancio J, Azevedo D, Saraiva F, Azevedo AI, Pires-Morais G, Leite-Moreira A, et al. Epicardial adipose tissue volume assessed by computed tomog‑ raphy and coronary artery disease: a systematic review and meta-analysis. Eur Heart J Cardiovasc Imaging. 2018;19:490–7. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Funding Thi d This study is supported by the National Natural Science Foundation of China (61773110), the Key Research and Development Program of Liaoning Prov‑ ince, China (2018225024). The funders play an important role in study design, data analysis, and preparation of the manuscript. 9. Yu WJ, Liu B, Zhang FF, Wang JF, Shao XL, Yang XY, et al. Association of epicardial fat volume with increased risk of obstructive coronary artery disease in Chinese patients with suspected coronary artery disease. J Am Heart Assoc. 2021;10:e018080. Author details 1 College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, People’s Republic of China. 2 Department of Radiology, General Hospital of Northern Theater Command, 83 Wenhua RoadLiaoning Province, Shenyang 110016, People’s Republic of China. 3 Key Laboratory of Cardiovascular Imaging and Research of Liaoning Province, Shenyang, People’s Republic of China. 4 Department of Cardiology, General Hospital of Northern Theater Command, Shenyang, People’s Republic of China. 5 Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK. 15. Camarena V, Sant D, Mohseni M, Salerno T, Zaleski ML, Wang G, et al. Novel atherogenic pathways from the differential transcriptome analysis of diabetic epicardial adipose tissue. Nutr Metab Cardiovasc Dis. 2017;27:739–50. 16. Margaritis M, Antonopoulos AS, Digby J, Lee R, Reilly S, Coutinho P, et al. Interaction between vascular wall and perivascular adipose tissue reveal novel roles for adiponectin in the regulation of endothelial nitric oxide synthase function in human vessels. Circulation. 2013;127:2209–21. synthase function in human vessels. Circulation. 2013;127:2209–2 17. Antonopoulos AS, Sanna F, Sabharwal N, Thomas S, Oikonomou EK, Herdman L, et al. Detecting human coronary inflammation by imaging perivascular fat. Sci Transl Med. 2017;9:eaal2658. Received: 8 December 2021 Accepted: 24 February 2022 Received: 8 December 2021 Accepted: 24 February 2022 18. Oikonomou EK, Antoniades C. The role of adipose tissue in cardiovascular health and disease. Nat Rev Cardiol. 2019;16:83–99. 19. Mancio J, Oikonomou EK, Antoniades C. Perivascular adipose tissue and coronary atherosclerosis. Heart. 2018;104:1654–62. 20. Liu ZH, Wang SJ, Wang YQ, Zhou NB, Shu J, Stamm C, et al. Association of epicardial adipose tissue attenuation with coronary atherosclerosis in patients with a high risk of coronary artery disease. Atherosclerosis. 2019;284:230–6. Consent for publication Not applicable. 14. Scanlon PJ, Faxon DP, Audet AM, Carabello B, Dehmer GJ, Eagle KA, et al. ACC/AHA guidelines for coronary angiography. A report of the American College of Cardiology/American Heart Association Task Force on practice guidelines (Committee on Coronary Angiography). Developed in col‑ laboration with the Society for Cardiac Angiography and Interventions. J Am Coll Cardiol. 1999;33:1756–824. Competing interests The authors declare that they have no competing interests. Authors’ contributions 5. Maron DJ, Hochman JS, Reynolds HR, Bangalore S, O’Brien SM, Boden WE, et al. Initial invasive or conservative strategy for stable coronary disease. N Engl J Med. 2020;382:1395–407. YS collected and analyzed the study population data, and was a major contributor in writing the manuscript. XGL contributed to the development of deep learning model used in this study and the interpretation of data. KX contributed to the design of this study and the analysis of data. JH, and MQ contributed to the collection and interpretation of data. HRY, RRZ, LBZ, LSX, and SEG contributed to the development of deep learning model. BQY contributed to the conception of the work and analysis of data, and was a major contributor in revising the manuscript. All authors read and approved the final manuscript. 6. Mazurek T, Opolski G. Pericoronary adipose tissue: a novel therapeu‑ tic target in obesity-related coronary atherosclerosis. J Am Coll Nutr. 2015;34:244–54. 7. Lu MT, Park J, Ghemigian K, Mayrhofer T, Puchner SB, Liu T, et al. Epicardial and paracardial adipose tissue volume and attenuation—association with high-risk coronary plaque on computed tomographic angiography in the ROMICAT II trial. Atherosclerosis. 2016;251:47–54. in the ROMICAT II trial. Atherosclerosis. 2016;251:47–54. 8. Thanassoulis G, Massaro JM, Hoffmann U, Mahabadi AA, Vasan RS, O’Donnell CJ, et al. Prevalence, distribution, and risk factor correlates of high pericardial and intrathoracic fat depots in the Framingham heart study. Circ Cardiovasc Imaging. 2010;3:559–66. Ethics approval and consent to participate The study protocol was approved by the ethics committee of the General Hospital of Northern Theater Command (Y [2021] 012) and the study com‑ plied with the Declaration of Helsinki. Informed consent was waived by the committee because of the retrospective nature of this study. 12. Li XG, Sun Y, Xu LS, Greenwald SE, Zhang LB, Zhang RR, et al. Auto‑ matic quantification of epicardial adipose tissue volume. Med Phys. 2021;48:4279–90. 13. Mosteller RD. Simplified calculation of body-surface area. N Engl J Med. 1987;317:1098. Consent for publication Not applicable. 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The effect of moderate-dose versus double-dose statins on patients with acute coronary syndrome in China: results of the CHILLAS trial. Atherosclerosis. 2014;233:707–12. 26. Zhao SP, Yu BL, Peng DQ, Huo Y. The effect of moderate-dose versus double-dose statins on patients with acute coronary syndrome in China: results of the CHILLAS trial. Atherosclerosis. 2014;233:707–12. References 1. Serruys PW, Kogame N, Katagiri Y, Modolo R, Onuma Y. Clinical outcomes of state-of-the-art percutaneous coronary revascularisation in patients with three-vessel disease: two-year follow-up of the SYNTAX II study. EuroIntervention. 2019;15:e244–52. 1. Serruys PW, Kogame N, Katagiri Y, Modolo R, Onuma Y. Clinical outcomes of state-of-the-art percutaneous coronary revascularisation in patients with three-vessel disease: two-year follow-up of the SYNTAX II study. EuroIntervention. 2019;15:e244–52. 21. Hedgire S, Baliyan V, Zucker EJ, Bittner DO, Staziaki PV, Takx RAP, et al. 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Makikallio T, Holm NR, Lindsay M, Spence MS, Erglis PA, Menown IBA, et al. Percutaneous coronary angioplasty versus coronary artery bypass grafting in treatment of unprotected left main stenosis (NOBLE): a prospective, randomized, open-label, non-inferiority trial. Lancet. 2016;388:2743–52. 23. Gorter PM, van Lindert AS, de Vos AM, Meijs MFL, van der Graaf Y, Doevendans PA, et al. Quantification of epicardial and peri-coronary fat using cardiac computed tomography; reproducibility and relation with obesity and metabolic syndrome in patients suspected of coronary artery disease. Atherosclerosis. 2008;197:896–903. 4. Hueb WA, Lopes N, Gersh BJ, Soares PR, Ribeiro EE, Pereira AC, et al. Ten-year follow-up survival of the medicine, angioplasty, or surgery study (MASS II): a randomized controlled clinical trial of 3 therapeutic strategies for multivessel coronary artery disease. Circulation. 2010;122:949–57. 24. Cavalcante JL, Tamarappoo BK, Hachamovitch R, Kwon DH, Alraies MC, Halliburton S, et al. Association of epicardial fat, hypertension, subclinical Page 10 of 10 Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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https://openalex.org/W3012964330
https://uu.diva-portal.org/smash/get/diva2:1443715/FULLTEXT01
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Two-Step Size-Exclusion Nanofiltration of Prothrombin Complex Concentrate Using Nanocellulose-Based Filter Paper
Biomedicines
2,020
cc-by
13,038
Received: 29 February 2020; Accepted: 21 March 2020; Published: 26 March 2020 Abstract: Coagulation Factor IX-rich protrhombin complex concentrate (FIX-PCC) is a therapeutic biologic product that consists of a mixture of several human plasma-derived proteins, useful for treating hemophilia B. Due to its complex composition, FIX-PCC is very challenging to bioprocess through virus removing nanofilters in order to ensure its biosafety. This article describes a two-step filtration process of FIX-PCC using a nanocellulose-based filter paper with tailored porosity. The filters were characterized with scanning electron microscopy (SEM), cryoporometry with differential scanning calorimetry, and nitrogen gas sorption. Furthermore, in order to probe the filter’s cut-offsize rejection threshold, removal of small- and large-size model viruses, i.e., ΦX174 (28 nm) and PR772 (70 nm), was evaluated. The feed, pre-filtrate, and permeate solutions were characterized with mass-spectrometric proteomic analysis, dynamic light scattering (DLS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analytical size-exclusion high-performance liquid chromatography (SEHPLC). By sequential filtration through 11 µm pre-filter and 33 µm virus removal filter paper, it was possible to achieve high product throughput and high virus removal capacity. The presented approach could potentially be applied for bioprocessing other protein-based drugs. Keywords: Mille-feuille filter; Cladophora cellulose; hemophilia B; protein aggregates; virus removal filtration biomedicines biomedicines Levon Manukyan 1 , Athanasios Mantas 1 , Mikhail Razumikhin 2, Andrey Katalevsky 3, Eugen Golubev 4 and Albert Mihranyan 1,* Levon Manukyan 1 , Athanasios Mantas 1 , Mikhail Razumikhin 2, Andrey Katalevsky 3 Eugen Golubev 4 and Albert Mihranyan 1,* 1 Nanotechnology and Functional Materials, Department of Materials Science and Engineering, Uppsala University, Box 534, 751 21 Uppsala, Sweden; levon.manukyan@angstrom.uu.se (L.M.); athanasios.mantas@angstrom.uu.se (A.M.) 2 Nacimbio JC, 10, 2nd Volkonsky lane, 127473 Moscow, Russia; razumikhin.m@gmail.com 3 Biopharmgarant LLC, Stancionnaya 45, 600901 Vladimir, Russia; andreyalexandr2003@yandex.ru 4 National Research Center for Hematology, Novyi Zykovskiy proezd 4, 125167 Moscow, Russia; ev-genius@bk.ru 2 Nacimbio JC, 10, 2nd Volkonsky lane, 127473 Moscow, Russia; razumikhin.m@gmail.com 3 Biopharmgarant LLC, Stancionnaya 45, 600901 Vladimir, Russia; andreyalexandr2003@yandex.ru 4 National Research Center for Hematology, Novyi Zykovskiy proezd 4, 125167 Moscow, Russia; ev-genius@bk.ru g * Correspondence: albert.mihranyan@angstrom.uu.se; Tel.: +46-18-471-7940 g * Correspondence: albert.mihranyan@angstrom.uu.se; Tel.: +46-18-471-7940 biomedicines biomedicines 1. Introduction p p gg g It is known that the levels of different impurities are highly dependent on the type of chromatographic separation that was used during plasma treatment [3,18,20]. The most extensively described impurities in vitamin K-dependent clotting factors include inter-α-trypsin-inhibitor (ITI), complement 4b binding protein (C4BP), and vitronectin (VN). C4BP is a large glycoprotein of 570 kDa [21] in the shape of an octopus that consists of seven α-chains connected to a single β-chain by disulfide bonds [22–24]. The α-chains are responsible for binding C4b, while the β-chain has high-affinity for VN (protein S), forming large complexes [22,25]. The molecular conformation of C4BP is highly dependent on the surrounding medium composition [11,19]. In the charged state the α-chains repel each other, thereby occupying much larger volume than the same molecule in the uncharged state. In its open structure form, C4BP would have a diameter of approximately 66 nm since each α-chain arm is 33 nm long [11,22]. Varying salt concentrations have been shown to affect the compactness of C4BP molecule and thereby the flux properties of the virus removal filter [19]. Another large Mw impurity present often in FIX products is ITI. ITI (225 kDa) is a large complex that consists of one light and several heavy chains (H1-H3) covalently linked by a chondroitin sulfate chain. The heavy chains of ITI proteins function as hyaluronic acid (HA) binding proteins, whereas the light chain, also called bikunin, functions as a serine protease inhibitor upon activation [26]. The third extensively described impurity is VN [27]. The monomeric form of VN has a cryptic hydrophobic pocket, which upon exposure and conformational changes exhibits heparin- and C5b-7 binding activity [28]. Normally, only 2% of VN in plasma shows heparin-binding activity but its fraction increases manifold during coagulation [29]. VN also presents a free thiol group capable of disulfide bonding [30]. When unfolded VN is highly prone to polymerization and may form aggregates with Mw up to 1000 kDa [28]. Studies on nanofiltration of FIX products where VN aggregates were detectable confirmed its role as a filter foulant [27]. Extensive coverage of various impurities at different intermediate stages during FIX manufacturing is discussed elsewhere [15,18]. So far successful virus removal filtration of FIX products has been described in the literature only for a limited number of commercial filters. 1. Introduction Replacement therapy using plasma-derived Factor IX (FIX) products is a life-saving treatment for patients with hemophilia B. Both recombinant and plasma-derived FIX show high efficacy in clinical trials [1]. Production of FIX normally involves multiple steps. High purity FIX is obtained from prothrombin complex concentrate (PCC), which is a mixture of vitamin K-dependent clotting factors, e.g., factor II (prothrombin), V, VII, IX, and X, and clotting inhibitors, e.g., protein C, Z, and S [2]. PCC preparation is a highly complex mixture of proteins and may contain up to 50% of proteins other than FIX [3]. Both highly purified FIX and PCC can be used for hemophilia B treatment [4]. Also, PCC preparation may be useful for prevention of bleeding due to overdose of oral anticoagulants and liver dysfunctions [2,3]. Biomedicines 2020, 8, 69; doi:10.3390/biomedicines8040069 www.mdpi.com/journal/biomedicines www.mdpi.com/journal/biomedicines 2 of 18 Biomedicines 2020, 8, 69 As it is with all plasma-derived products, the viral safety of FIX-rich PCC is a critical issue [5]. According to current regulations, at least two orthogonal virus clearance steps must be implemented to ensure viral safety of the final product [6]. The steps to mitigate virus contamination of FIX and PCC products include donor screening for known blood-borne viruses, i.e., HIV 1–2, HBV, HCV, HAV, and parvo B19; virus inactivation, such as solvent/detergent, mixed chemical inactivation (tri-n-butyl phosphate) and detergent (nonionic, polysorbate, and polyethylene oxide) treatment; and incubating intermediate product in controlled temperature (usually 6 h at 25 ◦C) [3,7,8]. Dry heat treatment may also be used after lyophilization, e.g., 100 ◦C for 1 h or 80 ◦C for 72 h. In the past, steam treatment at 60 ◦C (190 mbar) for 10 h or 80 ◦C (375 mbar) for 1 h was reported [3,7,8]. Presently, virus removal nanofiltration has become widely used as a robust and reliable method for ensuring viral safety. Nanofiltration is attractive because it is capable of physically removing all types of viruses from protein solution as opposed to virus inactivation. Several authors have described the application of virus removal nanofiltration for FIX industrial products using Planova 15/20/35N [9–15], Viresolve NFP [11,16–19], and Ultipor DV50 filters [2]. The conclusion of these studies is that filtration of plasma-derived FIX-rich products is challenging due to the presence of large molecular weight impurities and protein aggregates. 2.4. Filtration Setup Pre-filtration and filtration steps were performed in a 47 mm diameter Advantech KST 47 filter holder. Prior to filtration, the pre- and filter papers were wetted in order to extrude the air by running 20 mL of PBS. The pre-filtration steps with 6 and 11 µm pre-filters were performed at 1 bar transmembrane pressure, and the filtrations with 33 µm filters were carried out at 1 or 3 bar. The permeate solutions were collected and for filtrations of the larger volume, permeate was collected in one or three fractions and saved. 2.1. Materials Cladophora cellulose was provided by FMC Biopolymer (batch 3095-10; Newark, DE, USA). FIX-rich PCC was provided by National Center for Hematology, Moscow, Russia, as lyophilized powder. Coliphages ΦX174 (ATCC 13706-B1™) and PR772 (BAA-769-B1), and the host bacteria Escherichia coli (Migula) Castellani and Chalmers C (ATCC 13706) and K12 J53-1(R15) [HER 1221] (BAA-769) strains were obtained from ATCC (Manassas, VA, USA). Agar (214530) was obtained from BD (Franklin Lakes, NJ, USA). Tryptone (LP0042B) and yeast extract (Oxoid) (LP0021) were obtained from Thermo Fisher Scientific. Phosphate-buffered saline (P4417), 2-mercaptoethanol (M3148), sodium chloride (S5886), sodium phosphate dibasic (71640) and 2-mercaptoethanol (M3148) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Any kD™Mini-PROTEAN® TGX Stain-Free™ protein gels (4568125), tris/glycine/SDS running buffer (1610732), 4x Laemmli Sample Buffer (1610747), and Precision Plus Protein™unstained protein standards (1610363) were purchased from Bio-Rad (Hercules, CA, USA). 2.2. Filter Preparation Filters of different thickness were prepared from Cladophora cellulose dispersion (0.1 wt.%) made by microfluidization with 200 µm (twice) and 100 µm hole sized chambers at 1800 bar using LM20 Microfluidizer (Microfluidics, Westwood, MA, USA). Furthermore, the wet cake was made by draining the dispersion over a membrane (Durapore, 0.65 µm hydrophilic PVDF “DVPP”, Merck Millipore, Burlington, MA, USA) in a funnel, driven by vacuum. Obtained cellulose cakes were dried at 140 ◦C to produce pre-filters and 80 ◦C for filter papers using hot-press (Carver Model 4122CE, Carver, Wabash, IN, USA). 1. Introduction A novel type of virus removal filter paper was developed at Uppsala University, which is produced by adapting traditional paper making technology and consists of 100% cellulose nanofibers [31,32]. The pore size and flux properties of the filter paper can be controlled, which opens new opportunities to model fundamental aspects of bioprocessing [32–34]. The filter paper was previously validated in numerous studies to remove several large and small-size model viruses, including retroviruses (xMuLV, 100 nm) [35], parvoviruses (MVM, 20 nm) [32,36], and model phages (ΦX174, 28 nm) [37–39]. Recently, it was shown that this nanocellulose-based virus removal filter paper is useful for bioprocessing human plasma-derived IgG [40]. 3 of 18 Biomedicines 2020, 8, 69 In this article, for the first time the filtration of FIX-rich PCC using a nanocellulose-based virus removal filter paper is described. Furthermore, a two-step size-exclusion nanofiltration process is developed to remove foulants and ensure efficient virus removal filtration of FIX-rich PCC using nanocellulose-based virus removal filter paper. FIX-rich PCC was used as a model for a highly complex plasma-derived product to simulate industrial bioprocesses where impurities may greatly affect product yield and biosafety. 2.3. Dissolution of Factor IX-rich PCC Lyophilized FIX-rich PCC samples were reconstituted by dissolving in phosphate-buffered saline (PBS). No visible particles could be seen after reconstitution, and the solution was clear and transparent. Upon dissolution, the conductivity and pH values were 15.4 mS cm−1 and 7.4, respectively. 2.7. Dynamic Light Scattering Particle size distribution was obtained from dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, UK) instrument. All experiments were performed in triplicates. 2.8. Polyacrylamide Gel Electrophoresis 2.9. Analytical SEHPLC Samples were analyzed by size-exclusion high-pressure liquid chromatography using Hitachi Chromaster HPLC-UV system with bioZen 1.8 µm SEC-3 (Phenomenex, Torrance, CA, USA) analytical column. Chromatography was performed with 100 mM sodium phosphate, pH 6.8 mobile phase at 0.3 mL min−1 flow rate for 20 min. 2.5. Scanning Electron Microscopy (SEM) For top-view images samples were fixed onto aluminum stubs with double-adhesive carbon tape, and for cross-section images the samples were mounted onto aluminum sample holders with screw. Imaging was performed using Zeiss Merlin FEG-SEM instrument (Jena, Germany). To reduce charging 4 of 18 Biomedicines 2020, 8, 69 effects samples were sputtered with Au/Pd with a sputter coater (Polaron, Ashford, UK) was used. The sputtering settings were 4 × 102 mbar and 35 mA, and the sputtering time was 30 s. 2.6. Cryoporometry by Differential Scanning Calorimetry 2.6. Cryoporometry by Differential Scanning Calorimetry 2.8. Polyacrylamide Gel Electrophoresis Protein separation was performed by reducing polyacrylamide gel electrophoresis (SDS-PAGE). Samples were diluted (1:20 v/v) with PBS and Laemmli buffer, and boiled for 10 min. Electrophoretic separation was carried out at 270 V with Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, Hercules, CA, USA). Protein bands were detected with Gel Doc™EZ System (Bio-Rad, Hercules, CA, USA), and quantified using Image Lab 6.0 analysis software (Bio-Rad). 2.6. Cryoporometry by Differential Scanning Calorimetry Filter paper samples (1.5–2 mg) were soaked into deionized water overnight at room temperature. Water was decanted, and the samples were placed in aluminum crucibles with a lid. Samples were cooled down to 248.15 K (−25 ◦C) at a rate of 10 K min−1 followed by heating to 277.15 K (4 ◦C) at a rate of 0.7 K min−1. Measurements were performed in five replicates. The pore size was calculated according to Landry [41]: The pore size was calculated according to Landry [41]: ∆T = −19.082 rp −1.12 −0.1207 (1) (1) where rp is the radius of pore (nm) and ∆T is the difference between the peak maximum for melting of pore-confined water and peak value for melting of bulk water, experimentally determined at 0.6 ± 0.01 ◦C. where rp is the radius of pore (nm) and ∆T is the difference between the peak maximum for melting of pore-confined water and peak value for melting of bulk water, experimentally determined at 0.6 ± 0.01 ◦C. 2.10. LCMS Equal amounts (20 µg) of protein samples were taken out for digestion. After reduction and alkylation, the proteins were on-filter digested by trypsin using 3 kDa centrifugal filters (Millipore Tullagreen, Ireland) according to a standard operating procedure. Obtained peptides were dried using a speedvac system. Pellets were resolved in 60 µL of 0.1% formic acid and further diluted four times prior to nano-LCMS/MS. Tandem mass spectrometry was performed by applying HCD in the QEx-Orbitrap mass spectrometer (Thermo Finnigan, San Jose, CA, USA), equipped with a reversed-phase C18-column by 35 min long gradient. Database searches were performed using the Sequest algorithm, embedded in Proteome Discoverer 1.4 (Thermo Fisher Scientific, Waltham, MA, USA) against Homo Sapience proteome extracted from Uniprot, Release June 2019 with 95% confidence level per protein. 2.11. Bacteriophage Filtration and Titration 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC When the FIX-rich PCC at 20 L m−2 volumetric load was filtered through the 33 µm mille-feuille filter paper, a rapid flux decline was observed, e.g., from about 80 L m−2 h−1 to about 10 L m−2 h−1 at 3 bar overhead pressure. DLS analysis of the feed and permeate samples revealed that the feed sample showed widely distributed fraction of protein impurities above 70 nm, which were not detectable after filtration as shown in Figure 1. Notably, these large-size impurities could not be detected in the volume distribution profiles of the feed sample but only in the intensity distribution plots, which suggests that the original amount of the aggregates is small. In the permeate sample, no particle fractions above 40 nm were detected by DLS. When the FIX-rich PCC at 20 L m−2 volumetric load was filtered through the 33 μm mille-feuille filter paper, a rapid flux decline was observed, e.g., from about 80 L m−2 h−1 to about 10 L m−2 h−1 at 3 bar overhead pressure. DLS analysis of the feed and permeate samples revealed that the feed sample showed widely distributed fraction of protein impurities above 70 nm, which were not detectable after filtration as shown in Figure 1. Notably, these large-size impurities could not be detected in the volume distribution profiles of the feed sample but only in the intensity distribution plots, which suggests that the original amount of the aggregates is small. In the permeate sample, no particle fractions above 40 nm were detected by DLS. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 μm filter paper at 1 and 3 bar. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 µm filter paper at 1 and 3 bar. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 μm filter paper at 1 and 3 bar. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 µm filter paper at 1 and 3 bar. To investigate if significant changes were recorded in the protein molecular weight distribution in the permeate sample, SDS-PAGE analysis was performed, as shown in Figure 2. 2.11. Bacteriophage Filtration and Titration Coliphages PR772 and ΦX174 were spiked to the pre-filtered solutions to obtain final titer about 106 plaque forming units (PFU) mL−1 before filtration was performed. Bacteriophage titer was determined by PFU assay by double agar overlay method. Briefly, ten-fold serially diluted bacteriophage samples 5 of 18 Biomedicines 2020, 8, 69 were mixed with host E. coli strains and melted soft agar, and poured on the surface of prepared hard agar plate, followed by incubation at 37 ◦C for 5 h. Biomedicines 2020, 8, x FOR PEER REVIEW 5 of 18 were mixed with host E. coli strains and melted soft agar, and poured on the surface of prepared hard agar plate, followed by incubation at 37 ◦C for 5 h. Biomedicines 2020, 8, x FOR PEER REVIEW 5 of 18 p y Bacteriophage titer was calculated using Equation (1): B t i h tit l l t d i ti 1 Bacteriophage titer was calculated using Equation (1): log10  PFU mL−1 = log10  N V·d  (2) ulated using equation 1: 𝑜𝑔10(𝑃𝐹𝑈 𝑚𝑙−1) = 𝑙𝑜𝑔10 ( 𝑁 V ∙d ) (2) (2) (2) where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the dilution factor. where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the dilution factor where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the dilution factor. where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the dilution factor The virus retention was expressed as log10 reduction value (LRV): dilution factor. The virus retention was expressed as log10 reduction value (LRV): The virus retention was expressed as log10 reduction value (LRV): dilution factor. The virus retention was expressed as log10 reduction value (LRV): LRV = log10 PFUfeed PFUpermeate (3) 𝐿𝑅𝑉= 𝑙𝑜𝑔10 𝑃𝐹𝑈𝑓𝑒𝑒𝑑 𝑃𝐹𝑈𝑝𝑒𝑟𝑚𝑒𝑎𝑡𝑒 (3). (3) 3). 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC Additional proteomics analysis of the detected bands was not performed as it was outside of the scope of the present work. It is seen in Figure 2 that all major fractions in the permeate sample were reduced compared to the feed. The observed decrease in total protein fraction is concordant to that reported earlier for PCC product filtered through Planova 15N filter [14]. In all, it appears that the large molecular weight protein fractions are the main reason for the observed fouling. To investigate if significant changes were recorded in the protein molecular weight distribution in the permeate sample, SDS-PAGE analysis was performed, as shown in Figure 2. Additional proteomics analysis of the detected bands was not performed as it was outside of the scope of the present work. It is seen in Figure 2 that all major fractions in the permeate sample were reduced compared to the feed. The observed decrease in total protein fraction is concordant to that reported earlier for PCC product filtered through Planova 15N filter [14]. In all, it appears that the large molecular weight protein fractions are the main reason for the observed fouling. 6 of 18 6 of 18 Biomedicines 2020, 8, 69 Biomedicines 2020, 8, x FO Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper kDa 250 150 100 75 50 37 25 20 15 10 Feed Feed Permeate Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 Permeate 1 bar 3 bar Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 µm filter paper at 1 and 3 bar. Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper at 1 and 3 bar. kDa 250 150 100 75 50 37 25 20 15 10 Feed Feed Perme Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 Perme 1 bar 3 bar Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 µm filter paper at 1 and 3 bar. Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper at 1 and 3 bar. at 1 and 3 bar. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC Development and Validation of Two-Step Size-Exclusion Bioprocess for FIX-Rich PCC Nanofiltration Development and Validation of Two-Step Size-Exclusion Bioprocess for FIX-Rich PCC Nanofiltration 3.2. Development and Validation of Two-Step Size-Exclusion Bioprocess for FIX-Rich PCC Nanofiltration The nanocellulose-based filter paper platform provides the possibilities to relatively easily tailo the pore size distribution of the filter paper to a specific cut off value This could be achieved fo The nanocellulose-based filter paper platform provides the possibilities to relatively easily tailor the pore-size distribution of the filter paper to a specific cut-offvalue. This could be achieved for instance by varying the thickness of the filter paper. The nanocellulose-based filter paper platform provides the possibilities to relatively easily tailor the pore-size distribution of the filter paper to a specific cut-off value. This could be achieved for instance by varying the thickness of the filter paper. the pore-size distribution of the filter paper to a specific cut-off value. This could be achieved for instance by varying the thickness of the filter paper. Figure 3 shows the SEM images of the filters with varying thickness, including their top-view and cross-section It is seen from the images of the cross-sections of the filters that they indeed feature Figure 3 shows the SEM images of the filters with varying thickness, including their top-view and cross-section. It is seen from the images of the cross-sections of the filters that they indeed feature varying thicknesses. y y g p p Figure 3 shows the SEM images of the filters with varying thickness, including their top-view and cross-section. It is seen from the images of the cross-sections of the filters that they indeed feature varying thicknesses. varying thicknesses. varying thicknesses. Figure 3. SEM images of top-view and cross-sections of 6, 11, and 33 μm filter papers. Figure 3. SEM images of top-view and cross-sections of 6, 11, and 33 µm filter papers. Figure 3. SEM images of top-view and cross-sections of 6, 11, and 33 µm filter papers. 7 of 18 Biomedicines 2020, 8, 69 To derive information about the pore size of the filter cryoporometry analysis was performed. Figure 4 shows the typical CP-DSC curves of the studied samples and the boxplots of the derived pore width modes. Cryoporometry analysis has the benefit that it probes the pores in the wet state, and it is a relatively quick and highly automated and reliable method. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC In this method, the samples are first frozen to −40 ◦C and then slowly thawed. As the ice crystals start to melt, there is a detectable endotherm peak. When the water is located inside mesopores (i.e., 2–50 nm pore width), there will be a melting point depression as opposed to bulk water, present outside pores or in macropores (above 50 nm). In our experiments, bulk water melts at around 0.6 ◦C. The larger the melting point depression, the smaller are the pores. As seen from Figure 4 there is a trend of decreasing pore width mode with increasing thickness. Biomedicines 2020, 8, x FOR PEER REVIEW 7 of 18 To derive information about the pore size of the filter cryoporometry analysis was performed. Figure 4 shows the typical CP-DSC curves of the studied samples and the boxplots of the derived pore width modes. Cryoporometry analysis has the benefit that it probes the pores in the wet state, and it is a relatively quick and highly automated and reliable method. In this method, the samples are first frozen to −40 °C and then slowly thawed. As the ice crystals start to melt, there is a detectable endotherm peak. When the water is located inside mesopores (i.e., 2–50 nm pore width), there will be a melting point depression as opposed to bulk water, present outside pores or in macropores (above 50 nm). In our experiments, bulk water melts at around 0.6 °C. The larger the melting point depression, the smaller are the pores. As seen from Figure 4 there is a trend of decreasing pore width mode with increasing thickness. Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and 33 μm filter papers (n = 7). * p < 0.05, *** p < 0.01 Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and 33 µm filter papers (n = 7). * p < 0.05, *** p < 0.01. Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and 33 μm filter papers (n = 7). * p < 0.05, *** p < 0.01 Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and 33 µm filter papers (n = 7). * p < 0.05, *** p < 0.01. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC To assess the particle rejection cut-off for each filter, model probes with 2 different particle sizes were used in the form of bacteriophages, i.e., PR772 (70 nm) and ΦX174 (28 nm) phages, see Table 1 and 2, respectively. These probes provide a highly sensitive tool for assessing the size-dependent rejection capability of the filters with varying thickness, i.e., 6, 11, and 33 μm. The 33 μm mille-feuille filter paper shows the lowest hydraulic flux, i.e., 38 L m−2 h−1 bar−1, and the highest virus removal capacity for both small- and large-size viruses, i.e., LRV ≥5.7. The 6 μm filter in the series exhibits the fastest flux, i.e., 405 L m−2 h−1 bar−1, but poor virus removal capacity, i.e., LRV <1 and <2 for 28 nm and 70 nm model phages. The flux and virus removal properties of 11 µm filter are intermediate to the other two filters, wherein the 11 µm filter paper shows high clearance towards 70 nm virus, i.e., >5.7, and moderate clearance toward 28 nm one, i.e., LRV 3.5–4.5, and hydraulic flux of 125 L m−2 h−1 bar−1. Interestingly, the small-size virus removal capacity of 11 µm filter decreased with increasing load volume, whereas that of 33 µm filter remained unaffected under the experimental conditions. The latter could probably be due to redistribution of flow through the larger pores when the smaller pores become clogged in 11 µm filter paper. To assess the particle rejection cut-offfor each filter, model probes with 2 different particle sizes were used in the form of bacteriophages, i.e., PR772 (70 nm) and ΦX174 (28 nm) phages, see Tables 1 and 2, respectively. These probes provide a highly sensitive tool for assessing the size-dependent rejection capability of the filters with varying thickness, i.e., 6, 11, and 33 µm. The 33 µm mille-feuille filter paper shows the lowest hydraulic flux, i.e., 38 L m−2 h−1 bar−1, and the highest virus removal capacity for both small- and large-size viruses, i.e., LRV ≥5.7. The 6 µm filter in the series exhibits the fastest flux, i.e., 405 L m−2 h−1 bar−1, but poor virus removal capacity, i.e., LRV <1 and <2 for 28 nm and 70 nm model phages. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC The flux and virus removal properties of 11 µm filter are intermediate to the other two filters, wherein the 11 µm filter paper shows high clearance towards 70 nm virus, i.e., >5.7, and moderate clearance toward 28 nm one, i.e., LRV 3.5–4.5, and hydraulic flux of 125 L m−2 h−1 bar−1. Interestingly, the small-size virus removal capacity of 11 µm filter decreased with increasing load volume, whereas that of 33 µm filter remained unaffected under the experimental conditions. The latter could probably be due to redistribution of flow through the larger pores when the smaller pores become clogged in 11 µm filter paper. 8 of 18 Biomedicines 2020, 8, 69 Table 1. LRVs for 70 nm (PR772) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus clearance (LRV < 1). Thickness (µm) Load Volume 7.5 L m−2 15 L m−2 23 L m−2 6 1.6 ± 0.2 1.3 ± 0.1 1.1 ± 0.3 11 >5.5 ± 0.2 >5.5 ± 0.2 >5.5 ± 0.2 33 >5.7 ± 0.2 >5.7 ± 0.2 >5.7 ± 0.2 Table 2. LRVs for 28 nm (ΦX174) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus edicines 2020, 8, x FOR PEER REVIEW 8 Table 2. LRVs for 28 nm (ΦΧ174) bacteriophages filtered through 6, 11, and 33 μm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus clearance LRV>5; Yellow denotes moderate virus clearance (2<LRV<5); and pink denotes low virus l (LRV 2) Table 2. LRVs for 28 nm (ΦX174) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus clearance (LRV < 2). Table 2. LRVs for 28 nm (ΦΧ174) bacteriophages filtered through 6, 11, and 33 μm filter papers. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus clearance LRV>5; Yellow denotes moderate virus clearance (2<LRV<5); and pink denotes low virus clearance (LRV <2). Table 2. LRVs for 28 nm (ΦX174) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus clearance (LRV < 2). Table 2. LRVs for 28 nm (ΦΧ174) bacteriophages filtered through 6, 11, and 33 μm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus clearance LRV>5; Yellow denotes moderate virus clearance (2<LRV<5); and pink denotes low virus clearance (LRV <2). Thickness (µm) Load Volume 7.5 L m−2 15 L m−2 23 L m−2 6 0.7 ± 0.3 0.8 ± 0.3 0.8 ± 0.2 11 4.5 ± 0.5 3.7 ± 0.4 3.5 ± 0.7 33 >5.7 ± 0.4 >5.7 ± 0.4 5.7 ± 0.4 3 3 Two-Step 6 µm/33 µm Filtration of FIX-Rich PCC Thickness (μm) Load Volume 7.5 L m−2 15 L m−2 23 L m−2 6 0.7 ± 0.3 0.8 ± 0.3 0.8 ± 0.2 11 4.5 ± 0.5 3.7 ± 0.4 3.5 ± 0.7 33 >5.7 ± 0.4 >5.7 ± 0.4 5.7 ± 0.4 3 3 Two-Step 6 μm/33 μm Filtration of FIX-Rich PCC 3.3. Two-Step 6 µm/33 µm Filtration of FIX-Rich PCC 3.3. Two-Step 6 μm/33 μm Filtration of FIX-Rich PCC Figure 5 shows the permeate flux through the 6 µm/33 µm filtration sequence at 1 bar. Rapid flux decline was observed for the permeate after initial plateau. Please note that the flux of pre-filtrate was so fast that it was not recorded. Figure 5 shows the permeate flux through the 6 μm/33 μm filtration sequence at 1 bar. Rapid flux decline was observed for the permeate after initial plateau. Please note that the flux of pre-filtrate was so fast that it was not recorded. Figure 5. Observed permeate flux for two-step 6 μm/33 μm μm filtration at 1 bar. Figure 5. Observed permeate flux for two-step 6 µm/33 µm µm filtration at 1 bar. Figure 5. Observed permeate flux for two-step 6 μm/33 μm μm filtration at 1 bar. Figure 5. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC Figure 7 shows the results of SDS-PAGE analysis of the collected samples. It is seen from graph that all bands showed decreasing intensity. Even after 6 μm pre-filtration, some decline i band intensity was observed. The bands for lower Mw fractions, i.e., bands 4–6, were reduced greater extent after pre-filtration than those of the larg Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration throu Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 6 µm/33 µm filter paper at 1 bar. Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration throu Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 6 µm/33 µm filter paper at 1 bar. μm/33 μm filter paper at 1 bar. Overall, the results from the filtration with 6 μm/33 μm sequence suggest that the large impurities were not removed during the pre-filtration step and, subsequently, caused filter fo d h b l d i ld d i h d Overall, the results from the filtration with 6 µm/33 µm sequence suggest that the large Mw impurities were not removed during the pre-filtration step and, subsequently, caused filter fouling and thereby low product yield during the second step. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC 3.1. One-Step 33 μm Filtration of FIX-Rich PCC Observed permeate flux for two-step 6 µm/33 µm µm filtration at 1 bar. Figure 6 shows the DLS results for pre-filtrate and permeate samples for the 6 μm/33 μm filtration sequence at 1 bar. It was observed the fraction of large colloids was not removed by 6 μm pre-filtration. However, no aggregates were observed in the permeate sample after filtration through f l Figure 6 shows the DLS results for pre-filtrate and permeate samples for the 6 µm/33 µm filtration sequence at 1 bar. It was observed the fraction of large colloids was not removed by 6 µm pre-filtration. However, no aggregates were observed in the permeate sample after filtration through 33 µm filter. 9 of 18 y μ hrough Biomedicines 2020, 8, 69 pre-filtration. Howe 33 filt 3 μm filter. Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS profiles of feed, pre-filtrate, and permeate samples after 2-step filtration through 11 µm/33 µm filter paper at 1 bar. Biomedicines 2020, 8, x FOR PEER REVIEW Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS prof of feed, pre-filtrate, and permeate samples after 2-step filtration through 11 μm/33 μm filter pape OR PEER REVIEW Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS profiles of feed, pre-filtrate, and permeate samples after 2-step filtration through 11 µm/33 µm filter paper at 1 bar. Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS pr of feed, pre-filtrate, and permeate samples after 2-step filtration through 11 μm/33 μm filter pap Figure 7 shows the results of SDS-PAGE analysis of the collected samples. It is seen from the graph that all bands showed decreasing intensity. Even after 6 µm pre-filtration, some decline in the band intensity was observed. The bands for lower Mw fractions, i.e., bands 4–6, were reduced to a greater extent after pre-filtration than those of the larger Mw, i.e., bands 1–3. In the permeate sample all band intensities were further decreased. LCMS analysis suggested that key coagulation factors IX, X, V as well as prothrombin were not removed following the two-step 6 µm/33 µm filtration sequence, as shown in Appendix Tables A1–A3. 1 bar. and thereby low product yield during the seco 3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC 3.4. Two-Step 11 μm/33 μm Filtration of FIX-Rich PCC In another set of experiments, the pre-filtration was performed using 11-μm filter paper follo by filtration with 33 μm filter at 1 bar. Figure 8 shows the flux data of permeate for 11 μm/33 In another set of experiments, the pre-filtration was performed using 11-µm filter paper followed by filtration with 33 µm filter at 1 bar. Figure 8 shows the flux data of permeate for 11 µm/33 µm filtration sequence. Increasing the thickness of the pre-filter from 6 to 11 µm significantly affected the 10 of 18 Biomedicines 2020, 8, 69 results. The flux values for pre-filtration indicated rapid fouling as observed above for 33 µm filtration. However, in the second step of 11 µm/33 µm filtration sequence, i.e., through 33 µm filters, stable flux was observed for the entire processed volume, Figure 7. The results contrast starkly those observed for 6 µm/33 µm filtration sequence as shown in Figure 4. Biomedicines 2020, 8, x FOR PEER REVIEW 10 of 18 Biomedicines 2020, 8, x FOR PEER REVIEW 10 of 18 Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 µm/33 µm µm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 µm/33 µm µm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 9 shows the results of DLS analysis of the pre-filtrate and permeate samples. It is seen that the fraction of large-size impurities, which was clearly visible in the feed solution, was absent both in the pre-filtrate and permeate fractions of 11 μm/33 μm filtration sequence. The latter suggests that pre-filtration with 11 μm filter paper efficiently removes the large-size impurities, unlike pre- filtration with 6 μm filter paper. Additional SEHPLC analysis was performed on these samples as shown in Figure 8. It is seen in the graph that the peak retention times and relative intensities are similar in all three samples except for the early peak at 0.5 min in the feed sample. and thereby low product yield during the seco 3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC This peak, which corresponds to the largest protein fraction was not detectable in pre-filtrate and permeate samples. Figure 9 shows the results of DLS analysis of the pre-filtrate and permeate samples. It is seen that the fraction of large-size impurities, which was clearly visible in the feed solution, was absent both in the pre-filtrate and permeate fractions of 11 µm/33 µm filtration sequence. The latter suggests that pre-filtration with 11 µm filter paper efficiently removes the large-size impurities, unlike pre-filtration with 6 µm filter paper. Additional SEHPLC analysis was performed on these samples as shown in Figure 8. It is seen in the graph that the peak retention times and relative intensities are similar in all three samples except for the early peak at 0.5 min in the feed sample. This peak, which corresponds to the largest protein fraction was not detectable in pre-filtrate and permeate samples. Figure 9 shows the results of DLS analysis of the pre-filtrate and permeate samples. It is seen that the fraction of large-size impurities, which was clearly visible in the feed solution, was absent both in the pre-filtrate and permeate fractions of 11 μm/33 μm filtration sequence. The latter suggests that pre-filtration with 11 μm filter paper efficiently removes the large-size impurities, unlike pre- filtration with 6 μm filter paper. Additional SEHPLC analysis was performed on these samples as shown in Figure 8. It is seen in the graph that the peak retention times and relative intensities are similar in all three samples except for the early peak at 0.5 min in the feed sample. This peak, which corresponds to the largest protein fraction was not detectable in pre-filtrate and permeate samples. Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step filtration through 11 µm/33 µm filter paper at 1 bar. Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step filtration through 11 µm/33 µm filter paper at 1 bar. and thereby low product yield during the seco 3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11 µm/33 µm filter paper at 1 bar. medicines 2020, 8, x FOR PEER REVIEW 11 of Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11 /33 filt t 1 b Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11 μm/33 μm filter paper at 1 bar. Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11 µm/33 µm filter paper at 1 bar. Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11 Based on the above results, it was concluded that pre-filtration with 11 µm pre-filter removes the aggregates, which in turn greatly enhances the yield of the 33 µm filtration. To confirm the high virus removal capacity of 33 µm filter, the filter paper was loaded with much larger volume than that tested earlier, i.e., 175 L m−2. Figure 11 shows the result of the large load filtration. Following the filtration, no abrupt filter fouling was detected for the entire processed volume, although some flux decline could be observed (Figure 11a). Under the experimental conditions, it is estimated that Vmax of the process will be roughly around 500 L m−2, which is a drastic improvement from 20 L m−2 when filtering in a single-step process through 33 µm filter paper. Furthermore, the filter paper showed high model small-size virus removal capacity, wherein LRV was ≥ 5 in all collected fractions (Figure 11b). In particular, no detectable PFUs were observed at all up to 90 L m−2 load volume. In the last fractions only residual breakthrough (1–2 PFUs per agar plate, corresponding to 0.7 PFU mL−1) was detected. Thus, it was confirmed that the two-step 11 µm/33 µm filtration provides enhanced throughput and good capacity to remove small-size virus without abrupt fouling even when challenged with a relatively large load. Based on the above results, it was concluded that pre-filtration with 11 µm pre-filter removes the aggregates, which in turn greatly enhances the yield of the 33 µm filtration. To confirm the high virus removal capacity of 33 µm filter, the filter paper was loaded with much larger volume than that tested earlier, i.e., 175 L m−2. Figure 11 shows the result of the large load filtration. and thereby low product yield during the seco 3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC Following the filtration, no abrupt filter fouling was detected for the entire processed volume, although some flux decline could be observed (Figure 11A). Under the experimental conditions, it is estimated that Vmax of the process will be roughly around 500 L m−2, which is a drastic improvement from 20 L m−2 when filtering in a single-step process through 33 µm filter paper. Furthermore, the filter paper showed high model small-size virus removal capacity, wherein LRV was ≥5 in all collected fractions (Figure 11B). In particular, no detectable PFUs were observed at all up to 90 L m−2 load volume. In the last fractions only residual breakthrough (1–2 PFUs per agar plate, corresponding to 0.7 PFU mL−1) was detected. Thus, it was confirmed that the two-step 11 µm/33 µm filtration provides enhanced throughput and good capacity to remove small-size virus without abrupt fouling even when challenged with a relatively large load. μ / μ p p Based on the above results, it was concluded that pre-filtration with 11 µm pre-filter removes the aggregates, which in turn greatly enhances the yield of the 33 µm filtration. To confirm the high virus removal capacity of 33 µm filter, the filter paper was loaded with much larger volume than that tested earlier, i.e., 175 L m−2. Figure 11 shows the result of the large load filtration. Following the filtration, no abrupt filter fouling was detected for the entire processed volume, although some flux decline could be observed (Figure 11a). Under the experimental conditions, it is estimated that Vmax of the process will be roughly around 500 L m−2, which is a drastic improvement from 20 L m−2 when filtering in a single-step process through 33 µm filter paper. Furthermore, the filter paper showed high model small-size virus removal capacity, wherein LRV was ≥ 5 in all collected fractions (Figure 11b). In particular, no detectable PFUs were observed at all up to 90 L m−2 load volume. In the last fractions only residual br Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2 load volume at 3 bar (A) and LRV for ΦX174 phage (B). Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2 load volume at 3 bar (A) and LRV for ΦX174 phage (B) Figure 11. and thereby low product yield during the seco 3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC filtration through 11 μm/33 μm filter paper at 1 bar. The results of the SDS-PAGE analysis for 11 μm /33 μm filtration sequence are summarized in Figure 10. It is seen from the graph that the band intensities were reduced in the pre-filtrate and permeate samples as compared to the feed. It should be noted that in general the band intensities were reduced to a greater extent after pre-filtration with 11 μm filter than with 6 μm filter. The decrease of band intensity levels in the permeate sample passed through the 33 μm filter after 11 μm filtration through 11 μm/33 μm filter paper at 1 bar. The results of the SDS-PAGE analysis for 11 μm /33 μm filtration sequence are summarized in Figure 10. It is seen from the graph that the band intensities were reduced in the pre-filtrate and permeate samples as compared to the feed. It should be noted that in general the band intensities were reduced to a greater extent after pre-filtration with 11 μm filter than with 6 μm filter. The decrease of band intensity levels in the permeate sample passed through the 33 μm filter after 11 μm The results of the SDS-PAGE analysis for 11 µm /33 µm filtration sequence are summarized in Figure 10. It is seen from the graph that the band intensities were reduced in the pre-filtrate and permeate samples as compared to the feed. It should be noted that in general the band intensities were reduced to a greater extent after pre-filtration with 11 µm filter than with 6 µm filter. The decrease of band intensity levels in the permeate sample passed through the 33 µm filter after 11 µm filtration was much less drastic than that for 6 µm/33 µm filtration sequence. In particular, no significant changes 11 of 18 Biomedicines 2020, 8, 69 were observed for bands 1, 2, and 4. For bands 3, 5, and 6 some intensity reduction was further detected in the permeate sample. LCMS analysis suggested that key coagulation factors IX, X, V as well as prothrombin were not removed following the two-step 11 µm/33 µm filtration sequence (for details see Appendix Tables A1, A4 and A5). Biomedicines 2020, 8, x FOR PEER REVIEW 11 of 18 Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11 μm/33 μm filter paper at 1 bar. Figure 10. 4. Discussion In this article, the filtration of a highly challenging hematologic product was investigated. Considering that FIX-rich PCC inherently consists of many bioactive components and some impurities, the virus removal filtration of this product is difficult without fouling. The virus removal filtration of PCC was previously reported using Ultipor DV50 filters, which are dedicated for removal of large-size viruses but do not ensure viral safety against parvoviruses [2]. Filtration of PCC through small-size virus removal filters, e.g., Planova 15N, resulted in nearly 39% total protein loss and reduced FIX and FII activity, which was ascribed to presence of large-size complexes between clotting factors and high molecular weight impurities [14]. It was further reported in the same study that filtration of highly purified FIX through Planova 15N not only did not result in the decrease of FIX activity but also improved its purity [14]. Biomedicines 2020, 8, x FOR PEER REVIEW 12 of 18 of large-size viruses but do not ensure viral safety against parvoviruses [2]. Filtration of PCC through small-size virus removal filters, e.g., Planova 15N, resulted in nearly 39% total protein loss and reduced FIX and FII activity, which was ascribed to presence of large-size complexes between clotting factors and high molecular weight impurities [14]. It was further reported in the same study that f l f h hl f d I h h l l d d l h d f I In this work, in order to achieve high virus removal capacity combined with reduced fouling, a tailored two-step process of filtration with nanocellulose-based filter paper was developed. In particular, sacrificial pre-filters with a thickness of 6 and 11 µm were tested. The increased thickness of the filters resulted in tighter pore structure as detected by cryoporometry. The observed effect is explained as follows and illustrated in Figure 12. The mille-feuille filter paper consists of a stratified 3-dimensional network of cellulose nanofibers, producing a mesh-like stricture. The layered structure is illustrated in the side-view panel of Figure 12. Considering that the nanofibers are randomly distributed in each layer, the pores, which percolate throughout the entire depth of the filter, become tighter with increasing number of layers. The latter is reflected, e.g., in improved virus clearance properties with increased thickness or enhanced aggregate removal properties. 4. Discussion Based on the results of PFU titrations of 27 and 70 nm phage particles, it was concluded that the tested filters show varying particle size rejection threshold as the thickness of the filter is increased. Thus, the observed effect is due to the combination of the receding pore size and depth effects (increased tortuosity). The latter enables using pre-filters with tailored cut-offto remove protein aggregates, which eventually results in improved flux through the dedicated virus removal filter. filtration of highly purified FIX through Planova 15N not only did not result in the decrease of FIX activity but also improved its purity [14]. In this work, in order to achieve high virus removal capacity combined with reduced fouling, a tailored two-step process of filtration with nanocellulose-based filter paper was developed. In particular, sacrificial pre-filters with a thickness of 6 and 11 μm were tested. The increased thickness of the filters resulted in tighter pore structure as detected by cryoporometry. The observed effect is explained as follows and illustrated in Figure 12. The mille-feuille filter paper consists of a stratified 3-dimensional network of cellulose nanofibers, producing a mesh-like stricture. The layered structure is illustrated in the side-view panel of Figure 12. Considering that the nanofibers are randomly distributed in each layer, the pores, which percolate throughout the entire depth of the filter, become tighter with increasing number of layers. The latter is reflected, e.g., in improved virus clearance properties with increased thickness or enhanced aggregate removal properties. Based on the results of PFU titrations of 27 and 70 nm phage particles, it was concluded that the tested filters show varying particle size rejection threshold as the thickness of the filter is increased. Thus, the observed effect is due to the combination of the receding pore size and depth effects (increased tortuosity). The latter enables using pre-filters with tailored cut-off to remove protein aggregates, which eventually results in improved flux through the dedicated virus removal filter. Figure 12. Illustration of the mechanism of virus removal with increased thickness of nanocellulose- based filter paper. Yellow symbols represent large-size model virus and red symbols represent small- size viruses. Increased thickness of the filter results in tighter pores and enhanced virus clearance. and thereby low product yield during the seco 3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC Observed flux for FIX-rich PCC permeate for 2-step 11 µm/33 µm filtration with 175 L m−2 load volume at 3 bar (A) and LRV for ΦX174 phage (B). Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2 load volume at 3 bar (A) and LRV for ΦX174 phage (B). Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2 l d l b A d f h Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 µm/33 µm filtration with 175 L m−2 load volume at 3 bar (A) and LRV for ΦX174 phage (B). Biomedicines 2020, 8, 69 12 of 18 12 of 18 4. Discussion Overall, the two-step approach presented here is based on the size-exclusion principles and is the efo e obu t It ould thu fu the be ada ted i the a ufa tu i of othe otei ba ed Figure 12. Illustration of the mechanism of virus removal with increased thickness of nanocellulose-based filter paper. Yellow symbols represent large-size model virus and red symbols represent small-size viruses. Increased thickness of the filter results in tighter pores and enhanced virus clearance. Figure 12. Illustration of the mechanism of virus removal with increased thickness of nanocellulose- based filter paper. Yellow symbols represent large-size model virus and red symbols represent small- size viruses. Increased thickness of the filter results in tighter pores and enhanced virus clearance. Overall, the two-step approach presented here is based on the size-exclusion principles and is Figure 12. Illustration of the mechanism of virus removal with increased thickness of nanocellulose-based filter paper. Yellow symbols represent large-size model virus and red symbols represent small-size viruses. Increased thickness of the filter results in tighter pores and enhanced virus clearance. 13 of 18 Biomedicines 2020, 8, 69 Overall, the two-step approach presented here is based on the size-exclusion principles and is therefore robust. It could thus further be adapted in the manufacturing of other protein-based pharmaceutics, too, including recombinant proteins wherein impurities in the form of host cell proteins may greatly affect the final yield of the biologics during virus removal nanofiltration. Author Contributions: For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, A.M. (Albert Mihranyan) and M.R.; methodology, A.M. (Albert Mihranyan), L.M., A.M. (Athanasios Mantas); supply of material, E.G., A.K.; formal analysis, L.M.; investigation, L.M, A.M. (Athanasios Mantas); resources, A.M. (Albert Mihranyan); data curation, L.M. and A.M. (Athanasios Mantas); writing—original draft preparation, L.M., A.M. (Albert Mihranyan); writing—review and editing, A.M. (Albert Mihranyan) L.M., R.M., A.K.; visualization, L.M.; supervision, A.M. (Albert Mihranyan); project administration, A.M. (Albert Mihranyan); funding acquisition, A.M. (Albert Mihranyan). All authors have read and agreed to the published version of the manuscript. Funding: The project was funded by Knut and Alice Wallenberg Foundation (Bridge grant 2018.01141), Swedish Research Council [Vetenskapsrådet No. 2016-05715], and EU EIT Health Innovation by Idea program [VIREPAP 19104]. EIT Health is supported by the European Institute of Innovation and Technology (EIT), a body of the European Union that receives support from the European Union’ s Horizon 2020 Research and innovation program. 5. Conclusions A two-step process was developed to both enhance filtration capacity (25-fold) and achieve high clearance of small-size viruses (LRV >5) using appropriate pre-filter paper. Large-size aggregates were the main foulants in the feed solution, and by tailoring the properties of the pre-filters the foulants were efficiently removed. In particular, 11 µm/33 µm filtration was found most suitable. The presented approach could potentially be applied for bioprocessing other protein-based drugs, both derived from plasma and produced by recombinant approaches. The article further provides new insights regarding the mechanism of virus removal in the nanocellulose-based filter paper, highlighting the combined effect of size exclusion and tortuosity of pore network. Author Contributions: For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, A.M. (Albert Mihranyan) and M.R.; methodology, A.M. (Albert Mihranyan), L.M., A.M. (Athanasios Mantas); supply of material, E.G., A.K.; formal analysis, L.M.; investigation, L.M, A.M. (Athanasios Mantas); resources, A.M. (Albert Mihranyan); data curation, L.M. and A.M. (Athanasios Mantas); writing—original draft preparation, L.M., A.M. (Albert Mihranyan); writing—review and editing, A.M. (Albert Mihranyan) L.M., R.M., A.K.; visualization, L.M.; supervision, A.M. (Albert Mihranyan); project administration, A.M. (Albert Mihranyan); funding acquisition, A.M. (Albert Mihranyan). All authors have read and agreed to the published version of the manuscript. Funding: The project was funded by Knut and Alice Wallenberg Foundation (Bridge grant 2018.01141), Swedish Research Council [Vetenskapsrådet No. 2016-05715], and EU EIT Health Innovation by Idea program [VIREPAP 19104]. EIT Health is supported by the European Institute of Innovation and Technology (EIT), a body of the European Union that receives support from the European Union’ s Horizon 2020 Research and innovation program. Acknowledgments: For LCMS analysis, the services of the Mass Spectrometry Based Proteomics Facility in Uppsala were used. The authors thank Ganna Shevchenko, Alexander Falk, and Jonas Bergquist for assistance in performing LCMS analysis. Conflicts of Interest: The corresponding author (A.M. (Albert Mihranyan)) is the inventor behind the IP pertaining to virus removal filter paper. Acknowledgments: For LCMS analysis, the services of the Mass Spectrometry Based Proteomics Facility in Uppsala were used. The authors thank Ganna Shevchenko, Alexander Falk, and Jonas Bergquist for assistance in performing LCMS analysis. Appendix A Table A1. LCMS analysis of FIX-PCC feed solution. Table A1. LCMS analysis of FIX-PCC feed solution. Accession Description Score Coverage MW [kDa] calc. pI P0C0L5 Complement C4-B 1920.89 76.38 192.6 7.27 P0C0L4 Complement C4-A 1898.90 76.03 192.7 7.08 P00734 Prothrombin 1767.89 75.56 70.0 5.90 P19823 Inter-alpha-trypsin inhibitor heavy chain H2 917.12 53.59 106.4 6.86 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 685.72 55.76 101.3 6.79 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 257.18 40.22 99.8 5.74 P02760 Protein AMBP 199.11 27.56 39.0 6.25 P00740 Coagulation factor IX 134.15 47.29 51.7 5.47 P00742 Coagulation factor X 59.86 38.52 54.7 5.94 P02768 Serum albumin 48.49 28.41 69.3 6.28 P49747 Cartilage oligomeric matrix protein 41.93 14.27 82.8 4.60 P67936 Tropomyosin alpha-4 chain 32.67 39.11 28.5 4.69 P01857 Immunoglobulin heavy constant gamma 1 32.57 35.15 36.1 8.19 P01834 Immunoglobulin kappa constant 30.33 39.25 11.8 6.52 P07225 Vitamin K-dependent protein S 26.15 12.72 75.1 5.67 P0DOY3 Immunoglobulin lambda constant 3 21.63 78.30 11.3 7.24 14 of 18 Biomedicines 2020, 8, 69 Table A1. Cont. Table A1. Cont. Accession Description Score Coverage MW [kDa] calc. pI P51884 Lumican 21.32 19.53 38.4 6.61 B9A064 Immunoglobulin lambda-like polypeptide 5 19.49 41.12 23.0 8.84 P04004 Vitronectin 19.13 14.02 54.3 5.80 P01861 Immunoglobulin heavy constant gamma 4 19.09 20.18 35.9 7.36 Q08380 Galectin-3-binding protein 18.16 9.74 65.3 5.27 P07359 Platelet glycoprotein Ib alpha chain 17.06 8.28 71.5 6.29 P35443 Thrombospondin-4 16.88 6.66 105.8 4.68 P01876 Immunoglobulin heavy constant alpha 1 15.74 24.93 37.6 6.51 P01859 Immunoglobulin heavy constant gamma 2 13.53 21.78 35.9 7.59 P01871 Immunoglobulin heavy constant mu 8.22 4.64 49.4 6.77 Q14515 SPARC-like protein 1 7.09 6.93 75.2 4.81 P04070 Vitamin K-dependent protein C 5.36 7.16 52.0 6.28 O95810 Caveolae-associated protein 2 4.07 4.71 47.1 5.21 P16070 CD44 antigen 3.37 2.83 81.5 5.33 Q99436 Proteasome subunit beta type-7 2.23 8.30 29.9 7.68 P12259 Coagulation factor V 1.95 0.81 251.5 6.05 P07237 Protein disulfide-isomerase 1.69 3.35 57.1 4.87 P10909 Clusterin 1.53 4.01 52.5 6.27 Table A2. LCMS analysis of FIX-PCC pre-filtrate following filtration with 6 µm filter. Table A2. LCMS analysis of FIX-PCC pre-filtrate following filtration with 6 µm filter. Accession Description Score Coverage MW [kDa] calc. Appendix A pI P00734 Prothrombin 2759.75 73.31 70.0 5.90 P0C0L5 Complement C4-B 2411.85 78.61 192.6 7.27 P0C0L4 Complement C4-A 2379.43 75.80 192.7 7.08 P19823 Inter-alpha-trypsin inhibitor heavy chain H2 1480.56 56.77 106.4 6.86 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 1167.61 54.45 101.3 6.79 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 352.03 43.60 99.8 5.74 P02760 Protein AMBP 263.80 27.56 39.0 6.25 P00740 Coagulation factor IX 216.46 54.66 51.7 5.47 P00742 Coagulation factor X 90.46 37.09 54.7 5.94 P02768 Serum albumin 62.34 36.45 69.3 6.28 P01857 Immunoglobulin heavy constant gamma 1 47.83 39.09 36.1 8.19 P04004 Vitronectin 45.60 23.64 54.3 5.80 P49747 Cartilage oligomeric matrix protein 43.78 19.82 82.8 4.60 P51884 Lumican OS=Homo sapiens 43.31 42.90 38.4 6.61 P01834 Immunoglobulin kappa constant 40.16 49.53 11.8 6.52 P67936 Tropomyosin alpha-4 chain 39.83 40.32 28.5 4.69 P07359 Platelet glycoprotein Ib alpha chain 31.44 12.12 71.5 6.29 P0DOY2 Immunoglobulin lambda constant 2 29.10 75.47 11.3 7.24 Q08380 Galectin-3-binding protein 28.60 18.80 65.3 5.27 P01861 Immunoglobulin heavy constant gamma 4 28.41 22.02 35.9 7.36 P01876 Immunoglobulin heavy constant alpha 1 23.24 25.78 37.6 6.51 B9A064 Immunoglobulin lambda-like polypeptide 5 22.01 38.79 23.0 8.84 P07225 Vitamin K-dependent protein S 20.55 14.35 75.1 5.67 P01859 Immunoglobulin heavy constant gamma 2 19.80 24.23 35.9 7.59 Q14515 SPARC-like protein 1 19.72 9.19 75.2 4.81 P35443 Thrombospondin-4 14.73 4.79 105.8 4.68 P12259 Coagulation factor V 8.97 2.34 251.5 6.05 P13591 Neural cell adhesion molecule 1 8.03 4.55 94.5 4.87 P12814 Alpha-actinin-1 7.56 3.48 103.0 5.41 P0CG38 POTE ankyrin domain family member I 7.35 3.07 121.2 6.21 P01871 Immunoglobulin heavy constant mu 7.06 8.61 49.4 6.77 P04070 Vitamin K-dependent protein C 5.93 10.63 52.0 6.28 P16070 CD44 antigen 5.80 2.70 81.5 5.33 P61981 14-3-3 protein gamma 5.53 10.93 28.3 4.89 P07900 Heat shock protein HSP 90-alpha 3.08 3.42 84.6 5.02 P01023 Alpha-2-macroglobulin 2.56 1.76 163.2 6.46 Q14185 Dedicator of cytokinesis protein 1 1.99 1.66 215.2 7.56 Q562R1 Beta-actin-like protein 2 1.78 7.71 42.0 5.59 T bl A4 LCMS l i f FIX PCC filt t f ll i filt ti ith 11 filt A3. LCMS analysis of FIX-PCC permeate following two-step filtration with 6 µm/33 µm filters. Table A4. LCMS analysis of FIX-PCC pre-filtrate following filtration with 11 µm filter. Table A4. LCMS analysis of FIX-PCC pre-filtrate following filtration with 11 µm filter. Accession Description Score Coverage MW [kDa] calc. Appendix A pI P00734 Prothrombin 2584.88 71.86 70.0 5.90 P0C0L5 Complement C4-B 2441.20 77.69 192.6 7.27 P0C0L4 Complement C4-A 2393.34 74.89 192.7 7.08 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 1154.64 54.34 101.3 6.79 P19823 Inter-alpha-trypsin inhibitor heavy chain H2 1136.27 55.50 106.4 6.86 P02760 Protein AMBP 337.33 25.57 39.0 6.25 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 332.27 43.15 99.8 5.74 P00740 Coagulation factor IX 151.88 54.66 51.7 5.47 P00742 Coagulation factor X 81.34 41.19 54.7 5.94 P02768 Serum albumin 60.16 37.44 69.3 6.28 P01834 Immunoglobulin kappa constant OS=Homo sapiens 48.66 81.31 11.8 6.52 P51884 Lumican 42.56 35.50 38.4 6.61 P49747 Cartilage oligomeric matrix protein 41.14 16.12 82.8 4.60 P01857 Immunoglobulin heavy constant gamma 1 40.75 41.21 36.1 8.19 P67936 Tropomyosin alpha-4 chain 31.98 36.29 28.5 4.69 P01861 Immunoglobulin heavy constant gamma 4 30.25 28.13 35.9 7.36 P01860 Immunoglobulin heavy constant gamma 3 28.46 27.06 41.3 7.90 P04004 Vitronectin OS=Homo sapiens 25.83 17.99 54.3 5.80 P0DOY2 Immunoglobulin lambda constant 2 23.29 67.92 11.3 7.24 P07225 Vitamin K-dependent protein S 21.00 11.83 75.1 5.67 P07359 Platelet glycoprotein Ib alpha chain 20.03 8.28 71.5 6.29 Q08380 Galectin-3-binding protein 17.21 12.82 65.3 5.27 P01876 Immunoglobulin heavy constant alpha 1 15.47 19.55 37.6 6.51 P35443 Thrombospondin-4 14.04 4.79 105.8 4.68 P01859 Immunoglobulin heavy constant gamma 2 12.71 27.91 35.9 7.59 P01024 Complement C3 7.05 1.56 187.0 6.40 P12259 Coagulation factor V 6.08 1.57 251.5 6.05 Q14515 SPARC-like protein 1 5.71 6.93 75.2 4.81 P16070 CD44 antigen 5.36 2.70 81.5 5.33 P13591 Neural cell adhesion molecule 1 5.24 2.56 94.5 4.87 P04070 Vitamin K-dependent protein C 5.14 5.86 52.0 6.28 P07900 Heat shock protein HSP 90-alpha 4.64 4.92 84.6 5.02 P61981 14-3-3 protein gamma 3.79 7.29 28.3 4.89 P63104 14-3-3 protein zeta/delta 3.48 6.94 27.7 4.79 Q99436 Proteasome subunit beta type-7 2.11 8.30 29.9 7.68 P25786 Proteasome subunit alpha type-1 1.66 7.98 29.5 6.61 15 of 18 Biomedicines 2020, 8, 69 Table A3. LCMS analysis of FIX-PCC permeate following two-step filtration with 6 µm/33 µm filters. Accession Description Score Coverage MW [kDa] calc. Appendix A pI P00734 Prothrombin 2458.17 67.20 70.0 5.90 P0C0L5 Complement C4-B 2389.47 80.68 192.6 7.27 P0C0L4 Complement C4-A 2352.05 79.59 192.7 7.08 P19823 Inter-alpha-trypsin inhibitor heavy chain H2 1268.03 56.87 106.4 6.86 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 1122.57 59.50 101.3 6.79 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 352.56 40.00 99.8 5.74 P02760 Protein AMBP 285.56 27.56 39.0 6.25 P00740 Coagulation factor IX 182.33 57.27 51.7 5.47 P00742 Coagulation factor X 80.33 41.19 54.7 5.94 P01834 Immunoglobulin kappa constant 54.45 54.21 11.8 6.52 P49747 Cartilage oligomeric matrix protein 53.77 24.31 82.8 4.60 P01857 Immunoglobulin heavy constant gamma 1 45.54 43.33 36.1 8.19 P02768 Serum albumin 44.26 25.78 69.3 6.28 P07359 Platelet glycoprotein Ib alpha chain 33.83 16.56 71.5 6.29 P01860 Immunoglobulin heavy constant gamma 3 33.53 31.83 41.3 7.90 P0DOY3 Immunoglobulin lambda constant 3 33.11 70.75 11.3 7.24 16 of 18 Biomedicines 2020, 8, 69 Table A4. Cont. Table A4. Cont. Accession Description Score Coverage MW [kDa] calc. pI P01861 Immunoglobulin heavy constant gamma 4 30.81 23.85 35.9 7.36 P04004 Vitronectin 27.21 23.64 54.3 5.80 P51884 Lumican 26.62 35.50 38.4 6.61 P01876 Immunoglobulin heavy constant alpha 1 25.67 29.18 37.6 6.51 Q08380 Galectin-3-binding protein 25.15 17.78 65.3 5.27 P67936 Tropomyosin alpha-4 chain 25.00 44.35 28.5 4.69 P01859 Immunoglobulin heavy constant gamma 2 17.12 25.77 35.9 7.59 P07225 Vitamin K-dependent protein S 16.75 9.62 75.1 5.67 P35443 Thrombospondin-4 16.66 5.52 105.8 4.68 P12259 Coagulation factor V 10.18 1.98 251.5 6.05 P04070 Vitamin K-dependent protein C 7.36 5.86 52.0 6.28 P16070 CD44 antigen 5.64 2.70 81.5 5.33 P13591 Neural cell adhesion molecule 1 5.50 1.75 94.5 4.87 P22105 Tenascin-X 4.42 2.33 458.1 5.17 Q99436 Proteasome subunit beta type-7 4.03 8.30 29.9 7.68 P12814 Alpha-actinin-1 3.68 2.35 103.0 5.41 P27348 14-3-3 protein theta 3.53 6.53 27.7 4.78 P07900 Heat shock protein HSP 90-alpha 1.73 3.14 84.6 5.02 Table A5. LCMS analysis of FIX-PCC permeate following two-step filtration with 11 µm/33 µm filters. Table A5. LCMS analysis of FIX-PCC permeate following two-step filtration with 11 µm/33 µm filters. Accession Description Score Coverage MW [kDa] calc. References 1. EMA. Guideline on Clinical Investigation of Recombinant and Human Plasma-derived Factor IX Products. EMA/CHMP/BPWP/144552/2009; 2011. Available online: https://www.ema.europa.eu/en/documents/ scientific-guideline/guideline-clinical-investigation-recombinant-human-plasma-derived-factor-ix- products_en.pdf (accessed on 25 March 2020). 2. Josi´c, D.; Hoffer, L.; Buchacher, A.; Schwinn, H.; Frenzel, W.; Biesert, L.; Klöcking, H.-P.; Hellstern, P. Manufacturing of a Prothrombin Complex Concentrate Aiming at Low Thrombogenicity. Thromb. Res. 2000, 100, 433–441. [CrossRef] 2. Josi´c, D.; Hoffer, L.; Buchacher, A.; Schwinn, H.; Frenzel, W.; Biesert, L.; Klöcking, H.-P.; Hellstern, P. 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Appendix A pI P0C0L5 Complement C4-B 2266.18 76.26 192.6 7.27 P0C0L4 Complement C4-A 2219.29 76.26 192.7 7.08 P00734 Prothrombin 2011.89 71.54 70.0 5.90 P19823 Inter-alpha-trypsin inhibitor heavy chain H2 1452.50 55.81 106.4 6.86 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 1205.49 54.23 101.3 6.79 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 256.28 46.97 99.8 5.74 P02760 Protein AMBP 213.99 25.57 39.0 6.25 P00740 Coagulation factor IX 114.46 45.55 51.7 5.47 P00742 Coagulation factor X 74.31 36.07 54.7 5.94 P02768 Serum albumin 46.51 27.91 69.3 6.28 P01857 Immunoglobulin heavy constant gamma 1 45.77 43.33 36.1 8.19 P49747 Cartilage oligomeric matrix protein 34.16 20.08 82.8 4.60 P01834 Immunoglobulin kappa constant 32.23 49.53 11.8 6.52 P67936 Tropomyosin alpha-4 chain 27.70 37.90 28.5 4.69 P01860 Immunoglobulin heavy constant gamma 3 27.37 25.99 41.3 7.90 P51884 Lumican 25.59 39.94 38.4 6.61 P01861 Immunoglobulin heavy constant gamma 4 24.73 26.61 35.9 7.36 P01859 Immunoglobulin heavy constant gamma 2 23.14 32.82 35.9 7.59 P04004 Vitronectin 22.83 23.43 54.3 5.80 P07359 Platelet glycoprotein Ib alpha chain 21.45 9.82 71.5 6.29 P07225 Vitamin K-dependent protein S 20.38 10.65 75.1 5.67 P0DOY2 Immunoglobulin lambda constant 2 17.99 37.74 11.3 7.24 Q08380 Galectin-3-binding protein 17.44 15.73 65.3 5.27 P01876 Immunoglobulin heavy constant alpha 1 17.06 25.78 37.6 6.51 P35443 Thrombospondin-4 9.76 4.79 105.8 4.68 P12259 Coagulation factor V 8.36 2.11 251.5 6.05 P04070 Vitamin K-dependent protein C 7.60 8.89 52.0 6.28 P61981 14-3-3 protein gamma 5.93 10.53 28.3 4.89 P16070 CD44 antigen 4.96 2.70 81.5 5.33 Q99436 Proteasome subunit beta type-7 4.05 8.30 29.9 7.68 Q14515 SPARC-like protein 1 4.05 3.77 75.2 4.81 P07900 Heat shock protein HSP 90-alpha 2.46 2.60 84.6 5.02 P01023 Alpha-2-macroglobulin 1.97 1.49 163.2 6.46 P22105 Tenascin-X OS=Homo sapiens 1.66 0.66 458.1 5.17 P13591 Neural cell adhesion molecule 1 0.00 1.75 94.5 4.87 Q5UIP0 Telomere-associated protein RIF1] 0.00 1.62 274.3 5.52 17 of 18 17 of 18 Biomedicines 2020, 8, 69 References Johnston, A.; Macgregor, A.; Borovec, S.; Hattarki, M.; Stuckly, K.; Anderson, D.; Goss, N.H.; Oates, A.; Uren, E. 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Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics
BMC plant biology
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To cite this version: Clémence Chaintreuil, Xavier Perrier, Guillaume Martin, Joël Fardoux, Gwilym Lewis, et al.. Natu- rally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics. BMC Plant Biology, 2018, 18 (1), pp.54 (1-15). ￿10.1186/s12870- 018-1260-2￿. ￿hal-01850345￿ Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics Clémence Chaintreuil, Xavier Perrier, Guillaume Martin, Joël Fardoux, Gwilym Lewis, Laurent Brottier, Ronan Rivallan, Mario Gomez-Pacheco, Mickaël Bourge, Léo Lamy, et al. Distributed under a Creative Commons Attribution 4.0 International License Abstract Background: Among semi-aquatic species of the legume genus Aeschynomene, some have the unique property of being root and stem-nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the production of Nod factors. These species provide an excellent biological system with which to explore the evolution of nodulation in legumes. Among them, Aeschynomene evenia has emerged as a model legume to undertake the genetic dissection of the so-called Nod-independent symbiosis. In addition to the genetic analysis of nodulation on a reference line, natural variation in a germplasm collection could also be surveyed to uncover genetic determinants of nodulation. To this aim, we investigated the patterns of genetic diversity in a collection of 226 Nod-independent Aeschynomene accessions. Results: A combination of phylogenetic analyses, comprising ITS and low-copy nuclear genes, along with cytogenetic experiments and artificial hybridizations revealed the richness of the Nod-independent Aeschynomene group with the identification of 13 diploid and 6 polyploid well-differentiated taxa. A set of 54 SSRs was used to further delineate taxon boundaries and to identify different genotypes. Patterns of microsatellite diversity also illuminated the genetic basis of the Aeschynomene taxa that were all found to be predominantly autogamous and with a predicted simple disomic inheritance, two attributes favorable for genetics. In addition, taxa displaying a pronounced genetic diversity, notably A. evenia, A. indica and A. sensitiva, were characterized by a clear geographically-based genetic structure and variations in root and stem nodulation Conclusion: A well-characterized germplasm collection now exists as a major genetic resource to thoroughly explore the natural variation of nodulation in response to different bradyrhizobial strains. Symbiotic polymorphisms are expected to be found notably in the induction of nodulation, in nitrogen fixation and also in stem nodulation. Subsequent genetic analysis and locus mapping will pave the way for the identification of the underlying genes through forward or reverse genetics. Such discoveries will significantly contribute to our understanding of the molecular mechanisms underpinning how some Aeschynomene species can be efficiently nodulated in a Nod-independent fashion. Keywords: Aeschynomene, Diversity, Genotype, Legume, Nodulation, Ploidy, Species, Symbiosis Naturally occurring variations in the nod- independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics Clémence Chaintreuil1,8, Xavier Perrier2,3, Guillaume Martin2,3, Joël Fardoux1,8, Gwilym P. Lewis4, Laurent Brottier1,8, Ronan Rivallan2,3, Mario Gomez-Pacheco5, Mickaël Bourges5, Léo Lamy1,8, Béatrice Thibaud2,3, Heriniaina Ramanankierana6, Herizo Randriambanona6, Hervé Vandrot7, Pierre Mournet2,3, Eric Giraud1,8 and Jean-François Arrighi1,8* * Correspondence: jean-francois.arrighi@ird.fr 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM, Campus International de Baillarguet, F-34398 Montpellier, France 8LSTM, Univ. Montpellier, CIRAD, INRA, IRD, Montpellier SupAgro, Montpellier, France Full list of author information is available at the end of the article HAL Id: hal-01850345 https://hal.umontpellier.fr/hal-01850345v1 Submitted on 25 Nov 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Chaintreuil et al. BMC Plant Biology (2018) 18:54 https://doi.org/10.1186/s12870-018-1260-2 Background To broaden our understanding of the molecular mechanisms underlying the nitrogen- fixing symbiosis, there has arisen a fast growing interest in uncovering the diversity of nodulation processes that are found in other legume species [5, 6]. p g Forward genetics are now expected to allow the identi- fication of the specific molecular determinants of the Nod-independent process in A. evenia. To optimize re- search effort, a reference line was inbred and success- fully used to generate an SSR-based genetic map of A. evenia [9]. This genetic map uncovered the genome structure and the distribution of symbiotic genes. It also provides a basis for a genome sequencing project and paves the way of the genetic dissection of nodulation in this reference line. This does not exclude exploring the naturally occurring variations in nodulation, as a com- plementary genetic approach, in order to increase our understanding of symbiotic gene functions and of the genetic control of symbiosis as successfully performed in other legumes such as Medicago, Lotus and soybean [22]. But exploiting genetic diversity requires prior knowledge of the extent and structure of the variations occurring in the species of interest. Although the genetic relationships among the Nod-independent Aeschynomene species have been analysed using molecular markers, only two studies have included a small set of accessions for the diploid A. evenia and the related polyploid A. indica [17, 18]. As a consequence, the variations within and among the species of the Nod-independent clade remain largely uncharacterized. g In this line, the mainly tropical legume genus Aeschynomene represents a group of prime interest as it contains several original symbiotic features. The genus Aeschynomene was ori- ginally known for the ability of different species to develop stem nodules in addition to the typical root nodules. Stem nodulation is uncommon in legumes, being shared with a very few hydrophytic species of the genera Sesbania, Neptunia and Discolobium, but it is widespread among the semi-aquatic Aeschynomene species [7–9]. In addition, some bradyrhizobia isolated from Aeschynomene stem nodules exhibit a photosyn- thetic activity that was shown to play a key role in stem nodules by directly furnishing energy to the bacterium that can be used for biological nitrogen fixation [10, 11]. Background taxonomic revision of American Aeschynomene was pub- lished in 1955 by Rudd [15], but it predated phylogenetic studies in plants and included no species native outside the Neotropics. First molecular studies of Aeschynomene pointed to new cryptic taxa differing by their ploidy levels [16, 17]. The knowledge gained from the study of the Nod- independent clade was also used to select Aeschynomene evenia as a new model legume for the purpose of decipher- ing the molecular mechanisms of the Nod-independent sym- biosis [16, 18]. Key attributes of this species include its small, diploid genome (2n = 20, 415 Mb/1C), its selfing nature and its prolific seed production. Several tools have been devel- oped including artificial hybridization and the Agrobacterium rhizogenes-mediated root transformation, rendering this spe- cies ideal for molecular genetic studies. First insights were obtained from RNAseq analysis and reverse genetics by re- vealing that some symbiotic determinants identified in Medicago and Lotus are recruited in the Nod-independent process but several key genes involved in bacterial recogni- tion, symbiotic infection and nodule functioning were found not to be expressed during root nodulation [9, 19–21]. g The legume family (Leguminosae) accounts for ~ 27% of the world’s primary crop production and is second only to cereals in economic and nutritional value. It includes many crops of agronomic importance for grain production, pas- ture and agroforestry. Many legumes are pioneers plants improving soil fertility and moderating harsh environments. Such economic and ecological success of the legume family is, in large part, due to the ability of the vast majority of its 20,000 species to develop symbiotic interactions with nitrogen-fixing bacteria collectively referred as rhizobia [1]. In this symbiosis, the rhizobia produce signal molecules, the Nod factors, whose specific recognition by the host plant is necessary to activate the formation of root nodules that correspond to symbiotic organs where the rhizobia are hosted. Inside the nodules, the rhizobia reduce atmospheric nitrogen (N2) into ammonium (NH4 +), a form of nitrogen that is usable by the plant for its development. Historically, two model legumes, Medicago truncatula and Lotus japoni- cus, have been used to genetically investigate this nodula- tion process. Such studies have resulted in the identification and elucidation of the role of many genes that are essential for the different steps of nodule development and its infec- tion by the symbiont [2–4]. * Correspondence: jean-francois.arrighi@ird.fr 1 Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 2 of 15 Background Even more outstanding was the discovery that some of these photo- synthetic bradyrhizobia lack both the canonical nodABC genes required for the synthesis of the key Nod factors and a type III secretion system (T3SS) that is known in other rhizobia to activate or modulate nodulation [12–14]. This led to a new paradigm in nodulation studies in which an al- ternative symbiotic process between rhizobia and le- gumes efficiently triggers nodule formation in a Nod (factor)-independent fashion. To enable an efficient use of the natural variation in genetic studies of nodulation, we surveyed the genetic diversity occurring in a collection of 226 Nod-independent Aeschynomene accessions spanning the whole distributional range of this clade. As a first step, genetic relationships and differentiation between Nod-independent Aeschynomene taxa were established using a combination of molecular phylogenies, cytogenetics and hybridization experiments. Phylogenetic analysis of the genus Aeschynomene revealed that all the species endowed with a Nod-independent nodulation process cluster in a single clade where no species using a Nod-dependent symbiotic process are found [8]. A Page 3 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 This information then served as supportive data for the analysis of genotype data obtained for the germplasm col- lection using a set of 54 SSR markers. Patterns of microsat- ellite diversity illuminated the genetic basis of the Aeschynomene taxa and uncovered their genetic differenti- ation. The presence of an underlying genetic structure was then compared with geographical distribution data. with A. deamii, a third one comprising A. evenia and its sister species, and a fourth one containing A. sensitiva and related species (Fig. 1a). p g In this phylogenetic tree, a number of putative species displayed the same ITS sequence. In particular, two rare species, A. magna and A. pluriarticulata, tightly clus- tered with two widespread species, A. denticulata and A. evenia, respectively (Fig. 1a). Similarly, A. evenia and A. indica shared the same ITS signature but they were pre- viously shown to form a species complex containing three cytotypes (2×, 4× and 6×) [17]. To sort these, accessions of this species complex were genotyped with SSR markers that are indicators of their genome consti- tution [17]. This led to the additional discovery that the A. indica 6× accessions of African and Australian origin displayed different SSR profiles, justifying their distinc- tion in the phylogenic tree (Fig. 1a, data not shown). Species identification and relationships A collection of 233 accessions was developed to investi- gate the phylogenetic relationships and the genetic dif- ferentiation in the Nod-independent Aeschynomene clade (Additional file 1: Table S1). It included all the known species included in the clade and aimed to cover their distributional range [15, 23, 24]. The nuclear ribo- somal ITS region was used as a marker of the species identity and served to reconstruct a phylogeny of the whole group based on the Neighbor Joining (NJ) method (Additional files 2 and 3: Tables S2 and S3). To simplify the resulting tree, accessions showing less than 1% of di- vergence in their sequence were grouped in the same clade (Fig. 1a, Additional file 4: Doc. S1). The ITS tree was composed of four lineages: one grouping A. filosa, A. rostrata and A. tambacoundensis, a monospecific one a b Fig. 1 Phylogenetic and genetic relationships in the Nod-independent Aeschynomene clade. Phylogenetic reconstructions were obtained using the Neighbor Joining method. a ITS phylogeny. Accessions with ITS sequence divergence < 1% were clustered together. Numbers of accessions per taxon are indicated in brackets. b Phylogeny based on 5 concatenated low-copy nuclear gene fragments: CYP1, eiF1α, SUI1, SuSy and a gene homolog to Glyma.07G136800 and Glyma.18G187300. -A, -B and -C indicate the different copies found in polyploid species. The four gene pools are identified with a circled number and the A. evenia and A. sensitiva groups are framed in a box bordered with a dashed red line. Diploid taxa are in black and polyploid taxa in blue with ploidy level indicated. Numbers at nodes represent bootstrap values (% of 1000 replicates) b a b b Fig. 1 Phylogenetic and genetic relationships in the Nod-independent Aeschynomene clade. Phylogenetic reconstructions were obtained using the Neighbor Joining method. a ITS phylogeny. Accessions with ITS sequence divergence < 1% were clustered together. Numbers of accessions per taxon are indicated in brackets. b Phylogeny based on 5 concatenated low-copy nuclear gene fragments: CYP1, eiF1α, SUI1, SuSy and a gene homolog to Glyma.07G136800 and Glyma.18G187300. -A, -B and -C indicate the different copies found in polyploid species. The four gene pools are identified with a circled number and the A. evenia and A. sensitiva groups are framed in a box bordered with a dashed red line. Diploid taxa are in black and polyploid taxa in blue with ploidy level indicated. Background It is also noteworthy that two accessions did not fit well with the description of any known Aeschynomene species and so we refer to these as Aeschynomene sp. (328) and A. sp. (353) (Fig. 1a, Additional file 1: Table S1). To clarify the genetic status of these putative new taxa, they were both in- cluded in a flow cytometry analysis and a chromosome count (Additional file 1: Table S1, Additional file 5: Figure S1). Cytogenetic data were mapped onto the phylogeny, showing Genetic patterns and behaviours g To further uncover genetic relationships between taxa, five low copy nuclear genes -CYP1 (Cyclophilin 1), eiF1α (eukaryotic translation initiation factor α), SUI1 (transla- tion factor), SuSy (Sucrose Synthase) and a gene homolog to Glyma.07G136800 and Glyma.18G187300 identified in Glycine max - were cloned and sequenced in selected ac- cessions (Additional file 2: Table S2). For diploid species, single sequences were obtained, while for polyploid spe- cies homeologous sequences were isolated for almost all genes (Additional file 3: Table S3). The five genes treated separately gave similar NJ trees where the homeologous sequences for polyploid taxa could be differentiated based on the differential clustering with the sequences of diploid taxa (not shown). To provide a unique and well-resolved NJ phylogeny, the gene sequences were concatenated to- gether (Fig. 1b). In the resulting tree, the topology of the branches containing the diploid species was similar to that of the ITS tree, corroborating the distinctness of A. sp. (328) from other known Aeschynomene species (Fig. 1a,b). For the polyploid taxa, the different genome components were scattered different part of the phylogeny, revealing that the two taxa A. sp. (353) and A. virginica, and the African and Australian A. indica 6× had the same or a similar genomic constitution (Fig. 1b). To assess the gen- etic differentiation of these related taxa, they were manu- ally crossed: A. sp. (353) with A. virginica, the African A. indica 6× with the Australian A. indica 6× and A. indica 4× with A. indica 6× for comparison (Fig. 2). In all cases, hybrid plants were obtained but they greatly differed in their fertility compared to their respective parental acces- sions, as seen by a drastic reduction of both the number of developed pods per flowering axis and of seeds per pod. To determine the genetic structure of the genomes of dif- ferent taxa, we conducted a SSR genotyping of the collec- tion of Aeschynomene accessions. For this, we tested the set of 500 SSRs previously used when developing a genetic map for A. evenia [9]. These markers were screened for polymorphism in four samples that are genetically differ- ent: two accessions of A. evenia 2× (the reference line CIAT 22838 and the mapping parent CIAT 8232), one ac- cession of A. indica 4× (PI 196206) and one of A. indica 6× (LSTM19). Species identification and relationships Numbers at nodes represent bootstrap values (% of 1000 replicates) Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 4 of 15 As a result, these data suggest that the tested taxa are truly different Aeschynomene species. that Aeschynomene sp. (328) represented a new 2× taxon while A. sp. (353) was a 4× taxon, similar to the closely related A. virginica, and confirming the hexaploid status of the Australian A. indica 6× (Fig. 1a). Genetic patterns and behaviours sensitiva group was compensated by the use of an additional set of SSR markers. Of the 64 SSR markers, 54 generated clearly interpretable allele profiles in at least one of the two groups, 38 SSR markers for the A. evenia group and 46 SSR markers for the A. sensitiva group, with 30 of them in common (Additional file 6: Figure S2). Allelic diversity, estimated by the number of alleles Na, varied im- portantly among the SSR markers used with an average of 12.8 alleles per locus in the A. evenia group (n = 186 acces- sions) and 4.21 in the A. sensitiva group (n = 40 accession) (Additional file 10: Table S6). Observed heterozygosities were very low for each SSR (Ho < 0.1 with one exception at 0.2), indicating that they amplified at single locus for diploid accessions and homeologous loci for polyploid accessions (Additional file 10: Table S6). the Aeschynomene taxa, one of the highest being found in A. evenia (mean 5.2 alleles per SSR) (Table 1, Additional file 10: Table S6). These values reflected the genetic diversity of the taxa, but they were likely to have been influenced by the marked variation in sample sizes and by the non-random selection of the SSR markers. The mean number of alleles detected for the SSR markers in each taxon were congruent with their ploidy levels, with (i) 1.00 to 1.06 alleles observed for diploid taxa, (ii) 1.42 to 1.92 alleles for tetraploid taxa and (iii) 2.63 to 2.84 alleles for hexaploid taxa (Table 1, Additional file 12: Table S8). Accessions displaying more al- leles than expected from their ploidy level were considered as heterozygous (Additional files 1 and 13: Tables S1, S9). Selfing rates based on the observed heterozygosities, ranged from 94.5% to 100%, with an average 98.7% score for A. evenia (Table 1, Additional file 13: Table S9). This provided genetic support for previous observations that the Nod- independent Aeschynomene species are preferentially autog- amous [17, 18]. These results supported the appropriateness of these SSRs for investigating the genetic properties of species. Therefore they were analysed in a second step to characterize the taxa. Transferability level of the SSR markers from A. evenia, ranged from 92 to 100% for other taxa of the A. evenia group and from 72 to 78% for those of the A. sensitiva group (Table 1, Additional file 11: Table S7). Genetic patterns and behaviours Of these, 64 markers were selected using two main criteria: (1) the requirement to be polymorphic between the two A. evenia accessions so as to avoid the use of invariant SSRs, (2) to amplify a single allele in the 2× accessions, 2 and 3 alleles in the 4× and 6× accessions, respectively, with the assumption that the distinct alleles came from the different genomic components of the polyploid species (Additional files 6, 7 and 8: Figure S2, Tables S4 and S5). Forty nine out of the 64 selected markers were previously positioned on different A. evenia linkage groups, with their distribution representing a reasonable coverage of the A. evenia genetic map (Additional file 7: Table S4). Preliminary genotyping experiments revealed that the SSRs developed from A. evenia sequences had a transfer- ability rate qualified as (i) modest with A. deamii and A. tambacoundensis, (ii) good with A. sensitiva and A. pratensis and (iii) high with A. denticulata and A. scabra. These data were in accordance with the phylogenetic distance of the different gene pools relative to A. evenia (Fig. 1), but this prompted us to restrict our analysis to the A. evenia and A. sensitiva groups. The corresponding accessions were then subjected to high-throughput SSR Fig. 2 Hybridization experiments between related taxa. Manual crosses were performed between A. virginica and A. sp (353), A. indica 4× and A. indica 6× -Africa-, A. indica 6× -Africa- and A. indica 6× -Australia. Plant fertility was evaluated for the parental taxa and two independently obtained F1 hybrids based on the number of seeds produced per pod and the number of developed pods per flowering axis. Error bars represent s.d. (n = 30) Fig. 2 Hybridization experiments between related taxa. Manual crosses were performed between A. virginica and A. sp (353), A. indica 4× and A. indica 6× -Africa-, A. indica 6× -Africa- and A. indica 6× -Australia. Plant fertility was evaluated for the parental taxa and two independently obtained F1 hybrids based on the number of seeds produced per pod and the number of developed pods per flowering axis. Error bars represent s.d. (n = 30) Page 5 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 5 of 15 genotyping by capillary sequencing (Additional file 9: Figure S3). The absence of amplification for some of them in the A. Genetic patterns and behaviours Analysing the Na par- ameter revealed different levels of genetic variation among Geographical structure of the Aeschynomene species Geographical structure of the Aeschynomene species The Nod-independent Aeschynomene clade is mainly a trop- ical/subtropical group, but the species show distinct geo- graphical distributions [15, 23, 24]. Out of the 19 taxa considered here (Table 1), 13 are strictly American, while three taxa (A. evenia, A. indica 4× and A. sensitiva) have a wider distribution; two are African (A. indica 6× - Africa- and A. tambacoundensis) and one Australian (A. indica 6× - Australia-). This confirmed previous conclusions that Amer- ica is the centre of origin and diversification of this clade but that several outliers have subsequently evolved in other con- tinents [8]. Because some species showed a pronounced gen- etic differentiation and are part of polyploid species complexes, we investigated to what extent the corresponding cytotypes and genotypes are geographically structured. In the A. sensitiva-A. pratensis species complex, A. sensitiva has a transatlantic distribution. When mapping accessions globally, a clear geographical separation of the four A. sensitiva geno- types was observed. One occupies the Caribbean region, one found in Colombia, another one the central area of South America and, more noticeably a fourth one being present both in coastal East Brazil and in Africa, suggesting a recent dispersal event (Fig. 5a, Additional file 9: Figure S3, Additional file 1: Table S1). In contrast to A. sensitiva, the low genetic diversity observed in A. pratensis 4× revealed no consistent geographical pattern (Fig. 5A, Additional file 9: Figure S3, Additional file 1: Table S1). In a search for other relationships, a Factorial Ana- lysis (FA) was also carried out in DARwin v5 [25]. This approach is more informative regarding distances among different groups and it also allows comparison of accessions of different ploidy levels. We focused on A. evenia and A. sensitiva to get a more detailed view of the genetic relatedness between the 2× genotypes and the derived polyploid taxa. FA clearly distin- guished A. sensitiva and A. pratensis when using the factorial axes 1 and 2 (Fig. 4a). Factorial axes 2 and 3 separated the four A. sensitiva genotypes and but the central position of A. pratensis was interpreted has an absence of a preferential relationship with any of the A. sensitiva genotypes (Fig. 4b). Therefore, either the parental A. sensitiva genotype that contributed to the polyploid genome of A. pratensis was missing or A. In the pantropically distributed A. evenia-A. Genetic diversity and genotype delineation Based on the geographical distributions of the accessions making up the different Nod-independent Aeschynomene taxa, most of their genetic diversity and structure were ex- pected to be uncovered. Therefore, the genotyping data Table 1 Summary of the data obtained for the Aeschynomene taxa Taxa/ ploidy level n samples n genotypes Cross-species transferability N Table 1 Summary of the data obtained for the Aeschynomene taxa Table 1 Summary of the data obtained for the Aeschynomene taxa Taxa/ ploidy level n samples n genotypes Cross-species transferability NA n co-present alleles/SSR Ho Comment 2× taxa A. ciliata 5 100% 1.2 1.00 0.000 A. deamii 3 – – – – A. denticulata 24 3 92% 2.7 1.06 0.055 A. magna conspecific A. evenia ssp. evenia 44 7 – 5.2 1.01 0.013 A. pluriarticulata conspecific A. evenia ssp. serrulata 15 2 100% 2.3 1.02 0.019 A. filosa 2 – – – – A. rostrata 1 – – – – A. rudis 7 95% 1.9 1.00 0.004 A. scabra 8 100% 1.6 1.00 0.000 A. selloi 3 78% 1.1 1.00 0.000 A. sensitiva 27 4 78% 2.6 1.03 0.028 A. sp (328) 1 72% 1 1.00 0.000 new taxon A. tambacoundensis 1 – – – – 4× taxa A. indica 4× 41 6 100% 4.9 1.92 0.019 A. pratensis 9 78% 2.1 1.60 0.000 A. sp (353) 1 100% 1.6 1.66 0.000 new taxon A. virginica 1 100% 1.5 1.42 0.000 6× taxa A. indica 6× Africa 16 2 100% 4.8 2.84 0.005 A. indica 6× Australia 24 3 100% 6.9 2.63 0.002 new taxon Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 6 of 15 Page 6 of 15 obtained for the A. evenia and A. sensitiva groups were combined and used to estimate pair-wise distances be- tween all accessions and thereby generate a dissimilarity matrix in DARwin v5 [25]. From this, NJ trees were calcu- lated for the 2×, 4×, 6× ploidy levels separately. Indeed, al- leles of the genomic components of polyploid accessions cannot be analysed separately and so their inclusion in the analysis of the diploid accessions would result in their grouping with only one of the potential progenitors. Considering the 2× NJ tree, well-separated clades were evident and corresponded to the known species A. evenia, A. ciliata, A. denticulata, A. rudis, A. scabra, A. serrulata, A. selloi and A. sensitiva, as well as the newly identified A. sp. (328) (Fig. Genetic diversity and genotype delineation 3a,Additional file 9: Figure S3). Interestingly, A. pluriarticulata was found to be nested within the A. evenia accessions and the accession of A. magna tightly clustered with accessions of A. denticulata. This information together with the phylogenetic and genetic relationships makes their taxonomic distinctness uncertain. Conversely, the tree topology showed a clear separation of the two A. evenia subspecies, evenia and serrulata, and several 2× taxa (A. denticulata, A. evenia ssp. evenia, A. evenia ssp. serrulata and A. sensitiva) could be subdivided in different clusters de- lineating genotypes (Fig. 3a,Additional file 9: Figure S3). Among these, genetic diversity and differentiation was the highest for A. evenia ssp. evenia (here after A. evenia s.s. in the text), with 7 genotypes identified. Regarding the 4× tree, A. virginica and A. sp (353) were found to form sister line- ages and contrasted patterns of genetic differentiation were observed with a very low genetic variability noted for A. pratensis whereas A. indica 4× was composed of several well-diverged clusters (Fig. 3b,Additional file 9: Figure S3). In the 6× tree, the African and Australian A. indica 6× were genetically distant with the African set subdivided in two homogenous genotypes while the Australian set contained three genotypes that formed far more diverse assemblages, indicative of distinct evolutions (Fig. 3c, Additional file 9: Figure S3). pratensis was formed before the intraspecific differen- tiation of A. sensitiva as suggested by the sequence divergence of the nuclear genes (Fig. 1b). For A. evenia s.s., the FA separated the 7 identified geno- types but grouped the three A. indica taxa together when using factorial axes 1 and 2 (Fig. 4c). These polyploid taxa could be separated along factorial axis 4 and showed preferential affinity with the two un- separated genotypes (1) and (2) of A. evenia along factorial axis 2 (Fig. 4d). These observations suggested a common origin of the three A. indica taxa that would derive from the same A. evenia genome donor, this latter being potentially ancestral to the genotypes 1 and 2. obtained for the A. evenia and A. sensitiva groups were combined and used to estimate pair-wise distances be- tween all accessions and thereby generate a dissimilarity matrix in DARwin v5 [25]. From this, NJ trees were calcu- lated for the 2×, 4×, 6× ploidy levels separately. Genetic diversity and genotype delineation Indeed, al- leles of the genomic components of polyploid accessions cannot be analysed separately and so their inclusion in the analysis of the diploid accessions would result in their grouping with only one of the potential progenitors. Considering the 2× NJ tree, well-separated clades were evident and corresponded to the known species A. evenia, A. ciliata, A. denticulata, A. rudis, A. scabra, A. serrulata, A. selloi and A. sensitiva, as well as the newly identified A. sp. (328) (Fig. 3a,Additional file 9: Figure S3). Interestingly, A. pluriarticulata was found to be nested within the A. evenia accessions and the accession of A. magna tightly clustered with accessions of A. denticulata. This information together with the phylogenetic and genetic relationships makes their taxonomic distinctness uncertain. Conversely, the tree topology showed a clear separation of the two A. evenia subspecies, evenia and serrulata, and several 2× taxa (A. denticulata, A. evenia ssp. evenia, A. evenia ssp. serrulata and A. sensitiva) could be subdivided in different clusters de- lineating genotypes (Fig. 3a,Additional file 9: Figure S3). Among these, genetic diversity and differentiation was the highest for A. evenia ssp. evenia (here after A. evenia s.s. in the text), with 7 genotypes identified. Regarding the 4× tree, A. virginica and A. sp (353) were found to form sister line- ages and contrasted patterns of genetic differentiation were observed with a very low genetic variability noted for A. pratensis whereas A. indica 4× was composed of several well-diverged clusters (Fig. 3b,Additional file 9: Figure S3). In the 6× tree, the African and Australian A. indica 6× were genetically distant with the African set subdivided in two homogenous genotypes while the Australian set contained three genotypes that formed far more diverse assemblages, indicative of distinct evolutions (Fig. 3c, Additional file 9: Figure S3). Geographical structure of the Aeschynomene species indica species complex, a prominent geographical division be- tween the different taxa was observed. A. evenia s.s. (2×) grows both in America and Africa, A. indica 4× is wide- spread in Asia, including India, and it is also present in North Eastern America, while the two A. indica 6× taxa distinctly occur in Africa and Australia (Fig. 5b). At the intraspecific level, the identified genotypes were found to represent geographically defined groups. A. evenia displayed a high level of genetic structure with 5 Ameri- can and 2 African genotypes. As far as phylogenetic rela- tionships can be inferred from an SSR-based NJ tree, the Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 7 of 15 a b c Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk a b a a c c c Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk African genotypes appeared to have diverged after a re- cent transatlantic migration from the Neotropics (Fig. 5b, Additional file 9: Figure S3, Additional file 1: Table S1). A. Geographical structure of the Aeschynomene species Two pairss of factorial axes (with the percentage of variation they account for indicated in parenthesis) are used for each species complex so as to show genetic distinctness and relationships. Taxon colours and genotype numbers are the same as in Fig. 3 standpoint. For the polyploid taxa (4 tetraploids and 2 hexaploids), low-copy nuclear genes and SSR analysis supported an allopolyploid origin and several genome donors could be identified [17; this study]. This must fa- cilitate the identification and distinction of the different subgenomic components of the polyploid genomes and this indicated that these polyploid taxa must behave gen- etically as diploids. Segregation of molecular markers, however, has not been performed to date at a genome- wide scale and we therefore cannot exclude that they might behave as segmental allopolyploids just as recently reported for peanut and chrysanthemum [27, 28]. Such dual structure may be found notably in A. pratensis for which the two expected homeologous versions of the tested low-copy nuclear genes were not always detected. reactions characterized by the accumulation of brown compounds, most probably of polyphenol nature as already described for incompatible interactions [26], were obvious in nodules of A. indica 6× -Africa- inoculated with BTAi1 (Fig. 6b, Additional file 14: Figure S4a). This incompatibility was accompanied by a low nitrogen-fixing activity and an overall reduction in plant development (Fig. 6c, Additional file 14: Figure S4b). Geographical structure of the Aeschynomene species indica 4× contained 3 distinct Indian lineages, one genotype spanning Eastern Asia and the North Eastern America, a second widespread in Northern Australia but also occurring in Asia, and a third throughout South Asia and extending to the Pacific (Fig. 5b). The Australian A. indica 6× was found to have a wide distribution extend- ing from Eastern to Western Australia. Missing collec- tion data limited the analysis of the three 6× genotypes but they did not overlap in distributional area with A. indica 4× (Fig. 5b, Additional file 9: Figure S3, Additional file 1: Table S1). In contrast, distribution of the African A. indica 6× was restricted to the Subsahelian zone and the two identified genotypes clearly could be distin- guished based on their location, one in the western part of the zone, the other one with a more central position (Fig. 5b, Additional file 9: Figure S3, Additional file 1: Table S1). Noteworthy, at a macroscale, there exists an overlap in distribution of African A. indica 6× with A. evenia s.s, but the latter was more widespread in Africa. To test whether this genetic diversity could support poly- morphism in nodulation traits, a number of accessions of A. evenia s.s. and A. indica were submitted to root or stem in- oculation with two photosynthetic nodABC gene-lacking Bradyrhizobium strains, ORS278 and BTAi1. A high vari- ation in stem nodule development was observed in A. evenia when it was inoculated with ORS278 (Fig. 6a). Conversely, root nodulation was more homogenous but plant defense Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 8 of 15 b d a b c d Fig. 4 Factorial analysis of Nod-independent Aeschynomene taxa. (a) and (b) for the A. evenia-A. indica species complex, (c) and (d) for the A. sensitiva-A. pratensis species complex. Two pairss of factorial axes (with the percentage of variation they account for indicated in parenthesis) are used for each species complex so as to show genetic distinctness and relationships. Taxon colours and genotype numbers are the same as in Fig. 3 a a b a c d Fig. 4 Factorial analysis of Nod-independent Aeschynomene taxa. (a) and (b) for the A. evenia-A. indica species complex, (c) and (d) for the A. sensitiva-A. pratensis species complex. Discussion This in-depth characterization of a Nod-independent Aeschynomene germplasm collection identified four main lineages and uncovered genetic diversity and struc- ture at different scales: cytotypes, species and genotypes (as summarised in Table 1). Interestingly, this group of Aeschynomene is composed of mainly diploid taxa (13 out of 19), including the model legume A. evenia, which are expected to be the easiest to handle at a genetic In addition to shedding light on the genetic basis of the different Aeschynomene species, our analyses were used to identify different taxa and to delimit their Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 9 of 15 b a Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. 3 a a b Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. 3 b g. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. dica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 4 Table S1 and Fig re S4 Ta on colo rs and genot pe n mbers are the same as in Fig 3 b Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. Discussion 6 Variation of nodulation traits observed in accessions of A. evenia and A. indica. (a)Stem nodulation observed in various accessions of A. evenia, 3 weeks post inoculation with ORS278. (b) Root nodule development in accessions of A. evenia and A. indica following inoculation with Bradyrhizobium ORS278 and BTAi1. 14-dpi nodules were cut to observe the leghemoglobin color and reaction defense (arrow). (c) Comparison of plant growth (aerial part) after inoculation with Bradyrhizobium ORS278 and BTAi1, at 14 dpi. Scale bar in (a): 5 mm, in (b): 1 mm All these data taken together make our germplasm collection a valuable genetic resource for the Nod- independent Aeschynomene group. So how best to fur- ther exploit it? To decipher the molecular mechanisms underlying the different original nodulation properties found in the Nod-independent symbiosis, A. evenia was recently selected as a model species [16, 18]. The species is currently being subjected to full genome sequence analysis and an ongoing mutagenesis project is predicted to identify new symbiotic genetic determinants in the near future. Such approaches applied to the historical model legumes M. truncatula and L. japonicus led to major advances in the study of the nitrogen-fixing sym- biosis with the identification of a set of symbiotic genes involved in the recognition of rhizobial signals, transduc- tion, infection and nodule organogenesis [2, 4]. But mutants are usually screened for the loss of their ability to establish a symbiosis, due to the disruption of gene function, and they are developed in the frame of the study of a single plant line-rhizobial strain system. Conversely, the screening of natural populations with multiple rhizobial strains can reveal some symbiotic phe- notypes that depend on both the host genetic back- ground and the rhizobial strain. In fact, studying natural variation approach has been shown to be a powerful tool for gaining insights into the genetic basis underlying the specificity of the symbiotic interaction in three legumes, be reliably considered as a new species belonging to the A. sensitiva group, notwithstanding that this conclusion is based on a single accession of what is apparently a rare taxon. Given their high variability, the SSR markers are a power- ful tool to highlight putative subdivisions in different Aeschynomene species that served to define genotypes. Strik- ing is that these genotypes, delineated solely on the basis of marker data, corresponded to geographically based sub- groups. Discussion 3 were found to form sister clades in the ITS and low-copy nu- clear gene phylogenies (in accordance with their current grouping into a single species) but are clearly distinct entities in the SSR tree (Figs. 1,3). Concordant with this strong genetic differentiation, the two subspecies of A. evenia previ- ously were demonstrated not to be cross-compatible [18]. Similarly, A. virginica and A. sp. 353, along with the African and Australian 6× A. indica taxa, formed separate groups in the SSR tree and their hybridization generated F1 hybrids with a marked reduction in fertility (Figs. 2,3). This raises the question of whether these taxa should be treated at the subspecies level or as separate species. On the other hand, the situation was clear for Aeschynomene sp. (328); this taxon was consistently found to be divergent in the ITS, low-copy nuclear gene and the SSR trees (Figs. 1,3). Therefore, it can taxonomic boundaries. Indeed, the approach of genotyp- ing a germplasm collection already has shown to be a powerful tool resolving taxonomic issues and providing the basis of a good taxonomic classification in the leg- ume genus Lens that includes the cultivated lentil [29]. Here, the rare species Aeschynomene pluriarticulata and A. magna were found to have ITS and low-copy nuclear gene sequences very similar to those of A. evenia and A. denticulata, respectively (Fig. 1). The NJ SSR trees further revealed that these two pairs of taxa clustered closely together. This strongly suggested that A. pluriarticulata and A. magna are more likely morphological variants of A. evenia and A. denticulata than distinct species (Fig. 3). But for A. magna, living material is now necessary to confirm this. Conversely, A. evenia ssp. evenia and A. evenia ssp. serrulata Page 10 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 a b c Fig. 6 Variation of nodulation traits observed in accessions of A. evenia and A. indica. (a)Stem nodulation observed in various accessions of A. evenia, 3 weeks post inoculation with ORS278. (b) Root nodule development in accessions of A. evenia and A. indica following inoculation with Bradyrhizobium ORS278 and BTAi1. 14-dpi nodules were cut to observe the leghemoglobin color and reaction defense (arrow). (c) Comparison of plant growth (aerial part) after inoculation with Bradyrhizobium ORS278 and BTAi1, at 14 dpi. Scale bar in (a): 5 mm, in (b): 1 mm b c Fig. Discussion Thus, the genetic structure of the Aeschynomene taxa appeared to mirror the eco-geographic distribution of the associated genotypes, a situation also described for other plants including lentil, tomato, pigeonpea and switchgrass [30–33]. Although not a major aim of this study, such genetic structure can help to understand the origin and the migration of some populations. This is notably the case for A. indica whose natural distribution range is obscure. The identified genetic clusters, which most prominently corre- sponded with geographical distribution patterns, likely reflected real differences within each species. Among the species studied, A. evenia s.s. had the highest genetic diver- sity with 7 genotypes, some of them previously being shown to be fully cross-compatible [18]. It is also noteworthy that the different Aeschynomene taxa displayed very high selfing rates. Using lines that tend to be mostly homozygous facili- tates artificial hybridizations and analysis of confidently seg- regation patterns in the progeny in order to investigate the genetic determinism underlying the nodulation traits. Page 11 of 15 Page 11 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 genetic analysis of this differential susceptibility. Variations in symbiotic traits with naturally-nodulating photosyn- thetic Bradyrhizobium strains have not been thoroughly surveyed yet, but the observations made in the present study using the strains ORS278 and BTAi1 yield promis- ing preliminary results. By evaluating different genotype- rhizobial combinations, we predict that symbiotic poly- morphisms will be found, notably in the induction of nodulation, nitrogen fixation and also in stem nodulation. Crossable accessions exhibiting polymorphic symbiotic phenotypes can then be selected for hybridization experi- ments, subsequent genetic analysis and locus mapping. This will pave the way for the identification of the genes underpinning these symbiotic responses through forward or reverse genetics. To assist in these studies, genomic and genetic data, together with a number of molecular tools, are being accumulated for the model species A. evenia and it thus represents the easiest system to work with [16, 20]. Despite the high genetic diversity observed in A. evenia, the number of available germplasm samples remains relatively modest since it has not been as extensively sampled as other legumes of interest [22]. Therefore, the accessions of other Nod-independent taxa in the same gene pool or in the related one containing A. sensitiva, represent a good complement. In addition, the expected high level of microsynteny with A. Discussion evenia will facilitate a synteny-based positional approach, as has been successfully performed between Pea and Medicago to iden- tify the gene underlying the SYM2 locus [34, 45]. As a re- sult, genetic resources developed for the Nod-independent Aeschynomene clade can be fully exploited for the search of natural variation in nodulation with Bradyrhizobium. Medicago, Lotus or soybean. The identified symbiotic polymorphisms were mostly of two types: some plant- rhizobial strain combinations resulted in non-nodulating phenotypes (Nod−), others in the production of small white infected nodules proved to be defective in nitrogen fixation (Fix−) [34–38]. This indicated that the control of host-rhizobial strain compatibility occurred at two differ- ent levels in the symbiotic process. y p It is noteworthy that the genetic analysis of natural vari- ation shed light on the function of some key symbiotic genes. The LysM-RLK receptors, which were identified using the mutant approach to be the probable Nod factor receptors, represent a well-known example. Indeed, a synteny-based positional cloning identified LYK3 in Medi- cago as corresponding to the SYM2 gene that controls the symbiotic infection in a Nod factor structure dependent manner in the pea ‘Afghanistan’ ecotype [34, 38]. The diver- sity information has also been exploited in Lotus to further substantiate that the LysM-RLK receptors mediate specific recognition of Nod factors [39]. More recently, our current understanding of the symbiotic mechanisms has been chal- lenged for the supposed role of NCR peptides that were ini- tially shown as important effectors of endosymbiont’s differentiation to nitrogen-fixing bacteroids. Making use of the differential ability of Sinorhizobium meliloti Rm41 to form functional or non-nitrogen fixing nodules depending on the Medicago accession used, two genes, NFS1 and NFS2, were identified and shown to code for NCR peptides [36–40]. This broadened the role of the NCR peptides in the fixation stage by revealing that some of them also con- trol discrimination against incompatible microsymbionts. A thorough survey of naturally occurring variation also pro- vided ground for the discovery of new genetic determinants of nodulation. This was notably the case in soybean where this approach led to the identification of two dominant genes that restrict nodulation in a strain-specific manner, Rj2/Rfg1 encoding a TIR-NBS-LRR resistance (R) gene and Rj4 that codes for a thaumatin-like protein [41, 42]. Discussion This revealed that some host genotypes are able to trigger gene- for-gene resistance, which is found in plant-pathogen interactions, to selectively interact with certain symbiotic strains but to exclude others. Genome size estimation and chromosome counting g Flow cytometry measurement was performed on leaf material to estimate genome sizes of various accessions as already explained [16]. These estimates were based on the measurements of three plants per accession using Lycopersicum esculentum (Solanaceae) cv “Roma” (2C = 1.99 pg) as the internal standard. For chromosome num- ber counts, metaphasic chromosomes were prepared from root-tips, spread on slides and stained with DAPI (4′,6-diamidino-2-phenylindole). Chromosomes were counted from images obtained with a fluorescent micro- scope as previously described [16]. Plant DNA extraction underlying genetic determinants. Discoveries of alterna- tive functions in symbiotic genes identified in other model legumes or of new genes involved in the recogni- tion of the still unknown non-Nod bacterial signal, nod- ule functioning and in the restriction of compatibility would add a new dimension to our understanding of the genetic control of nodulation in the Nod-independent symbiosis. Genomic DNA was extracted from fresh leaves using the CTAB (Cetyl Trimethyl Ammonium Bromide) method improved by the addition of β-mercaptoethanol 2% and PVPP 2%. For herbarium material, a protocol adapted for fragmented DNA was used, with increased length of the incubation (90 min), centrifugation (20 min) and precipitation (15 min) steps was used. DNA quantity was evaluated by spectrophotometer and DNA samples normalized to a uniform concentration of 10 ng/μL. Gene sequencing and sequence analysis All the accessions of Aeschynomene used in this study, their geographical origin and source data are listed in Additional file 1: Table S1. Seeds were scarified with sulphuric acid for germination and plants were grown in pots filled with compost under greenhouse conditions (temperature: 26-36 °C, relative humidity: 70%-80%, insect-proof screens) as detailed [16]. Interspecific crosses were performed according to the protocol developed earl- ier and the nature of the resulting hybrids was checked using SSR markers (data not shown) [16]. Fertility of the F1 plants was assessed by recording the number of successfully developed pods per flowering axis and the number of seeds in each pod. The nuclear ribosomal internal transcribed spacer region (ITS: ITS1-5.8S rDNA gene-ITS2) and five low copy nuclear genes CYP1, eiF1α, Sucrose Synthase, SUI1 and a gene homolog to Glyma.07G136800 and Glyma.18G187300 identified in Glycine max were amplified with the primers listed in Additional file 2: Table S2. PCR amplifica- tions, cloning and sequencing of PCR products were performed as already described [17, 18]. The DNA se- quences generated in this study were deposited in Genbank (Additional file 3: Table S3) and additional sequences are available in Additional file 4: Doc. S1. For the phylogenetic analyses, the gene sequences were aligned in ClustalX, version 1.81b and the align- ments were checked in Genedoc v2.7. Phylogenetic reconstructions were performed with the MEGA v7 program using the Neighbor Joining approach and the Tamura 3-parameter model with a 1000 x bootstrap. SSR marker selection and genotyping SSR marker selection and genotyping A total of 500 primer pairs were initially defined to de- velop SSR markers for genetic mapping in A. evenia (Additional file 7: Table S4) [9]. In the present study, they were again tested on two polymorphic accessions of A. evenia and two cytotypes of A. indica (Additional file 8: Table S5). Forward primers all contained a 5′-end M13 tail (5′-CACGACGTTGTAAAACGAC-3′), enabling the tagging the PCR products during the PCR amplification with 4 M13 primer-fluorescent dyes 6-FAM™, NED®, VIC®, or PET® (Applied Biosystems, CA, USA). Amplicon sizes were analysed using an ABI 3700 automatic capillary se- quencer (Applied Biosystems) as previously described [9]. 54 SSR markers were organized in 4-SSR multiplexes and used to genotype the accessions belonging to the A. evenia and the A. sensitiva groups as detailed in Additional file 6: Figure S2. Allele scorings were analysed using GeneMapper 4.0 software (Applied Biosystems) and exported as data ta- bles for two groups of genotyping. Conclusions With the goal of discovering natural variation in the Nod-independent Aeschynomene legumes, we developed a large collection of 226 accessions that spans the geo- graphical distribution of the different taxa in this group, including the model species A. evenia. These accessions were subjected to combined analyses of gene sequen- cing, cytogenetics, hybridization experiments and SSR genotyping. This work resulted in the delineation of taxon boundaries and in the discovery of new genotypes. Taxa displaying a significant genetic diversity were char- acterized by a clear geographically-based genetic struc- ture. In addition, low-copy nuclear genes and patterns of microsatellite diversity illuminated the genetic basis of the Aeschynomene diploid and polyploid taxa that are all predominantly autogamous and have a predicted simple disomic inheritance, two attributes favorable for genet- ics. Such a well-characterized collection of accessions constitutes a major genetic resource for exploring the natural variation of nodulation in response to different Bradyrhizobia strains and for searching for their These illuminating examples show that there is a mileage to be gained from exploring the natural variation in nodulation in the Nod-independent Aeschynomene group. To screen our germplasm collection, many Bradyr- hizobium strains are available, including photosynthetic and non-photosynthetic ones [14, 43, 44]. A recent report revealed important variations in the ability of different Nod-independent Aeschynomene species to be nodulated in a T3SS-dependent fashion by the non-photosynthetic Bradyrhizobium strains STM6978 and USDA61 [14]. Such marked variations were also observed between different accessions of A. evenia s.s., providing the basis for a Page 12 of 15 Page 12 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Plant nodulation and ARA Nodulation tests were carried out using Bradyrhizobium sp. strains ORS278 and BTAi1 [12]. The strains were cultivated for 7 days in yeast-mannitol liquid medium at 34 °C. Root inoculation was performed in an in vitro growth chamber on 7-day-old plants using 1 mL of bac- terial culture with an optical density at 600 nm adjusted to 1. Stem inoculation was carried out in a greenhouse by wrapping the stem of 5-week-old plants with a paper soaked with bacterial culture for 24 h. Stem nodules were observed at 21dpi and root nodules at 14dpi. Nitrogen-fixing activity was estimated on the entire plant by measurement of acetylene reducing activity (ARA) and microscopic observations were performed using a stereo-microscope (Nikon AZ100, Champigny- sur-Marne, France) as previously published [46]. Competing interests Additional file 10: Table S6. Allelic diversity of the SSR markers (XLSX 22 kb) Additional file 10: Table S6. Allelic diversity of the SSR markers (XLSX 22 kb) Funding g This work was supported by a grant from the French National Research Agency (ANR-AeschyNod-14-CE19-0005-01) that served for the design of the study, experimentation and analysis of the data. Availability of data and materials The gene sequences generated in this study have been deposited in GenBank (accession numbers listed in Additional file 3: Table S3) and additional sequences are available in Additional file 4: Doc. S1. All the SSR marker characteristics are included as supplementary information files (Additional files 7, 8, 10, 11, 12 and 13: Table S4-S9). SSR data analysis Genotyping data files were assembled in a single database that was used to determine genetic relationships among the accessions. For this, a distance-based approach was Page 13 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 13 of 15 applied by calculating with a shared allele index a genetic dissimilarity matrix in DARwin v5 software [25]. Then, individual relations were separately ana- lyzed for each ploidy level (2×, 4×, 6×) with a tree construction based on an unweighted Neighbor Join- ing method while genetic affinities between diploid and polyploid species were investigated by a factorial analysis as implemented in DARwin v5. The genetic diversity was evaluated by computing the number of alleles per locus (Na) and the observed heterozygos- ities Ho for each SSR locus and for different accession groups (Additional files 12 and 13: Tables S8, S9). USDA (USA). We are also very grateful to the many herbaria and botanists who provided additional relevant plant material that significantly contributed our sampling. The present work has benefited from the facilities and expertise of the cytometry facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr/spip.php?rubri- que184) and of the molecular cytogenetic facilities of the AGAP laboratory (https:// umr-agap.cirad.fr/en/platforms/plateformes/genotyping). Authors’ contributions CC was responsible for obtaining the plant material and the SSR genotyping data, and together with LB carried out the gene sequencing. RR and PM participated in the SSR marker development and genotyping. XP, GM, LL and BT performed computational analysis of the SSR genotyping data. MGP and MB carried out cytometry analyses. JF and EG handled the nodulation tests. HV, H. Randriambanona and H. Ramanankierna organized field collection of plant material and contributed to the acquisition and analysis of diversity data. GPL provided expertise in legume taxonomy and biogeography, and together with EG reviewed the manuscript. J.F.A. conceived the study, analyzed the data and wrote the paper. All authors read and approved the final manuscript. Additional file 2: Table S2. Nuclear genes used for the phylogenetic analyses. (XLSX 11 kb) Additional file 3: Table S3. GenBank numbers for the sequences used in the phylogenetic analyses. (XLSX 12 kb) Additional file 4: Doc. S1. ITS sequences obtained for the Nod- independent Aeschynomene accessions. (DOCX 30 kb) Additional file 5: Figure S1. Chromosome numbers in new Aeschynomene taxa. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 1008 kb) Additional file 6: Figure S2. Schematic representation of the different steps of the genotyping process from marker selection to data treatment. (PPTX 64 kb) Additional files Additional file 1: Table S1. Accessions used in this study, origin and characteristics. (XLSX 23 kb) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 11: Table S7. Cross-species transferability of the SSR markers (XLSX 13 kb) Additional file 11: Table S7. Cross-species transferability of the SSR markers (XLSX 13 kb) Ethics approval and consent to participate The sources of the Aeschynomene accessions used in this study are indicated in Additional file 1: Table S1. In addition to seedbank and herbaria supply, some accession were collected for purpose of this study in Madagascar (with authorization from the Malagasy Ministry of Scientific Research) and in New-Caledonia (licence n°60,912 from the Northern Province). Collection and identification of these new accessions were performed by Heriniaina Ramanankierana, Herizo Randriambanona, Hervé Vandrot and Jean-François Arrighi. Additional file 7: Table S4. Origin, location and primer sequences for the SSR markers used for genotyping. (XLSX 13 kb) Additional file 8: Table S5. Repeat motif and allelic amplification profiles of the SSR selected for genotyping. (XLSX 13 kb) Additional file 9: Figure S3. Detailed NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 65 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly colored. Identified genotypes are marked with a red dot and numbered. Accessions are designated with their LSTM code mentioned in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb) Additional file 9: Figure S3. Detailed NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 65 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly colored. Identified genotypes are marked with a red dot and numbered. Accessions are designated with their LSTM code mentioned in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb) Author details 1 Additional file 12: Table S8. Scoring of mean allele number per SSR and species (XLSX 15 kb) Additional file 12: Table S8. Scoring of mean allele number per SSR and species (XLSX 15 kb) 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM, Campus International de Baillarguet, F-34398 Montpellier, France. 2CIRAD, Amélioration Génétique et Adaptation des Plantes Méditerranéennes et Tropicales, UMR AGAP, Campus de Lavalette, F-34398 Montpellier, France. 3AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France. 4Comparative Plant and Fungal Biology Department, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK. 5Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud. Université Paris-Saclay, 91198 Gif-sur-Yvette, France. 6Laboratoire de Microbiologie de l’Environnement/Centre National de Recherche sur l’Environnement, 101 Antananarivo, Madagascar. 7IAC, Laboratoire de Botanique et d’Ecologie Végétale Appliquée, UMR AMAP, 98825 Pouembout, Nouvelle-Calédonie, France. 8LSTM, Univ. Montpellier, CIRAD, INRA, IRD, Montpellier SupAgro, Montpellier, France. 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM, Campus International de Baillarguet, F-34398 Montpellier, France. 2CIRAD, Amélioration Génétique et Adaptation des Plantes Méditerranéennes et Tropicales, UMR AGAP, Campus de Lavalette, F-34398 Montpellier, France. 3AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France. 4Comparative Plant and Fungal Biology Department, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK. 5Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud. Université Paris-Saclay, 91198 Gif-sur-Yvette, France. 6Laboratoire de Microbiologie de Additional file 13: Table S9. Observed heterozygosity (Ho) in Aeschynomene species (XLSX 14 kb) Additional file 13: Table S9. Observed heterozygosity (Ho) in Aeschynomene species (XLSX 14 kb) Additional file 14: Figure S4. Comparison of the nodulation properties of A. evenia s.s. and A. indica. Different accessions were root inoculated with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a) Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb) Additional file 14: Figure S4. Comparison of the nodulation properties of A. evenia s.s. and A. indica. Different accessions were root inoculated with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a) Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb) 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. Acknowledgements f We are grateful to the different germplasm banks for provision of seeds: AusPGRIS (Australia), CIAT (Colombia), ILRI (Ethiopia), IRRI (Philippines), Kew Gardens (UK) and Page 14 of 15 Page 14 of 15 Page 14 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Received: 27 October 2017 Accepted: 6 March 2018 References Liu J, Yang S, Zheng Q, Zhu H. Identification of a dominant gene in Medicago truncatula that restricts nodulation by Sinorhizobium meliloti strain Rm41. BMC Plant Biol. 2014;14:167. 15. Rudd VE. The American species of Aeschynomene. Contributions of the United States National Herbarium. 1955;32:1–172. 37. Yang S, Wang Q, Fedorova E, Liu J, Qin Q, Zheng Q, et al. 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EMBO J. 2007;26(17):3923–35. 18. Arrighi JF, Cartieaux F, Chaintreuil C, Brown S, Boursot M, Giraud E. Genotype delimitation in the nod-independent model legume Aeschynomene evenia. PLoS One. 2013;8:e63836. 40. Wang Q, Yang S, Liu J, Terecskei K, Ábrahám E, Gombár A, et al. Host- secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula. Proc Nat Acad Sci (USA). 2017;114(26):6854–9. 19. Czernic P, Gully D, Cartieaux F, Moulin L, Guefrachi I, Patrel D, et al. Convergent evolution of endosymbiont differentiation in Dalbergioid and inverted repeat-lacking clade legumes mediated by nodule-specific cysteine-rich peptides. Plant Physiol. 2015;169:1254–65. 41. Yang S, Tang F, Gao M, Krishnan HB, Zhu HR. Gene-controlled host specificity in the legume-rhizobia symbiosis. Proc Natl Acad Sci U S A. 2010; 107(43):18735–40. 42. Tang F, Yang S, Liu J, Zhu H. Rj4, a gene controlling nodulation specificity in soybeans, encodes a thaumatin-like protein but not the one previously reported. Plant Physiol. 2016;170(1):26–32. 20. Fabre S, Gully D, Poitout A, Patrel D, Arrighi JF, Giraud E, et al. Nod factor-independent nodulation in Aeschynomene evenia required the common plant-microbe symbiotic toolkit. Plant Physiol. 2015;169(4): 2654–64. 43. Molouba F, Lorquin J, Willems A, Hoste B, Giraud E, Dreyfus B, et al. Photosynthetic bradyrhizobia from Aeschynomene spp. References Chaintreuil C, Arrighi JF, Giraud E, Miche L, Moulin L, Dreyfus B, Munive-Hernadez J, Villegas-Hernadez M, Béna G. Evolution of symbiosis in the legume genus Aeschynomene. New Phytol. 2013;200:1247–59. 8. Chaintreuil C, Arrighi JF, Giraud E, Miche L, Moulin L, Dreyfus B, Munive-Hernadez J, Villegas-Hernadez M, Béna G. Evolution of symbiosis in the legume genus Aeschynomene. New Phytol. 2013;200:1247–59. 31. 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Mtsym6, a gene conditioning Sinorhizobium strain-specific nitrogen fixation in Medicago truncatula. Plant Physiol. 2000; 123(3):845–51. 14. Okazaki S, Tittabutr P, Teulet A, Thouin J, Fardoux J, Chaintreuil C, et al. Rhizobium-legume symbiosis in the absence of nod factors: two possible scenarios with or without the T3SS. ISME J. 2016;10:64–74. 36. Received: 27 October 2017 Accepted: 6 March 2018 23. African Plant Database (version 3.4.0). Conservatoire et Jardin botaniques de la Ville de Genève and South African National Biodiversity Institute, Pretoria, http://www.ville-ge.ch/musinfo/bd/cjb/africa/. 24. Tropical Forages: an interactive selection tool. Cook, B.G., Pengelly, B.C., Brown, S.D., Donnelly, J.L., Eagles, D.A., Franco, M.A., Hanson, J., Mullen, B.F., Partridge, I.J., Peters, M. and Schultze-Kraft, R. CSIRO, DPI&F(Qld), CIAT and ILRI, Brisbane, Australia. 2005. http://www.tropicalforages.info References 1. Graham PH, Vance CP. Legumes: importance and constraints to greater use. Plant Physiol. 2003;131:872–7. 1. Graham PH, Vance CP. Legumes: importance and constraints to greater use. Plant Physiol. 2003;131:872–7. ILRI, Brisbane, Australia. 2005. http://www.tropicalforages.info 25. Perrier X, Jacquemoud-Collet JP. 2006. DARwin software http://DARwin. cirad.fr/DARwin 2. GED O. Speak, friend, and enter: signaling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol. 2013;11: 252–63. 26. Vasse J, de Billy F, Truchet G. Abortion of infection during the rhizobium meliloti-alfalfa symbiotic interaction is accompanied by a hy- persensitive reaction. Plant J. 1993;4:555–66. 3. Oldroyd GED, Murray JD, Poole PS, Downie JA. The rules of engagement in the legume-Rhizobial Symbiosis. Annu Rev Genet. 2011;45:119–44. 3. Oldroyd GED, Murray JD, Poole PS, Downie JA. The rules of engagement in the legume-Rhizobial Symbiosis. Annu Rev Genet. 2011;45:119–44. 27. Klie M, Schie S, Linde M, Debener T. The type of ploidy of chrysanthemum is not black or white: a comparison of a molecular approach to published cytological methods. Front Plant Sci. 2014;5:479. 4. Reid DE, Ferguson BJ, Hayashi S, Lin YH, Gresshoff PM. Molecular mechanisms controlling legume autoregulation of nodulation. Ann Bot. 2011;108(5):789–95. 4. Reid DE, Ferguson BJ, Hayashi S, Lin YH, Gresshoff PM. Molecular mechanisms controlling legume autoregulation of nodulation. Ann Bot. 2011;108(5):789–95. 28. Leal-Bertioli S, Shirasawa K, Abernathy B, Moretzsohn M, Chavarro C, Clevenger J, Ozias-Akins P, Jackson S, Bertioli D. Tetrasomic recombination is surprisingly frequent in allotetraploid Arachis. Genetics 2015;199(4):1093–105. 5. Sprent JI. Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation. New Phytol. 2007;174:11–25. 6. Sprent JI, James EK. Legume-rhizobial symbiosis: an anorexic model? New Phytol. 2008;179:3–5. 29. Wong MM, Gujaria-Verma N, Ramsay L, Yuan HY, Caron C, Diapari M, et al. Classification and characterization of species within the genus lens using genotyping-by-sequencing (GBS). PLoS One. 2015;10(3):e0122025. 7. Boivin C, Ndoye I, Molouba F, De Lajudie P, Dupuy N, Dreyfus B. Stem nodulation in legumes: diversity, mechanisms, and unusual characteristics. Crit Rev Plant Sci. 1997;16(1):1–30. 7. Boivin C, Ndoye I, Molouba F, De Lajudie P, Dupuy N, Dreyfus B. Stem nodulation in legumes: diversity, mechanisms, and unusual characteristics. Crit Rev Plant Sci. 1997;16(1):1–30. 30. Blanca J, Cañizares J, Cordero L, Pascual L, Diez MJ, Nuez F. Variation revealed by SNP genotyping and morphology provides insight into the origin of the tomato. PLoS One. 2012;7(10):e48198. 8. Chaintreuil et al. BMC Plant Biology (2018) 18:54 symbionts in tropical Africa and central America reveal that nod-independent stem nodulation is not restricted to photosynthetic bradyrhizobia. Environ Microbiol. 2010;12(8):2152–64. 45. Gualtieri G, Kulikova O, Limpens E, Kim DJ, Cook DR, Bisseling T, et al. Microsynteny between pea and Medicago truncatula in the SYM2 region. Plant Mol Biol. 2002;50(2):225–35. 46. Bonaldi K, Gargani D, Prin Y, Fardoux J, Gully D, Nouwen N, et al. Nodulation of Aeschynomene afraspera and A. indica by photosynthetic Bradyrhizobium sp. strain ORS285: the nod-dependent versus the nod- independent symbiotic interaction. MPMI. 2011;24:1359–71. symbionts in tropical Africa and central America reveal that nod-independent stem nodulation is not restricted to photosynthetic bradyrhizobia. Environ Microbiol. 2010;12(8):2152–64. 45. Gualtieri G, Kulikova O, Limpens E, Kim DJ, Cook DR, Bisseling T, et al. Microsynteny between pea and Medicago truncatula in the SYM2 region. Plant Mol Biol. 2002;50(2):225–35. 46. Bonaldi K, Gargani D, Prin Y, Fardoux J, Gully D, Nouwen N, et al. Nodulation of Aeschynomene afraspera and A. indica by photosynthetic Bradyrhizobium sp. strain ORS285: the nod-dependent versus the nod- independent symbiotic interaction. MPMI. 2011;24:1359–71. References are specific to stem-nodulated species and form a separate 16S ribosomal DNA restriction fragment length polymorphism group. Appl Environ Microbiol. 1999;65(7):3084–94. 21. Nouwen N, Arrighi JF, Cartieaux F, Gully D, Klopp C, Giraud E. The role of rhizobial (NifV) and plant (FEN1) homocitrate synthesis in Aeschynomene - photosynthetic Bradyrhizobium symbiosis. Sci Rep. 2017;7:448. 22. Gentzbittel L, Andersen SU, Ben C, Rickauer M, Stougaard J, Young ND. Naturally occurring diversity helps to reveal genes of adaptive importance in legumes. Front Plant Sci. 2015;6:269. 44. Miché L, Moulin L, Chaintreuil C, Contreras-Jimenez JL, Munive-Hernandez JA, Del Carmen Villegas-Hernandez M, et al. Diversity analyses of Aeschynomene Page 15 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 • 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 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 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 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:
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The Effect of Freezing on Non-invasive Prenatal Testing
Scientific reports
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Xiaolei Xie1,2, Fuguang Li1, Weihe Tan1, Weiguo Yin2, Feiyan Chen3 & Xiaoyan Guo1 Xiaolei Xie1,2, Fuguang Li1, Weihe Tan1, Weiguo Yin2, Feiyan Chen3 & Xiaoyan Guo1 Plasma cryopreservation is unavoidable in China, due to technical specifications requiring storage of additional plasma at −80 degrees for three years. However, the effect of freezing on non-invasive prenatal testing (NIPT) is still uncertain. We collected 144 euploid pregnant samples, 22 on trisomy 21, 4 on trisomy 13, and 3 on trisomy 18, by massively parallel sequencing before and after freezing. Compared with the success rate of 100% of fresh samples, the detection success rates of trisomy 21, trisomy 13 and euploidy in frozen samples by NIPT were 95.45%, 75% and 95.14%, respectively. Of these, 9 cases of frozen sample sequencing failed, with 8 cases being due to high GC content. The chromosome 21 (chr21) z-value of the frozen trisomy 21 samples was lower than that of fresh samples. Meanwhile, freezing reduced the male positive foetal cell-free DNA (cfDNA) fraction, which was accompanied by an increase in the Unimap-GC level in the massively parallel sequencing data and a decrease in the Unique reads/Total reads ratio. Laboratory freezing reduced the chr21 z-value of foetal trisomy 21, which can be explained by a reduction in the foetal cfDNA fraction and effective Unique reads for NIPT analysis. The Unimap-GC content of the serum samples after freezing was higher, which can lead to failure of NIPT detection. Received: 29 August 2018 Accepted: 10 April 2019 Published: xx xx xxxx Chromosomal foetal aneuploidies represent a major class of genetic defects, including trisomy 21, trisomy 18, trisomy 13 and sex chromosome aneuploidies1,2. Most notably, the incidence and hazard of trisomy 21 (Down syndrome) for families are much higher compared to trisomy 18 and 13, with the current occurrence rate for tri- somy 21 being one in 417 newborns2. Traditional serum screening is not effective for trisomy 21 detection3. With the development of next generation sequencing technology, non-invasive prenatal testing (NIPT) for foetal chro- mosomal aneuploidy serum screening has been validated by various clinical trials, and NIPT has been proven to be highly sensitive and specific for trisomy 214–6. g yi y Foetal cell-free DNA (cfDNA) is found in maternal plasma and serum7. One important clinical application is the use of cfDNA in maternal blood for non-invasive prenatal diagnosis8,9. Foetal trisomy determination of spe- cific chromosomes depends on a small increase in the proportion of the chromosome and is detected by a z-test. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Xiaolei Xie1,2, Fuguang Li1, Weihe Tan1, Weiguo Yin2, Feiyan Chen3 & Xiaoyan Guo1 The foetal cfDNA fraction is an important parameter to determine the z value for foetal chromosomal aneuploid assessment10,11.t During the NIPT laboratory testing process, the remaining maternal plasma samples are often stored at −80 °C for a long period of time. However, the effect of freezing on NIPT detection is still uncertain. In this study, we investigated the impact of freezing on the performance of NIPT testing, thus allowing us to understand the effect of laboratory treatment on trisomy 21 detection sensitivity. Received: 29 August 2018 Accepted: 10 April 2019 Published: xx xx xxxx Results The freezing time of each group was different, with the longest freezing time in the trisomy 13 group, with a median of 76 days, and the shortest freezing time in the euploid group, with a median of 13 days.ih and 25 weeks. The freezing time of each group was different, with the longest freezing time in the trisomy 13 group, with a median of 76 days, and the shortest freezing time in the euploid group, with a median of 13 days. h l ll l l fi d h f f l Th f l g p y g p g p y Among the 173 samples, all maternal plasma DNA first sequenced without freezing was successful. The foetal chromosomal aneuploidy or euploidy, including trisomy 21, trisomy 18, and trisomy 13, was accurately iden- tified with a detection sensitivity of 100% and a detection specificity of 100%. After freezing serum samples at −80 °C, the detection success rates of trisomy 21, trisomy 13 and euploidy on NIPT were 95.45%, 75% and 95.14%, respectively. Nine cases of plasma cfDNA for a second sequencing failed, including 1 case of trisomy 21, 1 case of trisomy 13, and 7 cases of euploidy. The median maternal age and gestational weeks for the nine cases in which sequencing failed were 34.5(range 27–44) and 16 (range 13–25), respectively. There was no specific distri- bution of maternal age or gestational week in these 9 failed cases. Among these cases, 8 cases were due to high GC content, and 1 case of trisomy 13 detection failed. All failed results were interpreted as redo by the BambniTest software. Details are shown in Table 2. Freezing reduces the z value of trisomy 21. The results of NIPT were consistent before and after freez- ing except for the detection failure of a few samples. Comparing the chromosome 21 (chr21) z value of NIPT between fresh samples and frozen samples, 80.95% (17/21) of fresh sample values were higher than frozen values, and 19.05% (4/21) of the values were lower than frozen values (Fig. 1a). Our data show that the chr21 z values of the fresh sample group were significantly higher than the values of the frozen group by using a paired t test (Fig. 1b). There was no significant difference between the chromosome 13 (chr13) z value and the chromosome 18 (chr18) z value before and after freezing (Fig. 1c,d). Results The normal euploid foetus exhibited no obvious difference in fresh and frozen samples (Fig. 1e). Comparing the sequencing data before and after freezing. We next investigated the sequencing data of 27 positive samples (21 trisomy 21, 3 trisomy13 and 3 trisomy18) before and after freezing. There were no sig- nificant differences in total reads and uniq-reads between the two groups (see Supplemental Fig. 1), but the ratios of Uniq-reads/Total-reads in fresh samples were higher than the ratios in frozen samples (Fig. 2a). This suggests that more effective sequencing data in fresh samples were used to evaluate the NIPT results. At the same time, the content of Unimap-GC upon freezing increased significantly (Fig. 2b).i p p g gi y ( g ) To confirm whether changes in foetal cfDNA fractions occur due to freezing, we further selected and com- pared the foetal fraction in 18 cases of male positive samples (14 trisomy 21, 2 trisomy13 and 2 trisomy18). The minimum chromosome Y (chrY) z-value of male positive samples was 22.47, and the foetuses were confirmed to be male by follow-up. The result showed that the foetal cfDNA fraction in fresh samples was slightly higher than the fractions in frozen samples by using the paired t test (Fig. 2c). Results Maternal plasma DNA sequencing before and after freezing. Maternal characteristics of the study population are presented in Table 1. We collected 144 euploid pregnant samples, 22 on trisomy 21, 4 on trisomy 13, and 3 on trisomy 18 for non-invasive prenatal cell-free DNA sequencing before and after freezing. The median gestational week for trisomy 21 was 17 (range 12–23), which is slightly older than the trisomy13 and 18 groups. In the clinical routine, traditional serum screening abnormalities would be required for NIPT, so the T21 gestational week was slightly older. Trisomies 13 and 18 were often accompanied by malformations that can be detected by B-ultrasound, suggesting early detection of gestational week. The gestational age of all samples was between 12 1Prenatal Diagnosis Center, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital, Qingyuan, Guangdong, 511518, China. 2Molecular Diagnosis Center, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital, Qingyuan, Guangdong, 511518, China. 3Guangzhou KingMed Company, Guangdong, China. Correspondence and requests for materials should be addressed to X.G. (email: 743090128@qq.com) Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 1 www.nature.com/scientificreports/ n Median, range Maternal age (years) Gestational weeks Freezing time (days) Trisomy 21 22 38.5 (15.0–44.0) 17.0 (12.0–23.0) 25.0 (12.0–88.0) Trisomy 13 4 30.0 (30.0–40.0) 16.0 (16.0–20.0) 76.0 (14.0–78.0) Trisomy 18 3 36.0 (35.0–41.0) 16.0 (15.0–20.0) 44.0 (38.0–45.0) Euploid 144 31.0 (16.0–43.0) 17.0 (12.0–25.0) 13.0 (9.0–18.0) Table 1. Characteristics of pregnant women within the study population. Table 1. Characteristics of pregnant women within the study population. Syndrome The detection success ratios of NIPT Fresh samples Frozen samples Trisomy 21 100% (22/22) 95.45% (21/22) Trisomy 13 100% (4/4) 75% (3/4) Trisomy 18 100% (3/3) 100% (3/3) Euploid 100% (144/144) 95.14% (137/144) Table 2. Summary of the syndromes detected before and after freezing. Syndrome The detection success ratios of NIPT Fresh samples Frozen samples Trisomy 21 100% (22/22) 95.45% (21/22) Trisomy 13 100% (4/4) 75% (3/4) Trisomy 18 100% (3/3) 100% (3/3) Euploid 100% (144/144) 95.14% (137/144) Table 2. Summary of the syndromes detected before and after freezing. Table 2. Summary of the syndromes detected before and after freezing. Table 2. Summary of the syndromes detected before and after freezing. and 25 weeks. Discussion Non-invasive prenatal testing by massively parallel sequencing has been reported since 200812. Numerous studies proved that NIPT was highly accurate for detecting foetal chromosomal aneuploidies2,5,6,8, which presents a new era of prenatal screening. Frozen serum samples are unavoidable in the process of NIPT detection in the labora- tory. The findings in this study demonstrated that the chr21 z-value of NIPT detection was reduced by laboratory freezing treatment. Meanwhile, freezing reduced the male foetal cfDNA fraction. It was also shown that the con- tent of Unimap-GC in frozen samples increased, whereas the Unique reads/Total reads ratio decreased.fh p p q Foetal cfDNA is a key determinant that affects the performance of foetal DNA-based prenatal tests11,13. The foetal fraction is derived from apoptotic trophoblastic cells in the placenta14. Samples with sufficient foetal frac- tions that pass quality control metrics can provide an accurate assessment of the chromosomes tested. A variety of methods are used to calculate foetal DNA concentration15,16. In our study, freezing led to a decrease in foetal DNA Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 2 www.nature.com/scientificreports/ Figure 1. Non-invasive prenatal testing of trisomy 21,13,18 or euploid foetus samples before and after freezing. (a,b) Comparison of the chr21 z-score of trisomy 21 before and after freezing. (c) Comparison of the chr13 z-score of trisomy 13 before and after freezing. (d) Comparison of the chr18 z-score of trisomy 18 before and after freezing. (e) Comparison of the chr21 z-score of the euploid foetus before and after freezing. Error bars are the SEM. **P < 0.01, paired t test. Figure 1. Non-invasive prenatal testing of trisomy 21,13,18 or euploid foetus samples before and after freezing. (a,b) Comparison of the chr21 z-score of trisomy 21 before and after freezing. (c) Comparison of the chr13 z-score of trisomy 13 before and after freezing. (d) Comparison of the chr18 z-score of trisomy 18 before and after freezing. (e) Comparison of the chr21 z-score of the euploid foetus before and after freezing. Error bars are the SEM. **P<0.01, paired t test. Figure 1. Non-invasive prenatal testing of trisomy 21,13,18 or euploid foetus samples before and after freezing. (a,b) Comparison of the chr21 z-score of trisomy 21 before and after freezing. (c) Comparison of the chr13 z-score of trisomy 13 before and after freezing. (d) Comparison of the chr18 z-score of trisomy 18 before and after freezing. Discussion (e) Comparison of the chr21 z-score of the euploid foetus before and after freezing. Error bars are the SEM. **P < 0.01, paired t test. Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a) Comparison of the ratios of Uniq-reads/Total-reads in 27 positive samples, including 21 trisomy 21, 3 trisomy 13and 3 trisomy 18. (b) Comparison of the content of Unimap-GC in 27 positive samples before and after freezing. (c) Comparison of the positive male foetal fraction before and after freezing. Error bars are the SEM. **P < 0.01, ****P < 0.0001, paired t test. Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a) Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a) Comparison of the ratios of Uniq-reads/Total-reads in 27 positive samples, including 21 trisomy 21, 3 trisomy 13and 3 trisomy 18. (b) Comparison of the content of Unimap-GC in 27 positive samples before and after freezing. (c) Comparison of the positive male foetal fraction before and after freezing. Error bars are the SEM. **P < 0.01, ****P < 0.0001, paired t test. Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a) Comparison of the ratios of Uniq-reads/Total-reads in 27 positive samples, including 21 trisomy 21, 3 trisomy 13and 3 trisomy 18. (b) Comparison of the content of Unimap-GC in 27 positive samples before and after freezing. (c) Comparison of the positive male foetal fraction before and after freezing. Error bars are the SEM. **P < 0.01, ****P < 0.0001, paired t test. concentration after cryopreservation. With the reduction of effective sequencing data for NIPT analysis, it is rea- sonable to speculate that the chr21 z-value decreases after freezing. Further analysis revealed that the 21-trisomy foetal cfDNA concentration did not decrease with the freezing time, suggesting that freezing may affect the chr21 z-value regardless of the length of freezing time. This reminds physicians and examiner to avoid freezing the NIPT plasma during transport or storage, especially plasma with low foetal cfDNA concentrations. The freezing time was different among groups. Trisomy 13 was frozen the longest and showed the worst performance. Discussion Due to limitation of the number of samples, we cannot make an inference regarding whether the long freezing time leads to a low detection success rate. Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 3 www.nature.com/scientificreports/ On average, the amount of foetal cfDNA in plasma from a pregnant woman is approximately 2–20%, but there is a large variance in the fraction of foetal cfDNA between patients17. The higher the percentage of foetal cfDNA, the more effective NIPT is at distinguishing foetal trisomy from a euploid foetus, especially for trisomy 2116. This study suggests that the high level of foetal cfDNA concentration and more effective data will increase the detection success rate of trisomy 21, especially the z-value in cut-off regions. It has been speculated that the foetal fraction should be at least 4% to allow for reliable detection of common trisomies18. Currently, the threshold we use for accurate detection of trisomy using Illumina’s NextSeq CN500 is a foetal cfDNA fraction of no less than 3.5%. In the 9 cases of NIPT detection failure after freezing, no sample was detected due to the cfDNA concen- tration being below 3.5%.f g Foetal cfDNA is affected by many factors, including the gestational age19, maternal weight16 and physical activ- ity20. The cfDNA in plasma increases as pregnancy progresses, but it decreases with maternal weight gain. There seemed to be a slight positive correlation between the foetal DNA fraction and the crown-rump length (CRL), log freeβ-hCG MoM and log PAPP-A MoM16. However, the nuchal translucency (NT) and serum prenatal screening results in the high-risk and low-risk groups showed no significant differences in the foetal cfDNA fraction21. In the samples positive for trisomies 13 and 18, the foetal fractions were significantly lower compared with that of the NIPT-negative cases and trisomy21, indicating that different foetal aneuploidies have varied effects on the foetal DNA fraction22,23.The higher foetus fractions in trisomy 21 may be one of the reasons that the detection performance for trisomy 21 was better than that of trisomy 13 or 18. These studies indicate that trisomy 21 is more susceptible to foetal fraction. Because of the limited number of trisomies 13 and 18, we did not see the effect of the foetal fraction changes in trisomies 13 and 18 after freezing in this study. Materials and Methods Study design and sample collection. Samples from 173 pregnant women were collected between April 2017 and July 2017 for this study. The research adhered to the tenets of the Declaration of Helsinki on research involving human subjects. The study design was approved by the ethics committee of the Six Affiliated Hospital of Guangzhou Medical University. All participants gave informed written consent. The pregnant women were enrolled as participants and were determined to be high risks for aneuploidy by conventional serum screen- ing, single serum index abnormalities, abnormal foetal ultrasound or maternal age (≥35 years). All partici- pants (range 12 to 25 weeks gestation) required NIPT detection to avoid foetal trisomies 21,18 and 13. One hundred seventy-three pregnant women bearing 99 male foetuses and 74 female fetuses participated in the study. Follow-up information was acquired by telephone. All positive results of NIPT detection were confirmed by pre- natal diagnosis for chromosome karyotype analysis. The foetus gender was confirmed by follow-up after birth. Ten milliliters of maternal peripheral blood was collected into cell-free DNA BCT tubes (Streck company, La Vista, USA) for NIPT detection. Maternal plasma processing, cfDNA extraction and sequencing. The peripheral maternal blood samples were sent to a clinical laboratory within 2 hours. The blood samples were centrifuged first at 1,600 g for 10 min at 4 °C. The plasma was transferred to microcentrifuge tubes and then centrifuged again at 16,000 g for 10 min to remove residual cells. Each blood sample was separated into three tubes of plasma (1.2 ml serum/ tube), with the fresh first tube used for a follow-up experiment and the rest stored at −80 °C. On the first day, we extracted cfDNA in 1.2 ml of serum from pregnant women using the Berry Genomics Nucleic Acid kit (Berry genomics company, Beijing, China). The cfDNA was dissolved in 40 μL of Tris-HCL solution (10 mM), with a concentration greater than 0.5 ng/μL. The next day, all cfDNA was used as the input DNA to create a library using the library prep kit from Berry Genomics. Plasma DNA libraries of 95 samples were indexed using 6 bp indexing oligos and quantitated by the SYBR fast qPCR kit from Kapa Biosystems (Woburn, MA, USA). The library con- centration of each sample must be greater than 10pM before sequencing. On the third day, each sample library was pooled and loaded into a NextSeq flow cell at 3pM DNA concentration. Discussion Future research should be exam- ined whether increased GC content in cfDNA after cryopreservation means that freezing more likely destroys the two hydrogen bonds between adenine and thymine, resulting in an increased GC content. y g y g In conclusion, this study determined that freezing decreased the chr21 z-value of NIPT detection. Laboratory freezing reduced the foetal cfDNA concentration, accompanied by an increase in the Unimap-GC content in the massively parallel sequencing data and a decrease in the Unique reads/Total reads ratio. The findings in this study provide one laboratory factor affecting the accuracy of NIPT detection, especially for trisomy 21, which will help to further understand cfDNA characteristics. Materials and Methods Clustering and sequencing were con- ducted according to Illumina’s NextSeq CN500 instructions using the single-ended 45 bp sequencing protocol. The time of the NIPT clinical reports for the fresh samples was no more than 7 working days. The procedure of NIPT detection in frozen samples was the same as in fresh samples. Sequencing data analysis The male foetal DNA fraction calculation based on unique read counts on the Y chromosome were reliable15. We calculated the proportion of male foetal DNA based on the calculation method published by Irena H et al.16.h The male foetal DNA fraction = (% chrY-female % chrY)/(male % chrY- female % chrY), where % chrY repre- sents the ratio of unique reads of the Y chromosome to the number of unique reads on all chromosomes; female % chrY is the average of % chrY in 200 pregnant women bearing euploid female foetuses; and male % chrY repre- sents the average of the ratio of % chrY of two adult male volunteers. Statistical analysis. Data were analysed using GraphPad Prism 6.0 software. Data between two groups were analysed by a two-tailed paired t test. Values are presented as the mean ± SEM. Statistical significance was set at **P < 0.01 and ****P < 0.0001. Statistical analysis. Data were analysed using GraphPad Prism 6.0 software. Data between two groups were analysed by a two-tailed paired t test. Values are presented as the mean ± SEM. Statistical significance was set at **P < 0.01 and ****P < 0.0001. References 1. Goldwaser, T., Klugman, S. & Cell-free, D. N. A. for the detection of fetal aneuploidy. FERTIL STERIL. 109, 195–200, https://doi. org/10.1016/j.fertnstert.2017.12.019 (2018). 1. Goldwaser, T., Klugman, S. & Cell-free, D. N. A. for the detection of fetal aneuploidy. FERTIL STERIL. 109, 195–200, https://doi. org/10.1016/j.fertnstert.2017.12.019 (2018). g j ( ) 2. Norton, M. E. et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 372, 1589–1597, https:/ org/10.1056/NEJMoa1407349 (2015). g J ( ) 3. Bianchi, D. W. et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 370, 799–808, https://doi org/10.1056/NEJMoa1311037 (2014). g 3. Bianchi, D. W. et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 370, 799–808, https://doi org/10.1056/NEJMoa1311037 (2014). g 4. McKanna, T. et al. Fetal fraction-based risk algorithm for non-invasive prenatal testing: screening for trisomies 13 and 18 and triploidy in women with low cell-free fetal DNA. Ultrasound Obstet Gynecol. 53, 73–79, https://doi.org/10.1002/uog.19176 (2019). g 4. McKanna, T. et al. Fetal fraction-based risk algorithm for non-invasive prenatal testing: screening for trisomies 13 and 18 and triploidy in women with low cell-free fetal DNA. Ultrasound Obstet Gynecol. 53, 73–79, https://doi.org/10.1002/uog.19176 (2019). 5. Allyse, M. A. & Wick, M. J. Noninvasive Prenatal Genetic Screening Using Cell-free DNA. JAMA. 320, 591–592, https://doi. p y y p g g 5. Allyse, M. A. & Wick, M. J. Noninvasive Prenatal Genetic Screening Using Cell-free DNA. JAMA. 320, 591–592, https://doi org/10.1001/jama.2018.9418 (2018). g j ( ) 6. Liao, C. et al. Noninvasive prenatal diagnosis of common aneuploidies by semiconductor sequencing. Proc Natl Acad Sci USA 111 7415–7420, https://doi.org/10.1073/pnas.1321997111 (2014). p g p 7. Lo, Y. M. et al. Presence of fetal DNA in maternal plasma and serum. LANCET. 350, 485–487, https://doi.org/10.1016/S0140 6736(97)02174-0 (1997). ( ) ( ) 8. Gil, M. M., Accurti, V., Santacruz, B., Plana, M. N. & Nicolaides, K. H. Analysis of cell-free DNA in maternal blood in screening for aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol. 50, 302–314, https://doi.org/10.1002/uog.17484 (2017). p p y y p g g 9. Valderramos, S. G. et al. Cell-free DNA screening in clinical practice: abnormal autosomal aneuploidy and microdeletion re Am J Obstet Gynecol. 215, 626.e1–626.e10, https://doi.org/10.1016/j.ajog.2016.06.039 (2016). y p g j j g 0. Le Conte, G. et al. Cell-free fetal DNA analysis in maternal plasma as screening test for trisomies 21, 18 and 13 in twin pregnancy Ultrasound Obstet Gynecol. Data Availability The data generated during and/or analysed during the current study are available from the corresponding author by reasonable request. y The data generated during and/or analysed during the current study are available from the corresponding author by reasonable request. y The data generated during and/or analysed during the current study are available from the corresponding author by reasonable request. References 52, 318–324, https://doi.org/10.1002/uog.18838 (2018).h y p g j j g 10. Le Conte, G. et al. Cell-free fetal DNA analysis in maternal plasma as screening test for trisom Ultrasound Obstet Gynecol. 52, 318–324, https://doi.org/10.1002/uog.18838 (2018).h y p g g ( ) 11. Burns, W. et al. The association between anticoagulation therapy, maternal characteristics, and a failed cfDNA test due to a low fetal fraction. Prenat Diagn. 37, 1125–1129, https://doi.org/10.1002/pd.5152 (2017). g p g p ( ) 12. Chiu, R. W. et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci USA 105, 20458–20463, https://doi.org/10.1073/pnas.0810641105 (2008). p p g p ( ) 3. Kim, S. K. et al. Determination of fetal DNA fraction from the plasma of pregnant women using sequence read counts. Prenat Diagn 35, 810–815, https://doi.org/10.1002/pd.4615 (2015). , , p g p ( ) 14. Hahn, S., Huppertz, B. & Holzgreve, W. Fetal cells and cell free fetal nucleic acids in maternal blood: new tools to study abnormal placentation? PLACENTA. 26, 515–526, https://doi.org/10.1016/j.placenta.2004.10.017 (2005). p p g j p 5. van Beek, D. M. et al. Comparing methods for fetal fraction determination and quality control of NIPT samples. Prenat Diagn. 37 769–773, https://doi.org/10.1002/pd.5079 (2017). p g p 6. Hudecova, I. et al. Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies PLOS ONE. 9, e88484, https://doi.org/10.1371/journal.pone.0088484. eCollection 2014 (2014). g j 17. Liang, B. et al. Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference. Sci Rep. 8, 17675, https://doi.org/10.1038/s41598-018-35738-0 (2018).h 18. Canick, J. A., Palomaki, G. E., Kloza, E. M., Lambert-Messerlian, G. M. & Haddow, J. E. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn. 33, 667–674, https://doi.org/10.1002/ pd.4126 (2013).f p 19. Wang, E. et al. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn. 33, 662–666, https://doi.org/10.1002/pd.4119 (2013).ht g 0. Schlutter, J. M. et al. The cell-free fetal DNA fraction in maternal blood decreases after physical activity. Prenat Diagn. 34, 341–344 https://doi.org/10.1002/pd.4306 (2014).hf p g p 21. Brar, H., Wang, E., Struble, C., Musci, T. J. & Norton, M. E. The fetal fraction of cell-free DNA in maternal plasma is not affected by a priori risk of fetal trisomy. J Matern Fetal Neonatal Med. Sequencing data analysis Sequencing data analysis. A analysis of sequencing reads was performed by the BambniTest software (Berry genomics company, Beijing, China). Sequencing reads were mapped to the non repeat-masked reference human genome (hg18) using the SOAP bioinformatic algorithm24. Unique reads were the only mapped one chro- mosome DNA sequences, which were analysed for follow-up counting. The number of unique sequence reads aligned to each chromosome was counted, and the Unimap-GC content was calculated. To offset the GC bias generated between runs, the final displayed Unimap-GC was corrected by the BambniTest25. If the Unimap-GC content was ≥42%, the software will interpret the sample as having high GC content. High GC content and sequencing detection failure were all defined as redo by the BambniTest software. q gi yt For each chromosome of each sample, the unique chromosome representation (% chrN) value was generated according to the following equation: % chrN = Unique count for chrN /Total unique count of the sequences Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 4 www.nature.com/scientificreports/ mapped to all the autosomes. The assessment of foetal aneuploidy risk was according to the formula chrN z-score = (% chrN- mean% chrNreference)/S.D.% chrNreference. The cut-off z-value was 312. Four hundred twelve pregnant women with euploidy foetuses were selected as the reference set. The mean (% chrNreference) and the standard deviation (% chrNreference) of the reference set were used for calculating z-scores. In this sequencing plat- form, the lowest foetal concentration for distinguishing trisomy effectively was 3.5%. Samples below this quality control standard were recommended to re-collect blood or abandon testing. mapped to all the autosomes. The assessment of foetal aneuploidy risk was according to the formula chrN z-score = (% chrN- mean% chrNreference)/S.D.% chrNreference. The cut-off z-value was 312. Four hundred twelve pregnant women with euploidy foetuses were selected as the reference set. The mean (% chrNreference) and the standard deviation (% chrNreference) of the reference set were used for calculating z-scores. In this sequencing plat- form, the lowest foetal concentration for distinguishing trisomy effectively was 3.5%. Samples below this quality control standard were recommended to re-collect blood or abandon testing. Foetal fraction calculation. The male foetal DNA fraction calculation based on unique read counts on the Y chromosome were reliable15. We calculated the proportion of male foetal DNA based on the calculation method published by Irena H et al.16.h Foetal fraction calculation. Author Contributions X.L.X. and X.Y.G. designed experiments, analysed and interpreted the data, and wrote the manuscript; F.G.L. and F.Y.C. performed NIPT detection; WH.T. performed genetic counselling for patients; X.L.X. and W.G.Y. analysed and interpreted the data. References 26, 143–145, https://doi.org/10.3109/14767058.2012.722731 (2013). 22. Rava, R. P., Srinivasan, A., Sehnert, A. J. & Bianchi, D. W. Circulating fetal cell-free DNA fractions differ in autosomal aneuploidies and monosomy X. CLIN CHEM. 60, 243–250, https://doi.org/10.1373/clinchem.2013.207951 (2014).f 3. Suzumori, N. et al. Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy. J HUM GENET. 61, 647–652, https:/ doi.org/10.1038/jhg.2016.25 (2016). g j g 4. Liu, C. M. et al. SOAP3: ultra-fast GPU-based parallel alignment tool for short reads. BIOINFORMATICS. 28, 878–879, https://doi org/10.1093/bioinformatics/bts061 (2012). g 25. Liang, D. et al. Non-invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing. Prenat Diagn. 33, 409–415, https://doi.org/10.1002/pd.4033 (2013). Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 5 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42980-7. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the 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/. © The Author(s) 2019 Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 6
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Identifying Relationships among Genomic Disease Regions: Predicting Genes at Pathogenic SNP Associations and Rare Deletions
PLOS genetics
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Abstract Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical method, Gene Relationships Among Implicated Loci (GRAIL), that takes a list of disease regions and automatically assesses the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally associated Crohn’s disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to disease risk). We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically robust approach to identifying functionally related genes from across multiple disease regions—that likely represent key disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/). Citation: Raychaudhuri S, Plenge RM, Rossin EJ, Ng ACY, International Schizophrenia Consortium, et al. (2009) Identifying Relationships among Genomic Disease Regions: Predicting Genes at Pathogenic SNP Associations and Rare Deletions. PLoS Genet 5(6): e1000534. doi:10.1371/journal.pgen.1000534 Received February 16, 2009; Accepted May 22, 2009; Published June 26, 2009 Copyright:  2009 Raychaudhuri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2009 Raychaudhuri et al. Abstract This is an open-access article distributed under the terms of the Creative C unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cred Funding: For this project, SR was supported by a T32 NIH training grant (AR007530-23), an NIH Career Development Award (1K08AR055688-01A1), an American College of Rheumatology Bridge Grant, and through the BWH Rheumatology Fellowship program, directed by Simon Helfgott. MJD is supported by a U01 NIH grant (U01 HG004171). MJD and RJX are supported by an R01 NIH grant (R01 DK083759). ACYN is supported through Research Fellowship Award from the Crohn’s and Colitis Foundation of America. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: soumya@broad.mit.edu (SR); mjdaly@broad.mit.edu (MJD) These disease regions often overlap multiple genes – though only one is typically relevant to pathogenesis and the remaining are spuriously implicated by proximity. The difficulty of this task is heightened by the limited state of cataloged interactions, pathways, and functions for the vast majority of genes. However, undefined gene relationships might often be conjectured from the literature, even if they are not explicitly described yet. Identifying Relationships among Genomic Disease Regions: Predicting Genes at Pathogenic SNP Associations and Rare Deletions Soumya Raychaudhuri1,2,3*, Robert M. Plenge1,3,4, Elizabeth J. Rossin1,2,5, Aylwin C. Y. Ng6,7, International Schizophrenia Consortium, Shaun M. Purcell2,8,9, Pamela Sklar2,8,9,10, Edward M. Scolnick 2,8,10, Ramnik J. Xavier6,7, David Altshuler1,2,11,12,13, Mark J. Daly1,2* 1 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, 2 Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 3 Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Harvard Medical School – Partners HealthCare Center for Genetics and Genomics, Boston, Massachusetts, United States of America, 5 Harvard-MIT Health Sciences and Technology, Cambridge, Massachusetts, United States of America, 6 Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 7 Gastroenterology Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 8 Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, 9 Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 10 Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 11 Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 12 Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, 13 Diabetes Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 Author Summary We propose, instead, to use a flexible metric of gene relatedness that not only captures clearly established close gene relationships, but also has the ability to capture potential undocumented or distant ones. Such a metric may be a more powerful tool to approach this problem rather then relying on incomplete databases of gene functions, interactions, or relationships. Encouraged by GRAIL’s ability to recognize biologically meaningful connections, we tested its ability to distinguish true disease regions from false positive regions in two practical applications in human genetics. First, in Crohn’s disease, we start with a long list of putative SNP associations from a recent GWA (genome-wide association) meta-analysis [24]. We demonstrate that a substantial fraction of these SNPs contain highly related genes—far beyond what can be expected by chance. We demonstrate that many of these SNPs subsequently validate in an independent replication genotyping experiment. Second, in schizophrenia, we previously identified an over-representation of rare deletions in schizophrenia cases compared to controls [8]. Despite the statistical excess, it is challenging to identify exactly which case deletions are causal, given the relatively high background rate of rare deletions in controls. Using GRAIL however, we are able to demonstrate that a subset of case deletions contain related genes. We further demonstrate that these genes are highly and significantly enriched for central nervous system (CNS) expressed genes. In stark contrast, GRAIL finds no excess relatedness among genes implicated by case deletions. To this end, we use established statistical text mining approaches to quantify relatedness between two genes – specifically, gene relatedness is the degree of similarity in the text describing them within article abstracts. The published literature represented in online PubMed abstracts encapsulates years of research on biological mechanisms. We and others have shown the great utility of statistical text mining to rapidly obtain functional information about genes, including protein-protein interactions, gene function annotation, and measuring gene-gene similarity [19–22]. Text is an abundant and underutilized resource in human genetics, and currently a total of 140,000 abstracts from articles that reference human genes are available through PubMed [23]. Additional valuable information can be seamlessly gained by including more than 100,000 references from orthologous genes; many important pathways have been more thoroughly explored in model systems than in humans. We have developed a novel statistical method to evaluate the degree of relatedness among genes within disease regions: Gene Relationships Among Implicated Loci (GRAIL). Identifying Common Function Across Disease Regions content across regions. We assume that each region contains a single pathogenic gene; therefore narrow regions with one or just a few genes are more informative than expansive regions with many genes, since they are likely to have many irrelevant genes. To take advantage of this, we have designed GRAIL to set a lower threshold in considering relatedness for those genes in narrow regions, allowing for more distant relationships to be considered; on the other hand it sets a more stringent threshold for genes located in expansive mutligenic regions and considers only the very closest of relationships. This strategy prevents large regions with many genes from dominating the analysis. Author Summary Modern genetic studies, including genome-wide surveys for disease-associated loci and copy number variation, provide a list of critical genomic regions that play an important role in predisposition to disease. Using these regions to understand disease pathogenesis requires the ability to first distinguish causal genes from other nearby genes spuriously contained within these regions. To do this we must identify the key pathways suggested by those causal genes. In this manuscript we describe a statistical approach, Gene Relationships Across Implicated Loci (GRAIL), to achieve this task. It starts with genomic regions and identifies related subsets of genes involved in similar biological processes—these genes highlight the likely causal genes and the key pathways. GRAIL uses abstracts from the entirety of the published scientific literature about the genes to look for potential relation- ships between genes. We apply GRAIL to four very different phenotypes. In each case we identify a subset of highly related genes; in cases where false positive regions are present, GRAIL is able to separate out likely true positives. GRAIL therefore offers the potential to translate disease genomic regions from unbiased genomic surveys into the key processes that may be critical to the disease. In this paper we apply GRAIL to four phenotypes. In each case GRAIL is able to identify a subsets of genes enriched for relatedness – more than expected by random chance. We demonstrate enrichment for relatedness among true disease regions rigorously based on both GRAIL’s theoretically derived p-value and also based on parallel analysis of either (1) carefully selected random regions matched for gene content and size or (2) experimentally derived false positive disease regions. GRAIL is able to identify subsets of highly related genes among validated SNP associations. First we use GRAIL to identify related genes from SNPs associated with serum lipid levels; GRAIL correctly identifies genes already known to influence lipid levels within the cholesterol biosynthesis pathway. In comparison to randomly selected matched SNP sets, the set of lipid SNPs demonstrate significantly more relatedness. Second, we use GRAIL to identify significantly related genes near height- associated SNPs; these genes highlight plausible pathways involved in height. In comparison to randomly selected matched SNP sets, the set of height SNPs also demonstrate significantly more relatedness. expression data, carefully constructed gene networks based on multiple information sources, predefined gene sets and pathways, and disease-related keywords. Author Summary Given only a collection of disease regions, GRAIL uses our text-based definition of relatedness (or alternative metrics of relatedness) to identify a subset of genes, more highly related than by chance; it also assigns a select set of keywords that suggest putative biological pathways. It uses no information about the phenotype, such as known pathways or genes, and is therefore not tethered to potentially biased pre-existing concepts about the disease. PLoS Genetics | www.plosgenetics.org Introduction An emerging challenge in genomics is the ability to examine multiple disease regions within the human genome, and to recognize a subset of key genes that are involved in a common cellular process or pathway. This is a key task to translate experimentally ascertained disease regions into meaningful understanding about pathogenesis. The importance of this challenge has been highlighted by advances in human genetics that are facilitating the rapid discovery of disease regions in the form of genomic regions around associated SNPs (single nucleotide polymorphisms) [1–6] or CNVs (copy number variants) [7–10]. y p y y The general strategy of using function to prioritize genes in disease regions has been substantially explored [11–18]. However, predicted disease genes have not, in general, been easily validated. Thus far, published approaches have utilized a range of codified gene information including protein-interaction maps, gene June 2009 | Volume 5 | Issue 6 | e1000534 1 June 2009 | Volume 5 | Issue 6 | e1000534 Identifying Common Function Across Disease Regions Figure 1. Gene Relationships Among Implicated Loci (GRAIL) method consists of four steps. (A) Identifying genes in disease regions. For each independent associated SNP or CNV from a GWA study, GRAIL defines a disease region; then GRAIL identifies genes overlapping the region. In this region there are three genes. We use gene 1 (pink arrow) as an example. (B) Assess relatedness to other human genes. GRAIL scores each gene contained in a disease region for relatedness to all other human genes. GRAIL determines gene relatedness by looking at words in gene references; related genes are defined as those whose abstract references use similar words. Here gene 1 has word counts that are highly similar to gene A but not to gene B. All human genes are ranked according to text-based similarity (green bar), and the most similar genes are considered related. (C) Counting regions with similar genes. For each gene in a disease region, GRAIL assesses whether other independent disease regions contain highly significant genes. GRAIL assigns a significance score to the count. In this illustration gene 1 is similar to genes in three of the regions (green arrows), including gene A. (D) Assigning a significance score to a disease region. After all of the genes within a region are scored, GRAIL identifies the most significant gene as the likely candidate. GRAIL corrects its significance score for multiple hypothesis testing (by adjusting for the number of genes in the region), to assign a significance score to the region. doi:10.1371/journal.pgen.1000534.g001 Figure 1. Gene Relationships Among Implicated Loci (GRAIL) method consists of four steps. (A) Identifying genes in disease regions. For each independent associated SNP or CNV from a GWA study, GRAIL defines a disease region; then GRAIL identifies genes overlapping the region. In this region there are three genes. We use gene 1 (pink arrow) as an example. (B) Assess relatedness to other human genes. GRAIL scores each gene contained in a disease region for relatedness to all other human genes. GRAIL determines gene relatedness by looking at words in gene references; related genes are defined as those whose abstract references use similar words. Here gene 1 has word counts that are highly similar to gene A but not to gene B. All human genes are ranked according to text-based similarity (green bar), and the most similar genes are considered related. (C) Counting regions with similar genes. Summary of statistical approach y pp GRAIL relies on two key methods: (1) a novel statistical framework that assesses the significance of relatedness between genes in disease regions (2) a text-based similarity measure that scores two genes for relatedness to each other based on text in PubMed abstracts. Details for both are presented in the Methods. The GRAIL statistical framework consists of four steps (see Figure 1). First, given a set of disease regions we identify the genes overlapping them (Figure 1A); for SNPs we use LD (linkage disequilibrium) characteristics to define the region. Second, for In addition to a flexible text-based metric of relatedness, GRAIL’s ability to successfully connect genes also leverages a statistical framework that carefully accounts for differential gene June 2009 | Volume 5 | Issue 6 | e1000534 2 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions each overlapping gene we score all other human genes by their relatedness to it (Figure 1B). In this paper we use a text-based similarity measure; alternative measures of relatedness, for example similarity in gene annotations or expression data, could be easily applied instead [25,26]. Third, for each gene we count the number of independent regions with at least one highly related gene (Figure 1C); here the threshold for relatedness varies between regions depending on the number of genes within them. We assign a p-value to that count. Fourth, for each disease region we select the single most connected gene as the key gene. We assign the disease region that key gene’s p-value after adjusting for multiple hypothesis testing (if there are multiple genes within the region) (Figure 1D). This final score is listed in this paper as pmetric where the metric is text, expression, or annotation based. Very low ptext scores for one region indicate that a gene within it is more related to genes in other disease regions through PubMed abstracts than Figure 1. Gene Relationships Among Implicated Loci (GRAIL) method consists of four steps. (A) Identifying genes in disease regions. For each independent associated SNP or CNV from a GWA study, GRAIL defines a disease region; then GRAIL identifies genes overlapping the region. In this region there are three genes. We use gene 1 (pink arrow) as an example. (B) Assess relatedness to other human genes. GRAIL scores each gene contained in a disease region for relatedness to all other human genes. GRAIL determines gene relatedness by looking at words in gene references; related genes are defined as those whose abstract references use similar words. Here gene 1 has word counts that are highly similar to gene A but not to gene B. All human genes are ranked according to text-based similarity (green bar), and the most similar genes are considered related. (C) Counting regions with similar genes. For each gene in a disease region, GRAIL assesses whether other independent disease regions contain highly significant genes. GRAIL assigns a significance score to the count. In this illustration gene 1 is similar to genes in three of the regions (green arrows), including gene A. (D) Assigning a significance score to a disease region. Identifying Common Function Across Disease Regions After all of the genes within a region are scored, GRAIL identifies the most significant gene as the likely candidate GRAIL corrects its significance score for multiple hypothesis testing (by adjusting for the number of genes in the region) Evaluating relationships between known associated SNPs: lipid levels and height expected by chance. Simulations on random groups of SNPs demonstrate that the ptext values approximately estimate Type I error rates, being approximately uniformly distributed under the null hypothesis (see Figure S1). However, we recommend the use of careful simulations or controls rather than actual theoretical p- values to reinforce the significance of GRAIL’s findings – as we do in the examples below. g We first applied GRAIL to a set of 19 validated SNPs associated with triglyceride, LDL, and/or HDL levels [5,6]. Since 14 SNPs (out of 19) are near genes that are known members of lipid metabolism pathways, we hypothesized that GRAIL should be able to identify these genes accurately. A total of 87 genes were implicated by the 19 associated SNPs. Of the 14 SNPs near compelling candidate genes, 13 obtained ptext scores,0.01 (Figure 2A, Table S1). GRAIL correctly identified those genes implicated in lipid metabolism from each of these 14 regions. To asses the significance of these findings, we applied GRAIL to 1000 random matched SNP sets; each set consisted of 19 SNPs randomly selected from a commercial genotyping array which implicated a similar total of 87610 genes. In contrast to lipid associated SNPs, not a single matched random set contained 13 SNPs that obtained ptext scores#0.01; on average matched sets had 0.26 (maximum 6) SNPs with ptext#0.01 (Figure 2A). Thus, there is substantial enrichment for highly connected genes captured by true lipid associated SNPs. p The text-based similarity metric is based on standard approach- es used in statistical text mining. To avoid publications that report on or are influenced by disease regions discovered in the recent scans, we use only those PubMed abstracts published prior to December 2006, before the recent onslaught of GWA papers identifying novel associations. This approach effectively avoids the evaluation of gene relationships being confounded by papers listing genes in regions discovered as associated to these phenotypes. In addition to including primary abstract references about genes listed in Entrez Gene, we augment our text compendium with references to orthologous genes listed in Homologene [23]; this increases the number of articles available per gene from 6 to 12 (see Table 1). We note that the distribution of articles per gene is skewed toward a small number of genes with many references; 0.4% of genes are referenced by .500 articles, while 26% of genes are referenced by ,5. Evaluating relationships between known associated SNPs: lipid levels and height In fact 2,034 genes could not be connected to any abstracts at all. For each abstract we convert free text into vectors of word counts [19]. For each gene we define a word vector that consists of averaged word counts from document references to it. Pairwise gene relatedness is then the correlation between the vectors of word counts between two genes. Two genes that are referenced by abstracts using the same sorts of words will receive a high similarity score, whereas two genes that have abstract references that largely use a different vocabulary will receive a low score (Figure 1B). Importantly, genes do not need to be co-cited in the same document to be identified as highly similar. Despite relatively comprehensive lipid biology annotation, GO does not identify relationships between regions as effectively as published text (Figure 2A). A total of 12 out of the 19 associated SNPs obtained pannotation,0.01. Relationships between highest scoring candidate genes are explained by several shared GO codes including: GO:0008203 (‘cholesterol metabolic process’), GO:0016125 (‘sterol metabolic process’), GO:0006629 (‘lipid metabolic process’), GO:0008202 (‘steroid metabolism’), and GO:0005319 (‘lipid transporter activity’). Gene expression does not identify relationships between regions as effectively as text, either (Figure 2A). A subset of 4 associated SNPs obtain pexpression,0.01. The regions with the most significantly connected genes have similar tissue-specific expression profiles. The highest expression is in four samples taken from adult and fetal liver tissues, known to play a major role in cholesterol metabolism. While associated SNPs are less connected with these alternative metrics, they do seem to leverage the appropriate functional variables and provide valuable phenotypic information. After regions are scored with GRAIL, PubMed text can be used to identify keywords that may provide insight into the underlying biological pathways. We define these keywords as those words that most strongly link the significant genes in each region, that is the words with overall greatest weight across all of the text vectors from those genes. We next applied GRAIL to 42 validated SNP associations to adult height in recent GWA studies [2–4]. This application tests GRAIL’s ability to connect genes in the absence of functional literature connecting the phenotype to the relevant pathways. In contrast to lipid metabolism, all associated common SNPs were identified in 2007 and 2008 and the underlying biological pathways involved in height are still poorly understood. For each gene in a disease region, GRAIL assesses whether other independent disease regions contain highly significant genes. GRAIL assigns a significance score to the count. In this illustration gene 1 is similar to genes in three of the regions (green arrows), including gene A. (D) Assigning a significance score to a disease region. After all of the genes within a region are scored, GRAIL identifies the most significant gene as the likely candidate. GRAIL corrects its significance score for multiple hypothesis testing (by adjusting for the number of genes in the region), to assign a significance score to the region. doi:10.1371/journal.pgen.1000534.g001 June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 3 Identifying Common Function Across Disease Regions Evaluating relationships between known associated SNPs: lipid levels and height The scatter plot on the right illustrates ptext values for actual SNPs associated with height (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green dots) and gene expression correlation (purple dots). On the right we list for each ptext threshold the number of expected SNPs less than the threshold based on matched sets, and the number of observed SNPs less than the threshold among height associated SNPs. doi:10.1371/journal.pgen.1000534.g002 Figure 2. SNPs associated with lipid metabolism and height contain genes related to each other. (A) 19 SNPs associated with lipid metabolism. The y-axis plots the ptext values on a log scale, with increasing significance at the top. The histogram on the left side of the graph illustrates values for matched SNP sets. 88.6% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for actual serum cholesterol associated SNPs (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green dots) and gene expression correlation (purple dots). (B) 42 SNPs associated with height. Similar plot for 42 height associated SNPs. The histogram on the left of the graph illustrates ptext values for random SNP sets carefully matched to height-associated SNP set. 86.5% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for actual SNPs associated with height (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green dots) and gene expression correlation (purple dots). On the right we list for each ptext threshold the number of expected SNPs less than the threshold based on matched sets, and the number of observed SNPs less than the threshold among height associated SNPs. doi:10.1371/journal.pgen.1000534.g002 The 42 height SNPs implicated a total of 185 genes (Table S2). Of these 42 regions, 13 obtained ptext scores,0.01 (Figure 2B). For comparison, we used GRAIL to score 1000 matched SNP sets; as before each set consisted of 42 SNPs randomly selected from a commercial array and implicated a total of 185610 genes. Not a single random set contained 13 SNPs that obtained ptext scores#0.01; on average matched sets had 0.77 (maximum 10) SNPs with ptext scores,0.01. Evaluating relationships between known associated SNPs: lipid levels and height This insures independence between association results and the functional literature from before 2007 that is mined in this study. In most cases the key genes are not yet known. Since the GRAIL framework can be easily used with any gene relatedness metric, we also devised and tested two alternative metrics derived from Gene Ontology (GO) annotations [27] and an mRNA expression atlas consisting of expression measurements across multiple human tissues (The Novartis Gene Expression Atlas) [28]. These metrics are described in greater detail in Methods. Table 1. Text resources. Refs/gene Genes Articles References mean median mean median Standard 18,690 137,395 260,658 13.9 6 1.9 1 Homologs 15,990 138,720 434,690 27.1 13 3.1 1 Combined 18,875 259,638 599,537 31.7 12 2.3 1 We obtained text from abstracts relevant to human genes from PubMed on December 2006. In the first row we list the number of human genes with any references listed, the total number of abstracts referencing them, and the total number of gene references. We then list the mean and median number of abstract references per gene, and also the mean and median number of gene references per abstract. We used Homologene to identify human gene orthologs, and obtained text for those genes; information about those genes and references is listed in the second row. We combined the two pools of gene references to create a large combined database of 18,875 genes with 599,537 references to 259,638 articles, described in the third row. doi:10.1371/journal.pgen.1000534.t001 Table 1. Text resources. We obtained text from abstracts relevant to human genes from PubMed on December 2006. In the first row we list the number of human genes with any references listed, the total number of abstracts referencing them, and the total number of gene references. We then list the mean and median number of abstract references per gene, and also the mean and median number of gene references per abstract. We used Homologene to identify human gene orthologs, and obtained text for those genes; information about those genes and references is listed in the second row. We combined the two pools of gene references to create a large combined database of 18,875 genes with 599,537 references to 259,638 articles, described in the third row. doi:10.1371/journal.pgen.1000534.t001 June 2009 | Volume 5 | Issue 6 | e1000534 4 Identifying Common Function Across Disease Regions Figure 2. Evaluating relationships between known associated SNPs: lipid levels and height Thus, we present clear statistical evidence that GRAIL identifies genes with non-random functional connections among associated loci. to prospectively identify true disease regions, based on the relatedness of genes within them, from false positive regions. We tested GRAIL’s ability to distinguish disease regions from a longer list of results containing a large number of false positive regions as well in two separate human genetics applications. A recent GWA meta-analysis in Crohn’s disease identified 74 independent SNPs as nominally significant (p,561025) [24]. While the excess beyond chance suggested many of these regions were likely true positives, up to half of these regions should by necessity be unrelated to Crohn’s and simply represent the tail of the null distribution. Thus we sought to explore whether GRAIL could identify a subset of these SNPs that implicate an inter- connected set of genes, and whether those represented true associations that could be validated. Strikingly, the top five keywords linking the genes were ‘hedgehog’, ‘histone’, ‘bone’, ‘cartilage’, and ‘growth’ (see Table S3 for a more complete list). Of note, ‘height’, does not emerge as a keyword since these genes had not been previously related to height. For comparison, the top five keywords for lipid metabolism associated SNPs were ‘lipoprotein’, ‘cholesterol’, ‘lipase’, ‘apoli- poprotein’, and ‘triglyceride’ (Table S3). These results are particularly noteworthy as this analysis uses only a simple list of SNPs implicated by GWA studies—no specific biological pathways or mechanisms or phenotype details are assumed. In a now published replication genotyping of the 74 SNPs, 30 replicated convincingly when tested in independent samples (defined as having one-tailed association p-values,0.0007 in replication samples and two tailed association p-values,561028 overall), confirming true positive associations, whereas 22 convincingly failed to replicate (defined as overall association p- value rising to .1024); the remaining 22 regions had intermediate levels of significance following replication (and can be considered as yet unresolved associations) [24]. Evaluating relationships between known associated SNPs: lipid levels and height SNPs associated with lipid metabolism and height contain genes related to each other. (A) 19 SNPs associated with lipid metabolism. The y-axis plots the ptext values on a log scale, with increasing significance at the top. The histogram on the left side of the graph illustrates values for matched SNP sets. 88.6% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for actual serum cholesterol associated SNPs (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green dots) and gene expression correlation (purple dots). (B) 42 SNPs associated with height. Similar plot for 42 height associated SNPs. The histogram on the left of the graph illustrates ptext values for random SNP sets carefully matched to height-associated SNP set. 86.5% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for actual SNPs associated with height (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green dots) and gene expression correlation (purple dots). On the right we list for each ptext threshold the number of expected SNPs less than the threshold based on matched sets, and the number of observed SNPs less than the threshold among height associated SNPs. doi:10.1371/journal.pgen.1000534.g002 Figure 2. SNPs associated with lipid metabolism and height contain genes related to each other. (A Figure 2. SNPs associated with lipid metabolism and height contain genes related to each other. (A) 19 SNPs associated with lipid metabolism. The y-axis plots the ptext values on a log scale, with increasing significance at the top. The histogram on the left side of the graph illustrates values for matched SNP sets. 88.6% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for actual serum cholesterol associated SNPs (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green dots) and gene expression correlation (purple dots). (B) 42 SNPs associated with height. Similar plot for 42 height associated SNPs. The histogram on the left of the graph illustrates ptext values for random SNP sets carefully matched to height-associated SNP set. 86.5% of those SNPs have ptext values that are .0.1. PLoS Genetics | www.plosgenetics.org Identifying Common Function Across Disease Regions In contast, we did not identify any regions with significantly related genes in the corresponding list of deletions; out of a total 124 regions, none obtained ptext scores,0.001 (see Table S8). This represents a significant enrichment within the cases (p = 0.01, one Fisher’s exact text). As a further test of GRAIL, we then evaluated the next most significant 74 associated SNPs that emerged from the Crohn’s disease GWA meta-analysis (association p-values ranging from 561025 to 261024). Out of the 75 regions, 8 are not near any gene, and we did not score them. The remaining 67 regions were tested with GRAIL for relationships to the 52 replicated and indeterminate regions that emerged following replication. Two emerge with highly significant GRAIL scores: rs8178556 on chromosome 21 (IFNAR1, ptext = 1.761024) and rs12928822 on chromosome 16 (SOCS1, ptext = 8.261024) suggesting these inde- pendent regions may lead to novel associated SNPs for Crohn’s disease (see Table S7). We then sought independent assessment of the biological relationship of the genes highlighted by GRAIL by examining the extent to which these genes demonstrate preferential expression in CNS tissues using a publicly available tissue atlas [30]. Here we define preferential expression as median CNS tissue expression significantly greater than in other tissues (p,0.01 by one-tailed Table 2. High scoring regions from a Crohn’s disease GWA meta-analysis. Table 2. High scoring regions from a Crohn’s disease GWA meta-analysis. Identifying Common Function Across Disease Regions any knowledge of the contemporaneous replication genotyping experiment. Each region contained between 1 and 34 genes, except for two regions that contained no genes and were not scored. GRAIL identified 13 regions as significant (achieving ptext scores,0.01), as with the previous examples far in excess of chance. We next applied GRAIL to recently published sets of rare deletions seen in schizophrenia cases and matched controls. Multiple groups have recently demonstrated that extremely rare deletions, many of which are likely de novo, are notably enriched in schizophrenia [8–10,29]. However, since rare deletions occur frequently in healthy individuals as well, many of these case deletions will also be non-pathogenic. In fact, we previously found that large (.100 kb), gene overlapping, singleton, deletions were present in 4.9% of cases but also in 3.8% of controls, suggesting that over two-thirds of these deletions are not relevant to disease [8]. We identified 165 published de-novo or case-only deletions of .100 kb overlapping at least one gene; a total of 511 genes are deleted or disrupted by these deletions [8,9,10]. Additionally, we identified 122 regions similar control-only deletions; a total of 252 genes are deleted or disrupted by these deletions. Of those 13 regions, 10 were among the set that convincingly validated in subsequent replication (Table 2)—the remaining three had indeterminate levels of significance. By contrast, only 20 of 63 SNPs remaining SNPs validated (Table S4). Disease regions that replicate have more significant GRAIL scores than those that failed (p = 0.00064, one-tailed rank-sum test, Figure 3A). As with randomly selected SNP lists, the distribution of scores for the 21 failed regions was indistinguishable from a random (uniform) distribution of p-values (Figure 3B). Using these Crohn’s results, we have compared GRAIL’s performance to four other competing algorithms that also use functional information to prioritize genes, and GRAIL’s perfor- mance is superior at predicting true positive associations (see Text S1, Figure S2, Table S5, Table S6). We applied GRAIL separately to both the case and control sets of deletions. In the case deletions, we identified a subset containing highly connected genes (Figure 4A). Specifically, 12 of the 165 regions obtain ptext scores,0.001 with text-similarity (Table 3). The top keywords suggest some common biological underlying functions: ‘phosphatase’, ‘glutamate’, ‘receptor’, ‘cadherin’, and ‘neurons’. Here we list a subset of the 74 regions that emerged from a Crohn’s disease GWA meta-analysis that GRAIL assigned the most compelling ptext scores to. The first three columns list information about the associated SNP. The fourth column lists the combined p-value of association from a GWA meta-analysis and subsequent replication. The fifth column indicates whether the region was validated, indeterminate, or failed in replication. Those regions that represent novel findings, not previously published are also indicated. The sixth column lists the number of genes in the disease region, and the seventh column lists the candidate gene identified by GRAIL. The eighth column lists the regions ptext score. doi:10.1371/journal.pgen.1000534.t002 Genetic associations to Crohn’s disease and schizophrenia—predicting disease regions After successfully applying GRAIL to validated associations for two phenotypes, we hypothesized that GRAIL could also be used We applied GRAIL prospectively to these 74 nominally associated SNPs. GRAIL was initially operated independent of PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 5 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions SNP Chr Position (HG17) passociation Replication Study Result N (genes) Implicated Gene ptext rs2066845 16 49314041 1.5E-24 VALIDATED 3 NOD2 0.00010 rs10863202 16 84545499 1.4E-05 INDETERMINATE 4 IRF8 0.00058 rs10045431 5 158747111 1.9E-13 VALIDATED-NOVEL 1 IL12B 0.00066 rs11465804 1 67414547 3.3E-63 VALIDATED 1 IL23R 0.00094 rs2476601 1 114089610 7.3E-09 VALIDATED-NOVEL 8 PTPN22 0.0014 rs762421 21 44439989 7.0E-10 VALIDATED-NOVEL 1 ICOSLG 0.0023 rs2188962 5 131798704 1.2E-18 VALIDATED 9 IRF1 0.0026 rs917997 2 102529086 1.1E-05 INDETERMINATE 5 IL18RAP 0.0027 rs11747270 5 150239060 1.7E-16 VALIDATED 3 IRGM 0.0032 rs2738758 20 61820069 2.7E-06 INDETERMINATE 10 TNFRSF6B 0.0038 rs9286879 1 169593891 7.7E-10 VALIDATED-NOVEL 4 TNFSF18 0.0042 rs2301436 6 167408399 5.2E-13 VALIDATED-NOVEL 3 CCR6 0.0052 rs4263839 9 114645994 1.3E-10 VALIDATED 2 TNFSF8 0.008 rs3828309 2 233962410 1.2E-32 VALIDATED 4 USP40 0.019 rs744166 17 37767727 3.4E-12 VALIDATED-NOVEL 2 STAT3 0.023 rs7758080 6 149618772 4.4E-06 INDETERMINATE 4 SUMO4 0.033 rs7161377 14 75071147 2.3E-05 INDETERMINATE 1 BATF 0.09 Here we list a subset of the 74 regions that emerged from a Crohn’s disease GWA meta-analysis that GRAIL assigned the most compelling ptext scores to. The first three columns list information about the associated SNP. The fourth column lists the combined p-value of association from a GWA meta-analysis and subsequent replication. The fifth column indicates whether the region was validated, indeterminate, or failed in replication. Those regions that represent novel findings, not previously published are also indicated. The sixth column lists the number of genes in the disease region, and the seventh column lists the candidate gene identified by GRAIL. The eighth column lists the regions ptext score. doi:10.1371/journal.pgen.1000534.t002 June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 6 Identifying Common Function Across Disease Regions Figure 3. GRAIL predicts Crohn’s disease SNPs. (A) Validated versus Failed SNPs. Prior to replication, GRAIL scored Crohn’s SNPs that emerged from a meta-analysis study. Results from follow-up testing either validated Crohn’s SNPs, or identified those SNPs that failed. We produce a scatter plot of the significance of text-based similiarty (ptext) for validated regions (green) versus regions that failed to replicate (red). Black horizontal lines mark the median ptext values. The distribution of scores for failed SNPs resembles a random distribution of p-values. The distribution of scores for validated SNPs is significantly different; almost K of these SNPs obtain ptext scores,0.1. (B) Histogram of text-based scores for Crohn’s disease candidate regions. Identifying Common Function Across Disease Regions Here we plot a histogram of ptext scores for 74 Crohn’s disease SNPs. Validated SNPs (green) have ptext values that are enriched for significant values. Indeterminate SNPs (yellow) have a subset of ptext values that are significant. Failed SNPs (Red) have all of their ptext scores.0.1. doi:10.1371/journal.pgen.1000534.g003 Figure 3. GRAIL predicts Crohn’s disease SNPs. (A) Validated versus Failed SNPs. Prior to replication, GRAIL scored Crohn’s SNPs that emerged from a meta-analysis study. Results from follow-up testing either validated Crohn’s SNPs, or identified those SNPs that failed. We produce a scatter plot of the significance of text-based similiarty (ptext) for validated regions (green) versus regions that failed to replicate (red). Black horizontal lines mark the median ptext values. The distribution of scores for failed SNPs resembles a random distribution of p-values. The distribution of scores for validated SNPs is significantly different; almost K of these SNPs obtain ptext scores,0.1. (B) Histogram of text-based scores for Crohn’s disease candidate regions. Here we plot a histogram of ptext scores for 74 Crohn’s disease SNPs. Validated SNPs (green) have ptext values that are enriched for significant values. Indeterminate SNPs (yellow) have a subset of ptext values that are significant. Failed SNPs (Red) have all of their ptext scores.0.1. doi:10.1371/journal.pgen.1000534.g003 This approach could have widespread application to follow-up GWA study results and offers a mechanism to prioritize the hundreds of SNPs that are expected to achieve an intermediately significant level of association (1025,p,1023). As far as we are aware – this is the first successful prediction of the outcome of a GWA validation study. rank-sum test). Considering the entire set, case-deletions are not enriched for genes preferentially expressed in the CNS (22% are preferentially expressed in the CNS, compared to 25% of control- deletion genes). However, considering the subset of genes indentified by GRAIL (ptext,0.01), 60% (9 of 15 genes) are preferentially CNS expressed. Furthermore, the fraction of genes with preferential CNS expression correlates inversely with the significance of the GRAIL score (Figure 4B). Regions that GRAIL assigns non-significant scores to, do not demonstrate any compelling enrichment for CNS expressed genes. GRAIL offers the greatest value in situations where disease regions are being considered that are difficult to validate, for example rare deletions. The ability to genetically validate any individual rare deletion is challenged given the limited power afforded by the size of available patient collections. Identifying Common Function Across Disease Regions In schizo- phrenia the excess of rare deletions has now been well documented – but it had been difficult to connect these rare deletions to a specific pathway. We identified a subset of related genes that have functions that are plausibly related to schizophrenia. As other diseases emerge where rare variants play a role in the genetic architecture, our approach may provide a crucial first step to put context to genetic findings. PLoS Genetics | www.plosgenetics.org Discussion We have presented an automated text-based strategy to take a list of disease regions and identify those regions with significantly inter-related genes. In the process it recognizes the likely candidate gene in each disease region. It makes no assumptions about the phenotype being studied or underlying pathways that might be presumed to be relevant to a disease state. While in principle a diligent investigator could potentially examine the literature related to all potentially associated genes and arrive at the same conclusions, they are unlikely in practice be able to work with the same efficiency and objectivity as the approach outlined here. In the schizophrenia application, for example, we objectively interpret and analyze the relationship between over 500 genes. PLoS Genetics | www.plosgenetics.org Identifying disease genes within a region SP110. IRGM is not readily connected to these genes in a well- defined pathway and, in-fact, is not referenced together with them in any abstracts; furthermore no IRGM interactions are listed in Entrez at all. Yet they all are involved in the host response to Mycobacterium and possibly other intracellular infections by macrophages. The top keywords describing the connections between IRGM and these genes were ‘macrophages’, ‘tuberculosis’ and ‘mycobacterium’. The IRGM gene has been shown exper- imentally to eliminate intracellular Mycobacterium tuberculosis via autophagy [31]. The IRF1 homolog studies in mouse have demonstrated its role in intra-cellular nitrous oxide production, necessary to fight Mycobacterium infections [32]. Individuals with loss of function IL12B mutations have been found with increased susceptibility to Mycobacterium infections [33] and knock out mice have demonstrated increased susceptibility to infection [34,35]. A SP110 mouse homolog has been shown to mediate innate immunity in fighting intra-cellular Mycobacterium tuberculosis infec- tion [36]. GRAIL is able to identify this common underlying similarity between these genes, and assign a significant score to IRGM, while at the same time revealing what may be an important pathway in Inflammatory Bowel Disease. Other strategies depending on interaction networks or functional databases may struggle to detect these relationships. GRAIL systematically identifies a single gene within a disease region as the likely disease gene. We highlight two interesting examples from the height data of previously unrecognized potentially causative genes. The first example is the rs42046 SNP on chromosome 7 region implicating five genes. The genetic studies that identified this region had suggested CDK6 as the likely causative gene [2–4]. However, GRAIL found greatest evidence in support of PEX1 (uncorrected ptext = 0.0084). When we compare the most compelling of these genes, PEX1, to candidates from the other 41 SNPs with our text-based metric, we found it to be most related to a gene in a height-associated SNP on chromosome 8, PEX2 (PXMP3). The protein products of both PEX1 and PEX2 are involved in peroxisome biogenesis and are implicated in a genetic disease associated craniofacial and skeletal abnormalities (Zellwe- ger’s syndrome) [37–39]. While it may be a coincidence that these two closely related genes are associated by chance, it is certainly possible that peroxisome biogenesis represents a previously unrecognized height pathway. Connecting seemingly unrelated genes through text Connecting seemingly unrelated genes through text The main strength of GRAIL is its ability to link genes through text that may not yet have an established common pathway or process. Consider the IRGM gene association to Crohn’s disease – for which GRAIL found strong evidence (uncorrected ptext = 0.0011). GRAIL’s text-based similarity metric recognize the significant connections between IRGM and four other validated or intermediate region genes: IRF1, IL12B, IRF8, and We present data that GRAIL can identify common SNPs that subsequently validate in replication genotyping. We have demon- strated superior performance in this application to other methods. June 2009 | Volume 5 | Issue 6 | e1000534 7 Identifying Common Function Across Disease Regions Figure 4. GRAIL identifies a subset of highly connected genes within rare deletions found in Schizophrenia cases. (A) Case deletions versus control deletions. Here we plot the results of the separate GRAIL analyses conducted on the deletions observed in schizophrenia cases and controls. Case deletion ptext scores are displayed in red; control deletion ptext scores are displayed in green. The line in each category in the middle of the box represents the median GRAIL ptext score. The box represents the 25–75% range. The bars represent the 5–95% range. Additional scores outside the range are individual plotted. (B) Text-based GRAIL significance score tracks with CNS specific expression. We partition case-only deletions by their GRAIL scores. For each range of GRAIL ptext scores, we assess the candidate genes selected by GRAIL for CNS expression. The upper portion of this plot illustrates the fraction of those candidate genes that demonstrate preferential CNS expression along with 95% confidence intervals. The blue line represents the total fraction of genes that are preferentially CNS expressed. For the most compelling GRAIL scores, the candidate genes are significantly enriched for CNS expression compared to what would be expected from a random group of genes. The lower portion of the plot is a histogram. doi:10.1371/journal.pgen.1000534.g004 Figure 4. GRAIL identifies a subset of highly connected genes within rare deletions found in Schizophrenia cases. (A) Case deletions versus control deletions. Here we plot the results of the separate GRAIL analyses conducted on the deletions observed in schizophrenia cases and controls. Case deletion ptext scores are displayed in red; control deletion ptext scores are displayed in green. The line in each category in the middle of the box represents the median GRAIL ptext score. Connecting seemingly unrelated genes through text The box represents the 25–75% range. The bars represent the 5–95% range. Additional scores outside the range are individual plotted. (B) Text-based GRAIL significance score tracks with CNS specific expression. We partition case-only deletions by their GRAIL scores. For each range of GRAIL ptext scores, we assess the candidate genes selected by GRAIL for CNS expression. The upper portion of this plot illustrates the fraction of those candidate genes that demonstrate preferential CNS expression along with 95% confidence intervals. The blue line represents the total fraction of genes that are preferentially CNS expressed. For the most compelling GRAIL scores, the candidate genes are significantly enriched for CNS expression compared to what would be expected from a random group of genes. The lower portion of the plot is a histogram. doi:10.1371/journal.pgen.1000534.g004 PLoS Genetics | www.plosgenetics.org Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions development and migration may contribute to the pathophysiol- ogy of schizophrenia [42]. Many of the highest scoring genes recovered by GRAIL within the deleted regions in cases (Table 3) are localized to the postsynaptic membrane/signaling complex that propagate signals resulting in changes synapse function and downstream gene expression/transcription. The DLG2 gene product interacts at postsynaptic sites to form a multi-meric scaffold for the clustering of receptors, ion channels, and associated signaling proteins. MAGI1 and MAGI2 both encode post-synaptic scaffolding molecules involved in cell adhesion and signaling [43,44]. Furthermore, glutamatergic neurotransmission is implicated through the selection of GRM1, GRM7, and GRM8. Many of the most significant candidate genes identified by GRAIL are involved in neuronal development, cell-cell adhesion and axon guidance. CNTN5 is an immunoglobulin super-family membrane- anchored neuronal protein that is also an adhesion molecule [45]. It may play a role in the developing nervous system [46]. The SDK1 gene expresses a synaptic adhesion protein [47] that guides axonal terminals to specific synapses in developing neurons. The PTPRM encodes a neuronally expressed protein tyrosine phos- phatase that mediates cell-cell aggregation and is involved in cell- cell adhesion [48,49]. Table 3. Rare or de novo schizophrenia case deletions. CHR Start Stop ptext Candidate Gene 7 77,788,564 78,591,795 0.0000013 MAGI2 11 *83,680,969 83,943,977 0.0000016 DLG2 3 65,781,878 65,975,330 0.0000057 MAGI1 11 99,153,400 99,286,239 0.000013 CNTN5 4 *87,919,851 88,032,640 0.000025 PTPN13 18 8,054,730 8,257,748 0.000035 PTPRM 3 *7,177,597 7,314,117 0.000087 GRM7 3 7,043,889 7,145,741 0.000087 GRM7 6 146,418,079 146,525,433 0.00013 GRM1 7 125,707,286 126,050,230 0.00015 GRM8 9 9,485,226 9,644,834 0.00024 PTPRD 7 3,759,288 4,087,229 0.00033 SDK1 3 *197,224,662 198,573,215 0.0011 DLG1 15 27,015,263 28,173,703 0.0014 TJP1 5 31,250,352 32,213,541 0.0033 PDZD2 19 10,231,490 10,493,592 0.0048 ICAM5 2 112,407,513 112,512,196 0.014 MERTK 5 19,570,562 19,843,415 0.018 CDH18 6 145,876,484 146,009,981 0.019 EPM2A 7 145,321,439 145,461,533 0.019 CNTNAP2 5 63,115,468 63,431,545 0.023 HTR1A 14 66,287,336 66,470,393 0.025 GPHN 18 56,109,430 56,255,536 0.029 MC4R 5 106,805,717 107,026,020 0.03 EFNA5 2 233,029,864 233,134,571 0.031 ALPPL2 20 2,923,491 3,618,945 0.034 PTPRA 1 72,287,807 72,439,333 0.037 NEGR1 7 94,306,868 94,497,412 0.044 PPP1R9A 7 157,378,450 157,569,847 0.046 PTPRN2 1 *144,943,150 146,292,286 0.047 ACP6 Here we list all of the deletions that GRAIL identified as most related to other deleted genes (ptext,0.05). Limitations to assessing gene relatedness with text g g While we have shown the promise of text-based similarity in identifying regions and the genes within them that are part of a larger biological pathway, we note that this strategy’s effectiveness is wholly contingent on the completeness of the scientific text. It could be biased towards subsets of genes and pathways that are particularly well studied, and against poorly studied ones. In many of the cases that we illustrate, there are regions that could not be connected – for example, GRAIL fails to connect 5 validated Crohn’s SNPs that obtain ptext scores.0.5 (Figure 3B). These regions might have been missed since the relevant gene is either poorly studied, or even if the gene is well studied, the relevant function of that gene is not well documented in the text. An alternative possibility is that the SNP is tagging non-genic regulatory elements. Additionally, the SNP may be the first discovered representative association for a critical pathway, not represented by other SNP associations – and therefore cannot be connected to them. In this case future discoveries will clarify the significance of that association. In fact, a mutation in the KY gene causes spinal scoliosis in a mouse model [40], and the KY protein product interacts with sarcomeric cytoskeletal proteins [41]. While these literature-based hypotheses may be obvious to a few specialized researchers, the strength of GRAIL is that it is able to suggest these connections in a systematic and objective manner from the entirety of the published literature. In fact, a mutation in the KY gene causes spinal scoliosis in a mouse model [40], and the KY protein product interacts with sarcomeric cytoskeletal proteins [41]. While these literature-based hypotheses may be obvious to a few specialized researchers, the strength of GRAIL is that it is able to suggest these connections in a systematic and objective manner from the entirety of the published literature. Competing methods The most critical technical difference between GRAIL and other strategies is that it does not use any strict definitions of gene functions or interactions, but rather uses a metric of relatedness that allows for a relatively broad range of freedom with which to connect genes. While GRAIL will certainly identify relationships between genes known to be in a common pathway, it goes beyond that, and can allow less strict evidence. In fact, it is even able to identify relatedness between genes that have no established common pathways or article co-citations! In contrast, other strategies start with static gene relationships—such as (1) pre- constructed molecular networks [12,16] or sets of gene with common function [11,15] or (2) a subset of functions identified as relevant to disease either by the user [17] or by mining the published text [14]. In a head to head match up against four other methods that we were able to obtain implemented versions of, GRAIL demonstrated superior performance in predicting Crohn’s associated SNPs (see Text S1, Figure S2, Table S5, and Table S6). Identifying Common Function Across Disease Regions For each deletion we list the chromosome, the range of the deletion, the GRAIL p-value for the region, and the best candidate gene in the region identified by GRAIL. Most genomic coordinates are listed in HG17. *HG18 coordinates. doi:10.1371/journal.pgen.1000534.t003 PLoS Genetics | www.plosgenetics.org Identifying disease genes within a region The second example is the rs10935120 SNP on chromosome 3, implicating three genes; the genetic study that had identified this gene had suggested ANAPC13 as the likely candidate in the region [4]. However, GRAIL identified the KY gene as the most likely disease gene (ptext = 0.04). June 2009 | Volume 5 | Issue 6 | e1000534 8 Scoring regions for functional relatedness We then proceed outwards in each direction to the nearest recombination hotspot [51]. The interval between those two hotspots, which would include the SNP of interest and all SNPs in LD, is defined as the disease region. The associated SNP could feasibly be tagging a stronger SNP signal from another SNP in that region. All genes that overlap that interval are considered implicated by the SNP. If there are no genes in that region, the interval is extended an additional 250 kb in either direction; we chose 250 kb as that distance since that is a range in which non-coding variants might express gene regulation [52]. For each query and seed CNV we define an interval that represents the deleted or duplicated region—all genes that overlap that interval are associated with the CNV for testing. After testing gene g across NSEED seed regions for related genes, the probability of a score exceeding cg under the null, pg, can be approximated: pg~p cwcg   ~ X Nseed nhit~0 p cwcg,nhit   ~ X Nseed nhit~0 p nhit ð Þp cwcg nhit j   Where nhit is the number of seed regions connected to gene g. Since under the null model the probability of a connected region by chance is always pf, we can estimate its probability distribution of nhit with a Poisson distribution: p nhit ð Þ~ NSEEDpf  nhite{NSEEDpf nhit! Step 2. Ranking gene relatedness. For each gene near a query region, we rank all human genes for relatedness. Ranking may be based on text similarity, or other metrics (see below for examples). Rank values range from 1 (most related) to NG (least related), where NG is the number of available human genes, in our application is 18,875 (see Table 1). Since, cg, is the sum of the log of nhit independent uniformly distributed values ranging from 0 to 1, for a fixed value of nhit we can calculate the distribution of cg with a cumulative gamma distribution: Since, cg, is the sum of the log of nhit independent uniformly distributed values ranging from 0 to 1, for a fixed value of nhit we can calculate the distribution of cg with a cumulative gamma distribution: Step 3. Scoring candidate genes against regions. To avoid double counting nearby regions, we first combine any seed regions sharing one or more genes. Genes deleted in schizophrenia suggest relevant neuronal processes We consider closely the subset of related genes identified by GRAIL from rare deletions in patients with schizophrenia. Schizophrenia is a disorder characterized by hallucinations, delusions, cognitive deficits and apathy. The molecular basis of the symptom complex associated with the disorder is largely unknown. However accumulating evidence suggest that dysregu- lation of synaptic activity and abnormalities in neuronal In cases where there is no apparent published connection between associated genes, other similarity metrics based on experimentally derived data, such as gene expression, protein- June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 9 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions testing: testing: protein interactions and transcription factor binding sites could also complement the text-based approaches presented here. In fact, we demonstrate how annotation-based metrics or gene expression-based metrics are able to identify a subset of the associated SNPs in lipid metabolism. As these and other metrics are optimized, they could be used in conjunction with the novel GRAIL statistical framework that we present here to help understand gene relationships. pg,s~1{ 1{pg,s,uncorrected  ns Now we identify those seed regions where pg,s is less than a pre- specified threshold pf as regions connected to gene g. For all applications presented here pf is arbitrarily set to 0.1. The number of seed regions containing at least one gene exceeding this threshold, nhit, can be approximated under a random model with a Poisson distribution. Scoring regions for functional relatedness The Gene Relationships Among Implicated Loci (GRAIL) has four basic steps that are outlined below. It has two input sets of disease regions: (1) a collection of NSEED seed regions (SNPs or CNVs) and (2) a collection of NQUERY query regions. Genes in query regions are evaluated for relationships to genes in seed regions, and query regions are then assigned a significance score. In most applications we are examining a set of regions for relationships between implicated genes, the query regions and the seed regions are identical. In other circumstances where we have a set of putative regions that are being tested against validated ones, the putative regions are defined as query regions, and the validated ones are defined as seed regions. wg,s~ {log pg,s pf   pg,sƒpf 0 pg,swpf ( ) Under a random model, if pg,s,pf , pg,s should range approximately uniformly from 0 to pf. Therefore, under these circumstances wg,s can be modeled approximately with a gamma distribution. For each candidate gene, g, we tally the number of seed regions that contain a highly related gene into a weighted count, cg: cg~ X i wsi,g~ X pg,si vpf {log psi,g pf   Step 1. Defining disease regions and identifying overlapping genes. For each query and seed SNP we find the furthest neighboring SNPs in the 39 and 59 direction in LD (r2.0.5, CEU HapMap [50]). We then proceed outwards in each direction to the nearest recombination hotspot [51]. The interval between those two hotspots, which would include the SNP of interest and all SNPs in LD, is defined as the disease region. The associated SNP could feasibly be tagging a stronger SNP signal from another SNP in that region. All genes that overlap that interval are considered implicated by the SNP. If there are no genes in that region, the interval is extended an additional 250 kb in either direction; we chose 250 kb as that distance since that is a range in which non-coding variants might express gene regulation [52]. For each query and seed CNV we define an interval that represents the deleted or duplicated region—all genes that overlap that interval are associated with the CNV for testing. Step 1. Defining disease regions and identifying overlapping genes. For each query and seed SNP we find the furthest neighboring SNPs in the 39 and 59 direction in LD (r2.0.5, CEU HapMap [50]). Methods We assign a greater weight to those cases where there is greater similarity; that is in the cases where pg,s is particularly small: Keywords To assign keywords to a collection of query regions, we first identify the single candidate genes with the best GRAIL ptext from each region. We then eliminate those regions where the uncorrected GRAIL score for the gene is ptext.0.2. We restrict Gene expression based relatedness wij~ 1zlog tfij    log2 NDOC dfi   tfijw0 0 tfij~0 ( ) wij~ 1zlog tfij    log2 NDOC dfi   tfijw0 0 tfij~0 ( ) To calculate gene relatedness based on expression we download- ed the Novartis Gene Expression Atlas [28]. The data set consists of measurements for 33,689 probes across 158 conditions. Probes were averaged into 17,581 gene profiles. Gene relatedness was calculated as the correlation between expression vectors. where NDOC is the total number of documents, and dfi (or document frequency) is the number of documents the term i appears in. This scheme emphasizes rare words, and de- emphasizes more common words. Lipid and height applications We applied GRAIL to score 19 lipid-associated SNPs and separately to score 42 height-associated SNPs. Specific SNPs are listed in Table S1 and Table S2. We used the SNP sets as both the seed and the query set to look for relatedness between genes across regions. We scored SNPs separately using text, annotation, and expression similarity metrics. We compiled the best candidate genes and scores for the SNP regions. For every gene, we define an averaged term-vector, which is an average of weighted term vectors from gene references and homologous gene references. Abstracts are weighted according to the number of genes they reference; articles referencing many genes are down-weighted to mitigate their influence: gik~ X j [ ref k ð Þ wij 1 1zlog2 nref ,j   Identifying Common Function Across Disease Regions keywords to those that appear in .500 documents, contain .3 letters, and have no numbers. For each term, i, we calculate a score which is the difference between averaged term frequencies among candidate genes and all genes: keywords to those that appear in .500 documents, contain .3 letters, and have no numbers. For each term, i, we calculate a score which is the difference between averaged term frequencies among candidate genes and all genes: Putting this together: Putting this together: Putting this together: pg~ X Nseed nhit~0 NSEEDpf  nhite{NSEEDpf nhit! X nhit{1 i~0 cg  i i! e{cg ! " # s ið Þ~ mean k [ candidate genes gik ð Þ{ mean k [ all genes gik ð Þ Step 4. Scoring regions. Finally, for each query region we identify the best scoring gene within it. A significance score for the query region, pq, is based on the p-value of that gene, pg, corrected for multiple hypothesis testing. Assuming the region has nq genes within it: The top twenty highest scoring terms are selected as keywords. The top twenty highest scoring terms are selected as keywords. Crohn’s disease application Prior to replication, we had access to 74 independent SNP regions that had emerged from a meta-analysis of Crohn’s Disease. All 74 SNPs were used as both the query set and as the seed set into GRAIL. We assessed whether those SNPs that replicated had different text-based significance values than those that fail to replicate. To identify additional regions of interest, we identified the next 75 most significant regions in the Crohn’s disease meta- analysis – they were used in GRAIL as a query set; for the seed set included all SNPs that did not fail in replication. where gik is a the weighted count of term i for gene k, j is a document reference for gene k, and document j references nref,j genes. For a given gene i these gik terms define a gene-text vector. The gene text vectors are normalized, so that their euclidean length is 1. Pairwise gene relatedness can be calculated as the dot product between two normalized term vectors for genes. Assessing gene relatedness with text-based similarity We measure relatedness between genes using similarity in published text from gene references. We first obtain article abstracts from Pubmed. We downloaded all abstracts on December 16, 2006. For each gene, we identified and downloaded abstract references listed in Entrez Gene [23]; additionally, we downloaded Entrez Gene abstract references for gene orthologs listed in Homologene [53]. We removed those articles referencing more than 10,000 genes. Only the title (TI) and abstract (AB) fields were included for further text processing. We defined a vocabulary consisting of only those terms appearing in 40 or more abstracts, and fewer than 130,000; this resulted in a vocabulary of 23,594 terms. For each abstract j we create a vector of term frequencies, tfij, representing the number of times each term i appears within it. Term frequencies are transformed into weights, wij, according to a standard inverse document frequency scheme [54]: gij~ log2 NG gfi   GOij~1 0 GOij~0 ( ) gij~ log2 NG gfi   GOij~1 0 GOij~0 ( ) where gij represented the weighted code i for gene j, NG is the total number of genes, and gfi (or GO frequency) is the number of genes annotated with the term i. Gene relatedness was the correlation between these weighted annotation vectors. Annotation based relatedness We defined a relatedness metric between genes based on similarity in Gene Ontology annotation terms [27]. We downloaded Gene Ontology structure and annotations on December 19, 2006. In addition to human gene GO annotations, we added orthologous gene annotations. Since GO is a hierarchically structured vocabulary, for each gene annotation we also added all of the more general ancestral terms. This resulted in a total of 843,898 annotations for 18,050 genes with 10,803 unique GO terms; this corresponds to a median of 40 terms per gene. We weighted annotations proportionally to the inverse of their frequency, so common annotations received less emphasis. We used a weighting scheme analogous to the one we used for word weighting: pq~1{ 1{pg  nq where pg~min pg’ g’ [ q j   Scoring regions for functional relatedness For a given gene g in a query region, we examine the degree of similarity to any of the ns genes in a given seed region s. To ensure independence, we only look at a seed region s, if it does not share a single gene with the query region that gene g is contained in. p cwcg nhit j   ~FGamma ?,nhit,1 ð Þ{FGamma cg,nhit,1   Since nhit is always an integer, the FGamma term can be simplified: FGamma cg,nhit,1   ~1{ X nhit{1 i~0 cg  i i! e{cg We identify in each region s, the rank of the most similar (or lowest ranking) gene in it to gene g, Rg,s. We convert the rank to a proportion: Therefore, we can be further simplified: Therefore, we can be further simplified: pg,s,uncorrected~Rg,s  NG p cwcg nhit j   ~FGamma ?,nhit,1 ð Þ{FGamma cg,nhit,1   ~ X nhit{1 i~0 cg  i i! e{cg To transform this proportion to a uniformly distributed entity under the null, we recognize that Rg,s was the lowest rank selected from ns genes – and we correct accordingly for multiple hypothesis June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 10 Identifying Common Function Across Disease Regions PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 Identifying Common Function Across Disease Regions were in cases only or in controls only, were at least 100 kb large, and included at least one gene. We obtained singleton deletions online published by the International Schizophrenia Consortium (2008) at [8]. We obtained de novo deletions published by Xu et al (2008) from Table 1 [10]. We obtained singleton deletions published in Walsh et al (2008) from Table 2 [9]. We identified a total of 165 case-only deletions and 122 control-only deletions. We applied the GRAIL algorithm separately to case and controls. We speculated that the case deletions might hit genes from a common pathway and GRAIL p-values may therefore be enriched for significant scores. On the other hand, we hypothesized that control deletions might be located effectively at random, and so no particular pathway or common function should necessarily be enriched in this collection. Table S1 19 Lipid regions scored with Text based GRAIL strategy. Here we scored 19 SNPs, associated with lipid metabolism. In the first three columns we list information about the SNP. In the fourth column we list the number of genes in the SNP associated regions. In the fifth column we list the highest scoring gene in the associated region based on GRAIL using a text-based metric. In the sixth column we list the ptext values for the associated regions. We have bolded those candidate genes that are known likely causative gene. The seventh and eight columns list similar results for GRAIL with an GO annotation-based metric. The ninth and tenth columns list similar results for GRAIL with an expression-based metric. Found at: doi:10.1371/journal.pgen.1000534.s003 (0.15 MB DOC) Table S2 42 Height regions scored with Text based GRAIL strategy. Here we scored 42 SNPs, associated with height. In the first three columns we list information of the SNP. In the fourth column we list the number of genes in the SNP associated regions. In the fifth column we list the highest scoring gene in the associated region for the SNP based on GRAIL using a text-based metric. In the sixth column we list the ptext values for the associated regions. The seventh and eight columns list similar results for GRAIL with an annotation- based metric. The ninth and tenth columns list similar results for GRAIL with an expression-based metric. Software An online version of this method is available (http://www. broad.mit.edu/mpg/grail/). Evaluation against other published methods We compared GRAIL’s performance in its ability to prospec- tively predict Crohn’s associations to five other published methods. The selection of these methods, and the evaluation is detailed in Text S1. Identifying Common Function Across Disease Regions Found at: doi:10.1371/journal.pgen.1000534.s004 (0.28 MB DOC) To examine genes for tissue specific expression in the CNS system, we obtained a large publicly available human tissue expression microarray panel (GEO accession: GSE7307) [30]. We analyzed the data using the robust multi-array (RMA) method for background correction, normalization and polishing [55]. We filtered the data excluding probes with either 100% ‘absent’ calls (MAS5.0 algorithm) across tissues, expression values ,20 in all samples, or an expression range ,100 across all tissues. To represent each gene, we selected the corresponding probe with the greatest intensity across all samples. The data contained expression profiles for 19,088 genes. We included expression profiles from some 96 normal tissues and excluded disease tissues and treated cell lines. We averaged expression values from replicated tissues averaged into a single value. To assess whether genes had differential expression for CNS tissues, we compared the 27 tissue profiles that represented brain or spinal cord to the remaining 69 tissue profiles with a one-tailed Mann-Whitney rank-sum test. Genes obtaining p,0.01 were identified as preferentially ex- pressed. Table S3 Keywords for Lipid and Height SNPs. We identified keywords associated with lipid and height associated SNPs; here we list the top 20. Table S3 Keywords for Lipid and Height SNPs. We identified keywords associated with lipid and height associated SNPs; here we list the top 20. Found at: doi:10.1371/journal.pgen.1000534.s005 (0.06 MB DOC) Found at: doi:10.1371/journal.pgen.1000534.s005 (0.06 MB DOC) Table S4 Crohn’s Disease SNPs from a meta-analysis of GWA studies. Here we list GRAIL results and summarize genotyping results for Crohn’s disease SNPs. These 74 SNPs emerged from a meta-analysis and as a result of replication genotyping, they were either validated (A), indeterminate (B), or failed (C). For each of the regions we list the SNP ID and the chromosome in the second and third column. In the fourth column we list the final combined association significance score of the SNP to the Crohn’s disease. In the fifth, sixth, and seventh columns we list GRAIL results including the number of genes in the region, the best candidate gene, and the text-based significance score for the region. Found at: doi:10.1371/journal.pgen.1000534.s006 (0.21 MB DOC) Schizophrenia application We identified singleton deletions or confirmed de novo deletions reported by one of three groups. We selected those deletions that June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 11 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions Supporting Information Pato11 Carlos Paz Ferreira27 Ben Pickard7 Jonathan Pimm6 Shaun M. Purcell1,2,3,4 Vinay Puri6 Digby Quested19¤ Douglas M. Ruderfer1,2,3,4 Edward M. Scolnick2,3 Pamela Sklar1,2,3,4 David St Clair12 Jennifer L. Stone1,2,3,4 Patrick F. Sullivan13 Emma F. Thelander9 Srinivasa Thirumalai18 Draga Toncheva15 Margaret Van Beck7 Peter M. Visscher14 John L. Waddington17 Nicholas Walker22 H. Williams5 Nigel M. Williams5 Table S7 Other promising regions in Crohn’s Disease GWA meta-analysis. Information about the top six regions identified by GRAIL from the next 75 most significant regions from the Crohn’s GWA study. All associations are indeterminate, and association p-values are taken from the GWA meta-analysis - these regions have not yet been replicated. Found at: doi:10.1371/journal.pgen.1000534.s009 (0.05 MB DOC) Table S8 Rare or de novo schizophrenia control deletions. Here we list all of the deletions that GRAIL identified as most related to other deleted genes (ptext,0.05). For each deletion we list the chromosome, the range of the deletion, the GRAIL p-value for the region, and the best candidate gene in the region identified by GRAIL. Most genomic coordinates are listed in HG17. * HG18 coordinates. Found at: doi:10.1371/journal.pgen.1000534.s010 (0.06 MB DOC) Text S1 A. Random SNP groups; B. Comparison of GRAIL to other related algorithms. Found at: doi:10.1371/journal.pgen.1000534.s011 (0.09 MB DOC) International Schizophrenia Consortium 3 International Schizophrenia Consortium Kristen Ardlie3 M. Helena Azevedo28 Nicholas Bass6 Douglas H. R. Blackwood7 Celia Carvalho11 Kimberly Chambert2,3 Khalid Choudhury6 David Conti11 Aiden Corvin8 Nick J. Craddock5 Caroline Crombie21 David Curtis20 Mark J. Daly2,3,4 Susmita Datta6 Stacey B. Gabriel3 Casey Gates3 Lucy Georgieva5 Michael Gill8 Hugh Gurling6 Peter A. Holmans5 Christina M. Hultman9,10 Ayman Fanous11 Gillian Fraser21 Elaine Kenny8 George K. Kirov5 James A. Knowles11 Robert Krasucki6 Joshua Korn3,4 Soh Leh Kwan12 Jacob Lawrence6 Paul Lichtenstein9 Antonio Macedo28 Stuart Macgregor14 Alan W. Maclean7 Scott Mahon3 Pat Malloy7 Kevin A. McGhee7 Andrew McQuillin6 Helena Medeiros11 PLoS Genetics | www.plosgenetics.org International Schizophrenia Consortium Kristen Ardlie3 M. Helena Azevedo28 Nicholas Bass6 Douglas H. R. Blackwood7 Celia Carvalho11 Kimberly Chambert2,3 Khalid Choudhury6 David Conti11 Aiden Corvin8 Nick J. Craddock5 Caroline Crombie21 David Curtis20 Mark J. Daly2,3,4 Susmita Datta6 Stacey B. Gabriel3 Casey Gates3 Lucy Georgieva5 Michael Gill8 Hugh Gurling6 Peter A. Holmans5 Christina M. Hultman9,10 Ayman Fanous11 Gillian Fraser21 Elaine Kenny8 George K. Kirov5 James A. Knowles11 Robert Krasucki6 Joshua Korn3,4 Soh Leh Kwan12 Jacob Lawrence6 Paul Lichtenstein9 Antonio Macedo28 Stuart Macgregor14 Alan W. Maclean7 Scott Mahon3 Pat Malloy7 Kevin A. Acknowledgments We would like to thank Drs. Eric Lander, Russ Altman , Chris Cotsapas, Joerg Ermann, Elizabeth W. Karlson, Kasper Lage, Guillaume Lettre, Roland Nilsson, and Ayellet Segre for insightful feedback and comments. We also thank Jesse Ross for assistance in constructing the web server. We also thank Jesse Ross for assistance in constructing the web server. 1 Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, Frank Middleton23 Vihra Milanova16 Christopher Morley23 Derek W. Morris8 Walter J. Muir7 Ivan Nikolov5 N. Norton5 Colm T. O’Dushlaine8 Michael C. O’Donovan5 Michael J. Owen5 Carlos N. Pato11 Carlos Paz Ferreira27 Ben Pickard7 Jonathan Pimm6 Shaun M. Purcell1,2,3,4 Vinay Puri6 Digby Quested19¤ Douglas M. Ruderfer1,2,3,4 Edward M. Scolnick2,3 Pamela Sklar1,2,3,4 David St Clair12 Jennifer L. Stone1,2,3,4 Patrick F. Sullivan13 Emma F. Thelander9 Srinivasa Thirumalai18 Draga Toncheva15 Margaret Van Beck7 Peter M. Visscher14 John L. Waddington17 Nicholas Walker22 H. Williams5 Nigel M. Williams5 2 Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, Supporting Information Table S5 Algorithms to prioritize candidate genes. Our search of the literature identified nine algorithms that could be used to prioritize genes for replication. Four methods require no user- specified disease information (supervised), and five require some disease information from the user. We list in each row the name of the disease, the website, the necessary genetic data, the functional data used to prioritize genes, the disease-specific information that must be included, and the availability of the method. Found at: doi:10.1371/journal.pgen.1000534.s007 (0.09 MB DOC) Figure S1 GRAIL p-value scores for random SNPs. We scored 100 random groups of 50 SNPs with GRAIL. The y-axis is the fraction of SNPs in the group with values below the threshold, the x- axis lists the specific threshold. For each threshold, we plot the distribution of the fraction of the 50 SNPs below that threshold as a box plot. The bar is the median - the mean value is explicitly listed below the box-plot. The box at each threshold lists the 25%–75% range. The error-bars line depicts the 1.5 inter-quartile range. The black dots illustrate outliers outside the 1.5 inter-quartile range. Found at: doi:10 1371/journal pgen 1000534 s001 (0 39 MB PDF) Table S6 Performance measures for prioritization algorithms. We used five algorithms (column 1) to score putatively associated SNPs from the Crohn’s meta-analysis. After calculating an ROC curve for each algorithm, we calculated the AUC (column 2). We also calculated a p-value with a one-tailed rank-sum test comparing the median rank of the validated SNPs to the median rank of the failed SNPs (column 2). Found at: doi:10.1371/journal.pgen.1000534.s001 (0.39 MB PDF) Figure S2 Sensitivity versus specificity for prioritization algorithms. We used 5 algorithms to score the 74 most promising putative SNP associations from the Crohn’s meta-analysis study. We assessed each algorithm’s ability to predict those SNP associations that ultimately validated in follow-up genotyping. For each algorithm, we created a received-operator curve (ROC). Found at: doi:10.1371/journal.pgen.1000534.s008 (0.04 MB DOC) Found at: doi:10.1371/journal.pgen.1000534.s002 (0.40 MB PDF) PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 12 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions Frank Middleton23 Vihra Milanova16 Christopher Morley23 Derek W. Morris8 Walter J. Muir7 Ivan Nikolov5 N. Norton5 Colm T. O’Dushlaine8 Michael C. O’Donovan5 Michael J. Owen5 Carlos N. References Perez-Iratxeta C, Bork P, Andrade MA (2002) Association of genes to genetically inherited diseases using data mining. Nat Genet 31: 316–319. 37. 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(2008) Large recurrent microdeletions associated with schizophrenia. Nature 455: 232–6. 7. References Weiss LA, Shen Y, Korn JM, Arking DE, Miller DT, et al. (2008) Association between Microdeletion and Microduplication at 16p11.2 and Autism. N Engl J Med 358: 667–75. 30. Roth RB, Hevezi P, Lee J, Willhite D, Lechner SM, et al. (2006) Gene expression analyses reveal molecular relationships among 20 regions of the human CNS. Neurogenetics 7: 67–80. g 8. International Schizophrenia Consortium (2008) Rare chromosomal deletions and duplications increase risk of schizophrenia. Nature. pp 237–241. 31. Singh SB, Davis AS, Taylor GA, Deretic V (2006) Human IRGM induces autophagy to eliminate intracellular mycobacteria. Science 313: 1438–1441. 9. Walsh T, McClellan JM, McCarthy SE, Addington AM, Pierce SB, et al. (2008) Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science 320: 539–543. 32. 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Identifying Common Function Across Disease Regions 27 Department of Psychiatry, Sao Miguel, Azores, Portugal, 27 Department of Psychiatry, Sao Miguel, Azores, Portugal, 19 West London Mental Health Trust, Hammersmith and Fulham Mental Health Unit and St Bernard’s Hospital, London, United Kingdom, 28 Department of Psychiatry, University of Coimbra, Coimbra, Portugal, 28 Department of Psychiatry, University of Coimbra, Coimbra, Portugal, 28 Department of Psychiatry, University of Coimbra, Coimbra, Portugal, 20 Queen Mary College, University of London and East London and City Mental Health Trust, Royal London Hospital, Whitechapel, London, United Kingdom, ¤ Current address: Department of Psychiatry, University of Oxford, Warneford Hospital, Headington, Oxford, United Kingdom ¤ Current address: Department of Psychiatry, University of Oxford, Warneford Hospital, Headington, Oxford, United Kingdom ¤ Current address: Department of Psychiatry, University of Oxford, Warneford Hospital, Headington, Oxford, United Kingdom 21 Department of Mental Health, University of Aberdeen, Aberdeen, United Kingdom, Author Contributions 22 Ravenscraig Hospital, Inverkip Road, Greenock, United Kingdom, Conceived and designed the experiments: SR RMP EJR SMP PS DA MJD. Performed the experiments: SR EJR ACYN International Schizophrenia Consortium. Analyzed the data: SR ACYN EMS RJX MJD. Contributed reagents/materials/analysis tools: SR International Schizophrenia Consortium EMS DA MJD. Wrote the paper: SR RMP EJR ACYN SMP PS EMS RJX DA MJD. Critically read and contributed to the final manuscript: SR RMP SJR ACYN SMP PS EMS RJX DA MJD. 23 State University of New York – Upstate Medical University, Syracuse, New York, United States of America, 24 Washington VA Medical Center, Washington D. C., United States of America, 25 Department of Psychiatry, Georgetown University School of Medicine, Washington D. C., United States of America, 26 Department of Psychiatry, Virginia Commonwealth University, Richmond, Virginia, United States of America, International Schizophrenia Consortium 3 McGhee7 Andrew McQuillin6 Helena Medeiros11 PLoS Genetics | www.plosgenetics.org 1 Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, 1 Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, 2 Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, 3 Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 4 Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America, p 5 School of Medicine, Department of Psychological Medicine, School of Medicine, Cardiff University, Cardiff, United Kingdom, 5 School of Medicine, Department of Psychological Med Medicine, Cardiff University, Cardiff, United Kingdom, 6 Molecular Psychiatry Laboratory, Department of Mental Health Sciences, University College London Medical School, Windeyer Institute of Medical Sciences, London, United Kingdom, 7 Division of Psychiatry, School of Molecular and Clinical Medicine, University of Edinburgh, Edinburgh, United Kingdom, 8 Neuropsychiatric Genetics Research Group, Department of Psychiatry and Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland, 9 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden, 10 Department of Neuroscience, Psychiatry, Ullera˚ker, Uppsala University, Uppsala, Sweden, y, pp , , 11 Center for Genomic Psychiatry, University of Southern California, Los Angeles, California, United States of America, 12 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom, 13 Departments of Genetics, Psychiatry, and Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 14 Queensland Institute of Medical Research, Brisbane, Queensland, Australia, 15 Department of Medical Genetics, University Hospital Maichin Dom, Sofia, Bulgaria, 16 Department of Psychiatry, First Psychiatric Clinic, Alexander University Hospital, Sofia, Bulgaria, y p g 17 Molecular and Cellular Therapeutics and RCSI Research Institute, Royal College of Surgeons in Ireland, Dublin, Ireland, 7 Molecular and Cellular Therapeutics and RCSI Research I 18 West Berkshire NHS Trust, Reading, United Kingdom, June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 13 References Shiratsuchi T, Futamura M, Oda K, Nishimori H, Nakamura Y, et al. (1998) Cloning and characterization of BAI-associated protein 1: a PDZ domain- containing protein that interacts with BAI1. Biochem Biophys Res Commun 247: 597–604. 22. Krallinger M, Valencia A (2005) Text-mining and information-retrieval services for molecular biology. Genome Biol 6: 224. PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 14 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions 48. Del Vecchio RL, Tonks NK (2005) The conserved immunoglobulin domain controls the subcellular localization of the homophilic adhesion receptor protein- tyrosine phosphatase mu. J Biol Chem 280: 1603–1612. 46. Ogawa J, Lee S, Itoh K, Nagata S, Machida T, et al. (2001) Neural recognition molecule NB-2 of the contactin/F3 subgroup in rat: Specificity in neurite outgrowth-promoting activity and restricted expression in the brain regions. J Neurosci Res 65: 100–110. 47. Yamagata M, Sanes JR, Weiner JA (2003) Synaptic adhesion molecules. Curr Opin Cell Biol 15: 621–632. Identifying Common Function Across Disease Regions 44. Hirao K, Hata Y, Ide N, Takeuchi M, Irie M, et al. (1998) A novel multiple PDZ domain-containing molecule interacting with N-methyl-D-aspartate receptors and neuronal cell adhesion proteins. J Biol Chem 273: 21105–21110. 49. Sugino K, Hempel CM, Miller MN, Hattox AM, Shapiro P, et al. (2006) Molecular taxonomy of major neuronal classes in the adult mouse forebrain. Nat Neurosci 9: 99–107. 50. (2005) A haplotype map of the human genome. Nature 437: 1299–1320 45. Kamei Y, Takeda Y, Teramoto K, Tsutsumi O, Taketani Y, et al. (2000) Human NB-2 of the contactin subgroup molecules: chromosomal localization of the gene (CNTN5) and distinct expression pattern from other subgroup members. Genomics 69: 113–119. 51. Myers S, Bottolo L, Freeman C, McVean G, Donnelly P (2005) A fine-scale map of recombination rates and hotspots across the human genome. Science 310: 321–324. 46. Ogawa J, Lee S, Itoh K, Nagata S, Machida T, et al. (2001) Neural recognition molecule NB-2 of the contactin/F3 subgroup in rat: Specificity in neurite outgrowth-promoting activity and restricted expression in the brain regions. J Neurosci Res 65: 100–110. 52. Stranger BE, Nica AC, Forrest MS, Dimas A, Bird CP, et al. (200 52. Stranger BE, Nica AC, Forrest MS, Dimas A, Bird CP, et al. (2007) Population genomics of human gene expression. Nat Genet 39: 1217–1224. 53. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, et al. (2008) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 36: D13–21. 47. Yamagata M, Sanes JR, Weiner JA (2003) Synaptic adhesion molecules. Curr Opin Cell Biol 15: 621–632. 54. Manning CM, Schutze H (1999) Foundations of Statistical Natural Language Processing. Cambridge: The MIT Press. 48. Del Vecchio RL, Tonks NK (2005) The conserved immunoglobulin domain controls the subcellular localization of the homophilic adhesion receptor protein- tyrosine phosphatase mu. J Biol Chem 280: 1603–1612. Processing. Cambridge: The MIT Press. 55. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, et al. (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31: e15. PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 15
https://openalex.org/W1982691950
https://zenodo.org/records/2270450/files/article.pdf
Italian
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Sugli spazii che ammettono un gruppo continuo di movimenti
Annali di matematica pura ed applicata
1,903
public-domain
17,221
(*) Memorie della Soeiet~ Italiana delle Seienze, 1897: Sugli syazii a trc dbnznsioni, che ammettonQ, ece. Ques~a Memoria sara in seguito indieata con (A). Avverto pure ehe la elassiea Theorie der Trans/ormationsgruppen del LIF~-ENGI,:L sar£ indicata in seguito soltanto col home di Lm. Sugli spazii che un gruppo continuo ammettono di movimenti. (Di GUIDO FuB~NJ, ~t Pisa.) (2) Come seppi, dope che questo lavoro era gi£ terminate, in una Memoria, che io non conoscevo, del BtscoNcI•I, inserita nel Nuovo Cimento (Aprile 1901) : Suuna classifi- cazione dei proble~ni dinamiei, quesLo teorema era gi£ state enunciate: perb la dimostra- zione del BIS0ONClNI in reMt'£ vorrebbe invece prorate che(< ogni gruppo continue con p ~rasformazioni infiniLesime [ndipendenLi si pub ridurre a un gruppo su¢ variabili>> ed percib completamente sbagliata (cfr. LIE; vo]. I, § 109), perch6 questa proposizione non vera in generale. Le mie co:~siderazioai rendono rigorosi gli altri bei risultati del BI- SCONCINI, MEMORIA I. Questo lavoro e un altro che mi riservo di pubblicare fra breve trat. tano della teoria generale degli spazii che ammettono un gruppo continuo di movimenti, delle proprietb, di questi gruppi, dei loro sottogruppi finiti diseon- tinui, e della determinazione degli spazii a quattro dimensioni che ammettono un tale gruppo di movimenti. I metodi di cut il prof. BiA~cm si servt (*) per determinare tutti gli spazii a tre dimensioni con un gruppo continuo di mo- vimenti non illuminano abbasianza sul problema generale: uno dei risultati del presente lavoro b appunto quelIo di dare un metodo generale per risol- vere con sole quadrature il problema, e che, applieato al caso particolare di spazii a tre dimensioni, permetterebbe di trovare rapidamente i risultati del prof. Bix~cm. Ma nb il metodo generale, nb il metodo del prof. Btx~cm generalizzato possono pot condurre senza una interminabile serie di ealeoli all'effettiva determinazione di tali spazii quando il numero delle loro dimen- sioni ~ maggiore di tre: la Memoria seguente svolgerh per il easo di quattro dimensioni un metodo assai pih rapido, e eomodo. p p Lo studio infine dei sottogruppi finiti diseontinui di movimenti ammessi da tall spazii condurr~, fra l'altro, a notevoli rappresentazioni degli spazii dei tipi (Viii) e (IX) del prof. BiA:~cm sulla sfera e sulla pseudosfera. 4O Fubini: Sugli spazii cite ammettono Un altro risultafo del presente lavoro che spore possa presentare qual- che interess% ~ di dare le condizioni necessarie e sufficienti affinch~ un gruppo si possa considerate come gruppo di movimenti: condizioni~ da cui si pub trarre qualche proprietk degna di nora per taluni di tali gruppi. Mi sia permesso infine di ringraziare qui vivamente l'illustre prof. LEvi- CIWTA, che acconsentl a leggere questo lavoro, prima che fosse pubblicato. § 1. Noi vogliamo trovare anzitutto dei teoremi generali relativi alla ricerca degli spazii che ammettono un gruppo continue di movimenti: prin- ciple della nostra ricerca sar~ di distinguere il problema in due parti: ]a prima relativa alla rieerca dei gruppi, che si possono considerare come gruppi di movimenti di uno spazi% la seconda re]ativa alla determinazione di uno spazio, di cui sia note il gruppo corrispondente. MEMORIA I. Il teorema fondamentale della prima parte delle nostre ricerche ~ il se- guente : Se un gru~vpo Gm trasforma in s~ uno spazio Sn~ e sele variet~ mi- time invarianti sono delle V,,-t,, ognuna di queste Vn-~ ~ pure trasformata i~ s~ da un gruppo proprio ad m parametri, eil gruppo G., si pub, con un opportune cangiamento di variabili, ridurre a un gruppo transitive sun -- k variabili (*). ,'t) Per dimostrare il nostro teorema, prendiamo una varietk V,,_~ composta di Vn-~ invariantij tale cio~ che per ogni punto di V,~L1 passi almeno una Vn-h invariante contenuta nella it., --,,-1 e scegliamo come coordinata (n k+ 1) esima di un punto A di S~ la distanza geodetica ynJ,+, dal punto stesso alla --.-iv(~) in discorso. Le y~_~÷, = cost. sono delle varieth. Vn-, geodeticamente parallele alla zr,~) invarianti, ossia composte di variet~ --,,-1 e quindi sono come la ~'(~) ~'~t -- 1 minime invarianti. Immaginiamo era nella _V('._ ~ scelta una .Tr(~),,_~ composta pure di V,~-k invarianti e consideriamo ]a varieth V (~,,,-1 generata dalle geodetiche dello Tr~) nei punti di V (~' Le trasfcrmazioni di G., spazio ambiente nermali a v n_ 1 n-2. TT(D , pure mutando la V ('),~-1 e ]a V `~',~-2 in s~ stesse, e le geodetiche normali a .~_. un gruppo continuo di movlmenti. 41 lr(~) ~ ben eerto che]a V (~ ~ una variet~ in- in geodetiche normali a -n-l, ,,-1 variant% ossia che una V~_~ invariante ehe passa per un suo punto giace tutta in eqsa. La V~,)_~ dovr~ quindi tagliare ogni variet~ y~-k+,---~ coat. pure in variet~ iuvarianti. Posto questo, osserviamo che per determinare la posi- zione di un punto di S~, baster~ conoscerne la proiezione geodetica Ao) su W(1) ,,_, e la coordinata y~_~+,. E not siamo cosl ricondotti per determinare un sistema di coordinate in S~, a cercare un sistema di coordinate per ]a VI~)_~, la quale varie~ ~, come lo spazio ambient% trasformata in s~ dal gruppo G,~. E noi percib opere- remo sulla V~'~)_I, come abbiamo operato in S~. MEMORIA I. Not determineremo cio~ la posizione di un punto Ao) di p'.-1Tr(') nel seguente modo: Prenderemo una va- T/C1) riet~t --._~lz(~, tutta contenuta in --,,_~ e composta di variet~ minime invarianti e individueremo un punto A(0 di V~L1 dando la sua distanza geodetiea y,--k+~ da]la V(,~L~ (cio~ ]a distanza misurata qulla geodetica di V~L, tirata dal punto in discorso normalmente alla V (~) ~ .-2j e la posizione della qua pro- iezione A(~) sul]a VI~L2 stessa. E come coordinata (n-- k + 2) esima di un punto quatunque A diS, prenderemo appunto ]a y,,_~+~ test~ definit% reIa- tiva alla qua proiezione Ao) sulla V~L~. Or% per un ragionamento gik usato, ben chiaro the ]e y,-k+~ = coat. sono nella W(~)~ 1 variet~t invarianti, perch~ geodeticamente parallele alla VC~)_2 entro TT~, --~,_,; e per quanto abbiamo no- tato saranno pure invarianti quelle variet~ di S, generate dalle geodetiche di S, normali a V(,~L1 net punti di una di queste variet~ y,,-.~+2 = cost, ossia anehe in tutto lo spazio ambienle S. le y,-a+~ ----- cost. sono variet~ (a n--1 dimensioni) invarianti. Ci siamo dunque ridotti a determinare la posizione di un punto A(~) di V'~) E anche qui, procedendo con lo stesso metodo, prenderemo una --.-a, n - 2 • TT~I~ ~,on~enuta nella V~I>o, e invariante; e definiremo la posizione di un punto Ao) di V(+~>o dandone la distanza geodetica yn-~+~ (cio~ misurata lungo una geodetica di v-<, o normale alla r/-(,) ~ dalla v'c, ~,_o -,~_~ -~_~ stessa e la posizione della sua proiezione A(~) sulla V<,',)_a • E definiremo pot come coordinata (n -- k + 3) e~im~ di un punto qualunque A di S~ la y,~-~+s, test~ definit% relativa alla pro- iezione A(~) su V<~'2_~. della proiezione Ao) del punto A sulla V¢+~)_~. Per una osservazione gi~ usat% le y~_n+~ ~ cost. (che nella --n-2TT(I) sono variet~ inva- rianti, perch~ geodeticamente parallele alla V(~l~)__s) sono varietY, pure inva- rianti in T~-~) --n-~ e quindi anche nello spazio ambiente S~. Cosl si prosegue fino ad arrivare a una VI~)._7~+~ invariante, in cut la posizione di un suo punto qualunque A(~-') si definisee dandone la dis~anza Annali di Matematica, Serie III, tomo VIII. 6 Fu b in i: Sugli spazii che ammettono 42 TT(1) Tr(1) invariante della v,-~+l e la posizione della sua geodetica y, da una ~,,-k proiezione A(~) sulla V',1/_1~ stessa. MEMORIA I. E si definisce come coordinata n e~i'~ di un punto A di S~ lay,,, testb definita, relativa a quel punto A(g-o di VC,~2x.+~, a eui si perviene mediante le successive proiezioni considerate. Nella Tz¢~, rn--k si assume poi un sistema qualsiasi di coordinate y,, y~,.., y,-k; e per le prime n -- k coordinate di un punto A di S, si scelgono appunto le y,... y,-~ relative al corrispondente punto A ¢~) di V~,IL~ a cui si arriva mediante suc- cessive proiezioni. p Le osservazioni da noi fatte ci dicono senz'altro che le y,-~+i = cost. (i ~ 1, 2,..., k) sono in S, varieth invarianti, e che quindi le yn-~+J ----- COSt. ~... ~ yn ~ cost. sono precisamente le varieth minime invarianti. sono precisamente le varieth minime invarianti. Quale aspetto assume ora il nostro gruppo con questo sistema di varia. bill? Intanto, siccome le y,_h+i--cosf, sono, come si diss% varietK inva- rianti ~ certo cheit nostro gruppo non trasforma le y~-k~, y,-k~.~7-.. Y- ossia che se le trasformazioni di Gm sono date dalle: y't ~- ft (y, .. • yn-k y.-h+, • • • y.) (t = 1, 2,.... n) (*) y't ~- ft (y, .. • yn-k y.-h+, • • • y.) (t = 1, 2,.... n) (*) (dove left conterranno m parametri arbitrarii, sar~ certamente: y't ~- ft (y, .. • yn-k y.-h+, • • • y.) (t = 1, 2,.... n) (* (dove left conterranno m parametri arbitrarii, sar~ certamente: (dove left conterranno m parametri arbitrarii, sar~ certamente: f,-k+i= y,-l,+i (i = 1, 2,... k). Ma noi diciamo di pih che le f,...f,-~ non contengono y,-I,+t.., y, e dipendono ciob soltanto (oltre ehe dai parametri del gruppo) dalle variabili y,.. • y,-~. In altre parole: noi dimostreremo che le prime ,, n--/c ,, coordinate y'~...y',k di quel punto Ao, dove un punto A di coordinate y,...y,-k y,_~,+,.., y, ~ condotto da una trasformazione F qualsiasi di G,~ non di- pendono dai valori di y,,-h~-,.., y,~ ossia cher conduce il punto che ha per prime coordinate proprio y,...y,~_h~ e ehe ha tutte le altre coordinate uguali a zero, nel punto, le cui prime coordinate sono y'~...y',-k e di cui tutte le altre coordinate sono nulle. (*) Dove indichiamo con Y't i valori trasformati delle yt. (*) Ricordiamo perb esplicitamente (a scanso di ogni equivoco) che, come faremo ve- dere piO tarJi~ queste condizioni sono semi)re soddisfatte da un tale gruppo. MEMORIA I. Posto questo, not ora procederemo a dare a]cune proprieth di un gruppo, che sono condizioni necessarie e sufi3cienti, affinch~ esso si possa considerare come un gruppo di movimenti. E comineieremo anzitutto dalla considerazione dei gruppi a trasformazioni infinitesime linearmente indipen- denti; e rammenteremo che il Prof. BIA:NcltI dimostrb (e pih sotto io ne darb una dimostrazione intuitiva, chg ne spiega l' intima ragione di essere) ehe ogni gruppo G~ transitivo su n variabili g ammesso da qualehc spazio a n dimensioni; not dimostreremo anzi che eib g veto per qualsiasi gruppo a n variabili con trasformazioni infinitesime linearmente indipendenti. Not per ora, a proposito di questi gruppi, non possiamo che dare il teorema seguente: Condizione necessaria e sufficiente affinch~ un gruppo Gm a trasforma- zioni i~finitesime linearmente indipendenti sun + m variabili si possa con- siderare come gruppo di movimenti di uno spazio ad n + m dimensioni ~ ehe il gruppo sia simile a un gru?po semplicemente transitivo sum lettere ; cib chesi pub anche esprimere dicendo che Gm'deve essere un sottogruppo di un gruppo semplicemente transitivo con n + m lettere ed n + m parametri (*). La condizione g evidentemente necessaria; infatti il gruppo Gm ha come variet~ minime invarianti delle V,~: e~ se S~+,~ g uno spazio the ammette G~, si vede tosto prendendo in esso quel sistema di coordinate, the fu deft- nito al paragrafo precedente che il gruppo Gm sar~ trasformato in un gruppo simile ehe opera transitivamente sum lettere. Che questa condizione sia anche sufficiente, si dimostra facilmente cos): Se G~ g simile a un gruppo the opera transitivamente sum variabili y, y~... y,~, si introducano come coordinate appunto le y, y~... ym insieme ad altre n funzioni qualsiasi delle coordinate iniziali ym+,...ym+~ tali the y,...ym ym+,...y,~,~ siano indipendenti tra § 2. Posto questo, not ora procederemo a dare a]cune proprieth di un gruppo, che sono condizioni necessarie e sufi3cienti, affinch~ esso si possa considerare come un gruppo di movimenti. E comineieremo anzitutto dalla considerazione dei gruppi a trasformazioni infinitesime linearmente indipen- denti; e rammenteremo che il Prof. BIA:NcltI dimostrb (e pih sotto io ne darb una dimostrazione intuitiva, chg ne spiega l' intima ragione di essere) ehe ogni gruppo G~ transitivo su n variabili g ammesso da qualehc spazio a n dimensioni; not dimostreremo anzi che eib g veto per qualsiasi gruppo a n variabili con trasformazioni infinitesime linearmente indipendenti. A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h) di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E v~,) sar~ infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1 17-(1) portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~ V(t) portato nella proiezione B(')di B su rn_l; C0SI A (~) sarg portato nella T/-(t) C0Si eontinuando arriveremo alla di- proiezione B(2) di B(') su --,~-2 ece.; e mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti B', B(~) coincidono. MEMORIA I. O in altre parole io dimostrerb chese Ao) ~ la proiezione di un punto A di S, sulla g (' A(O la proiezione di .4(0 sulla --,,-2TT(~), ..., A (k) quella di n--1 un gruppo continuo di movimenti. 43 A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h) di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E v~,) sar~ infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1 17-(1) portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~ V(t) portato nella proiezione B(')di B su rn_l; C0SI A (~) sarg portato nella T/-(t) C0Si eontinuando arriveremo alla di- proiezione B(2) di B(') su --,~-2 ece.; e mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti B', B(~) coincidono. A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h) di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E v~,) sar~ infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1 17-(1) portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~ V(t) portato nella proiezione B(')di B su rn_l; C0SI A (~) sarg portato nella T/-(t) C0Si eontinuando arriveremo alla di- proiezione B(2) di B(') su --,~-2 ece.; e mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti B', B(~) coincidono. A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h) di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E v~,) sar~ infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1 17-(1) portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~ V(t) portato nella proiezione B(')di B su rn_l; C0SI A (~) sarg portato nella T/-(t) C0Si eontinuando arriveremo alla di- proiezione B(2) di B(') su --,~-2 ece.; e mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti B', B(~) coincidono. , ( ) § 2. MEMORIA I. Not per ora, a proposito di questi gruppi, non possiamo che dare il teorema seguente: Condizione necessaria e sufficiente affinch~ un gruppo Gm a trasforma- zioni i~finitesime linearmente indipendenti sun + m variabili si possa con- siderare come gruppo di movimenti di uno spazio ad n + m dimensioni ~ ehe il gruppo sia simile a un gru?po semplicemente transitivo sum lettere ; cib chesi pub anche esprimere dicendo che Gm'deve essere un sottogruppo di un gruppo semplicemente transitivo con n + m lettere ed n + m parametri (*). La condizione g evidentemente necessaria; infatti il gruppo Gm ha come variet~ minime invarianti delle V,~: e~ se S~+,~ g uno spazio the ammette G~, si vede tosto prendendo in esso quel sistema di coordinate, the fu deft- nito al paragrafo precedente che il gruppo Gm sar~ trasformato in un gruppo simile ehe opera transitivamente sum lettere. Che questa condizione sia anche sufficiente, si dimostra facilmente cos): Se G~ g simile a un gruppo the opera transitivamente sum variabili y, y~... y,~, si introducano come coordinate appunto le y, y~... ym insieme ad altre n funzioni qualsiasi delle coordinate iniziali ym+,...ym+~ tali the y,...ym ym+,...y,~,~ siano indipendenti tra Fubini: Sugli spazii che ammettono 44 loro. Le trasforma.zioni infinitesime di Gm insieme alle trasformazioni genereranno un gruppo Fro+,,, semplicemente transitiv% y,~+t ' " ' " ' ~ y.~+~ e di cui Gm ~ un sottogruppo. Per il citato teorema del prof. BiA~cm F,,~., ammesso da qualche S,,+~, che ammetter~ percib anche G~. q p Nella seconda parte di questa dimostrazione abbiamo visto incidental- mente che se un G,~ ~ simile a un gruppo transitivo surn lettere, esso anehe sottogruppo di un F,,~ transitivo in S,,+,,. ¥ieeversa se G~ ~ sotto- gruppo di un F,,+, transitivo in S,,+~, esso sar~ certo un gruppo di movimento di ciaseuno degli spazii, che ammettono F,,+,~ e quindi, per quanto abbiamo dimostrato, sarh simile a un gruppo semplicemente transitivo sum lettere. Noi pia tardi dimostreremo che ogni gruppo a trasformazioni infinite- sime linearmente indipendenti pub essere considerato come gruppo di movi- menti: il presente teorema ci dar~ pcrcib atcune interessanti proprietY, ge- nerali di gruppi siffatti; cib che permette di .~gevolarne di molto ]a ricerca. § 3. MEMORIA I. Abbiamo cosl studiato i gruppi a trasformazioni infinitesime li- nearmente indipendenti, che posstmo essere considerati (tutti)come gruppi di movimenti e abbiamo visto che in fin dei conti essi si riducono tutti ai gruppi transitivi, in cui it numero dei parametri eguaglia quello de]le variabili. Ora ci proporremo l~ questione di riconoscere quando un gruppo a trasformazioni infinitesime linearmente dipendenti pub essere considerato come gruppo di movimenti. Noi ora daremo una prima risoluzione di ql, esto problema, av- vertendo perb ehe noi vi ritorneremo pih oltre, con altri metodi, e ottenendo una risoluzione pih elegante ed intuitiva. p g Anzitutto osserveremo che per il teorema fondamentale del § 1, seil nostro gruppo ~ un gruppo G~+~ a ,, n + m ,, parametri con sole n trasfor- mazioni infinitesime linearmente indipendenti, esso (avendo delle V~ per va- riet~ minime invarianti) sarh certo simile a un gruppo transitivo su n varia- bill, e si potr~ considerare come gruppo di movimenti di una varietY, ad n dimensioni. Noi supporremo senz'altro che il gruppo sia gi~ stato ridotto sotto questa forma, che diremo forma normale del gruppo, con un opportuno cangiamento di variabiIi. Siano dunque Xt=~/)(x, . x,) ~ (t=l, 2,...n+m) r:i " " ~ Xr le trasformazioni infinitesime del nostro gruppo Gn+m e sia le trasformazioni infinitesime del nostro gruppo Gn+m e sia n ds'~ ~a~dxidx~ un gruppo continuo di movimenti. 45 un gruppo continuo di movimenti. 45 l'elemento lineare di uno spazio S,, che ammetta G,+,~. Le formule di Kir.- Ia~G danno che ' ~" /=0 (l, r= 1, 2,..., n). (1) (1) E noi le seriverem% ponendo E noi le seriverem% ponendo (l) v =:, av,=: . alr~ +ak~-] (l) v =:, av,=: . alr~ +ak~-] sotto la forma W ~l) O_ ai~ + a~. -~- 0, ossia Xz (aik) "q- a']~ = 0. (1) Ox,. (1) I1 prof. Bia~crxI osservb the in virth delle (1)' si ha the I1 prof. Bia~crxI osservb the in virth delle (1)' si ha the Xl [X, (aik) + -'"' .ikl--Xt[Sl(aik)+a'[~] (1, t=l, 2,..., n+m) (2) Xl [X, (aik) + -'"' .ikl--Xt[Sl(aik)+a'[~] (1, t=l, 2,..., n+m) (2) (2) identicamente nullo: cib ehe del resto ~ intuitivo perch~ se un S,, ammette due trasformazioni infinitesime Xt, Xt esso deve ammettere anehe la (Xt, X,). Per ipotesi m delle trasformazioni infinitesime del gruppo sono combinazione lineare delle altre n, le quali sono poi linearmente indipendenti. MEMORIA I. :Noi ammet- teremo cite queste ultime sieno le X,~ X,,..., X,~ e porrem% indieando con ?~il delle funzioni di x,, x,,.., zn st X,,+i = ~=' ~,I" X, (i = 1, 2,... m). E il sistema delle equazioni di Klr,r,i~(~ sarh da noi scritto nella forma: Xl (aik) + -i~-(" --~- 0 (l = 1, 2,... n) (3) "'+"- = 2,... m). (4) Xn+t (aik) -- Z ~i ° Xl (aik) 2r- am ,*ik = 1=1 (3) (4) Cib ~ chiaramente lecito perch~ se alla (4) aggiungiamo Cib ~ chiaramente lecito perch~ se alla (4) aggiungiamo st ~,b 1 rc, ft) ,,-fill st ~,b 1 rc, ft) ,,-fill somm~ evidentemente nulla in causa delle (3), otieniamo appunto l'equazione di Kit, r~xs(~ relativa a X,,+~ ed a ai~, coma 5 scritta nella (1)', E ora poich~ identicamente somm~ evidentemente nulla in causa delle (3), otieniamo appunto l'equazione di Kit, r~xs(~ relativa a X,,+~ ed a ai~, coma 5 scritta nella (1)', E ora poich~ identicamente tl -- "~ "' Xt (aik) 0 x.+, (..O = Fubini: Sugli spazii che ammettono 46 le (4) diventano le (4) diventano ossia ossia n *'dn+t) l~=l a (l) 0 (4)' r:l ~ Xk r7> + i" (4)" ^ "^~*)\ n) ,~,. ai~ ~a, ~ ~7 ~ ~}, 6 ~, | (t, i, k, r ~- 1, 2,... ,,.~+ah,.. ~-)=0 (t=l, 2,... m). (4)'" (4)' ossia r:l ~ Xk r7> + i" (4)" (4)" ossia ^ "^~*)\ n) ,~,. ai~ ~a, ~ ~7 ~ ~}, 6 ~, | (t, i, k, r ~- 1, 2,... ,,.~+ah,.. ~-)=0 (t=l, 2,... m). (4)'" (4)'" Il nostro sistema di equazioni resta cosl mutato nel sistema di equazloni formato dalle (3) 0 dalle (4)"'. Ora dalle (3), essendo il determinante delle ~, (r, 1 1, 2,... n) differente da zero si possono trarre le derivate delle aih in funzione delle alk stesse; e, per l'osservazione tests citata del pro- fessore Bxx~cm si vede ehe affineh~ il sistema delle" (3)~ (4)'" sia integrabile basta ehe 1. ° Le (4)"' considerate come equazioni lineari a]gebriche per le aik ammettono in un punto almeno una soluzione (che ci dar~ i valori iniziali delle aln). Si dovrb~ poi solo esaminare (volendoci restringere a spazii reali) se le solite disuguaglianze sono soddisfatte. g g 2. MEMORIA I. ° I primi membri delle (4)'" derivate rispetto a x,, x~,.., x,~ suc- cessivamente dieno per le (3) e le (4)"' stesse risultati identicamente nulli. Questa seconda condizione equivale anehe (essendo il determinante delle ~ diverso da zero) alt'altra eondizione ehe applicando ai primi membri de]!e (4)"' le X,... Xn si ottenga in virth delle (3) e delle (4)'" stesse un risultato iden- ticamente nullo. Noi ora vogliamo far vedere che questa seeonda eondizione h sempre soddisfatta. Infatti per l'osservazione del prof. Bra~rcm ~ sempre per le (3) e le (4)" identieamente : _ Xn t [Xz(aik) + alk j ~ 0 It=l, 2,... m] [i, k, l=l, 2,... n] ossia ossia Xt [X,,,t (aih)] -- X~+, [Xl (ai~)] + Xz (a~.~ +°) -- -v- /'ae) xxn+~ k iki ----- O. (5) (5) Xt [X,,,t (aih)] -- X~+, [Xl (ai~)] + Xz (a~.~ +°) -- -v- /'ae) xxn+~ k iki ----- O. (5) un gruppo continuo di mov6nenti. 47 Si ha pure identieamente: Si ha pure identieamente: p Xz [X~ (a~,)] -- X,. [Xz (.i~,)] + X, [ai'/.] -- X~ r..,,~ = 0 Lt~ik] (I~ r~ i~ k~---1~ 2~...~ n) a ] (I~ r~ i~ k~---1~ 2~...~ n) ossia • It) -- Ct) fr) ~,. Xz [x~ (a;k)] ~, x,, [xz (a~)] + ¢' X~ (a,~) -- ~' X~ (a'f~) = o ossia : • It) -- Ct) fr) ~,. Xz [x~ (a;k)] ~, x,, [xz (a~)] + ¢' X~ (a,~) -- ~' X~ (a'f~) = o ossia : • It) -- Ct) fr) ~,. Xz [x~ (a;k)] ~, x,, [xz (a~)] + ¢' X~ (a,~) -- ~' X~ (a'f~) = o ossia : Xz [~o;' X¢ (ai~)] XI (77)) X~ (aik) -- _(t, • - v,. X~ [Xt (a~k)] + '~' ~7' X~ (a~i) -- o. E, poich~ per le (3) sarh pure identicamente (in. virth delle (3) e delle (4)"') sarh pure identicamente (in. virth delle (3) e delle (4)"') Xz ~ " X~ [Xt (ai~)] + Xl " ('' -- .~G r~,,,~ 0 t?,. X. (aik)] 97' "' -- t~ a',7] ~,. w,kJ = e quindi anehe identicamente: [Xz ~'~ X, [X~ (a,k)] + Xz r,,,w,,~ / L~,' u,:k] -- ~(~)X,, (aTe) 0.. (6) r=l [Xz ~'~ X, [X~ (a,k)] + Xz r,,,w,,~ / L~,' u,:k] -- ~(~)X,, (aTe) 0.. (6) r=l (6) Sottraendo dalla (5) la (6) troviamo l'identith: Xl Xn+t(a/k)-- ~rXr(a/k) -- Xn+t-- ~ ~(t,,~ X~ Xl(aik) -~- r=l r-=l r=l r=l E poieh~ ~ identicamente: E poieh~ ~ identicamente: r=l troveremo senz'altro identieamente: / tt \ \ r----I / che ~ appunto quanto si voleva dimostrare. (Si ricordi che i primi membri delle (4)' e delle (4)"' sono identici.) Prima ora di venire a parlare della 1) eondizlone vogliamo fare un'os- servazione generale. 48 Fubini: Sugli spazii che ammettono Se un gruppo F qualsiasi si pub considerare come gruppo di movimenti di uno spazio a n dimensioni, esso pub essere anche considerato come gruppo di movimenti di uno qualche spazio a un numero n + t di dimensioni (t in- tero qualsiasi). ossia q Supposto infaifi il gruppo ridotto a forma normale in n o rneno varia- bili~ e se d s 2 ~- ~ aik d x~ d ~k (i, k = 1, 2,... n) i,k l'elemento di uno spazlo a n dimensioni che ammette il nostro gruppo, al- lora~ come tosto si verifiea l'elemento di uno spazlo a n dimensioni che ammette il nostro gruppo, al- lora~ come tosto si verifiea ds'---- ~aikdxid x~ + ~almdxzdx,n i,k Im (i, k-----l, 2,... n) (/, m-----n+ 1,... n+t) x~ + ~almdxzdx,n Im (i, k-----l, 2,... n) (/, m-----n+ 1,... n+t) dove le al~, non dipendono dalle x,, x2,.., x~ ~ l'elemento lineare di uno spazio a n + t dimensioni che ammette il nostro gruppo. Dunq ue : Affinch$ un dato gruppo, gih ridotto a forma normale in n variabili, possa essere ammesso come gruppo di movimenti da uno spazio (che natu- ralmente non potrh avere meno di n dimensioni) D condizione necessaria e sufficiente che le (4)'" considerate come equazioni lineari algebriche helle aik sieno in un punto risolubili rispetto alle ail,. Dunque questa condizione si esprime semplicemente con l'annullarsi di una matrice. Ora osserviamo il prim() membro delle (4)"' e vediamo fosto ehe il ter- ~(l, ~ 77 ~ - ~ un termine de'l determinante che si ottiene moltiplicando mine ai~ ~,. ~ xk con le note regole, in modo opportuno i] determinante delle aik per il deter- minante delle ~(l'e per l'Iaeobiano delle ~(~). Quindi abbiamo: Condizione necessaria e sulficiente affinchD il nostro gruppo gi& ridotto a forma normale possa essere considerato come gruppo di movimenti ~ che si possa trovare un determinante non nullo simmetrico [ ai~ [, il prodotto del quale per il determinante [ ~'~'[ , l = 1 7 2,... n) e per l'Iacobiano delle • ,~,!) (r = 1, 2,... n) ottenuto in modo opportuno con le note regole sia, almeno in un punto, semisimmetrico. E cib per ciascun valore particolare di t. in un punto, semisimmetrico. E cib per ciascun valore particolare di t. Se ora n ~ dispari, il determinante semisimmetrieo cosi ottenuto ~ hullo; • • • . * ¢~ (t) ='= 0 (x, x, . . . x,) non sono funzioni indipendenti. Se ora n ~ dispari, il determinante semisimmetrieo cosi ottenuto ~ hullo; • • • . * ¢~ (t) ='= 0 (x, x, . . . Annag eli Matematica~ Serle III, tomo VIII. ossia x,) non sono funzioni indipendenti. un gruppo continuo di movimenti. 49 Quindi in particolare: l h Quindi in particolare: Se un S~ ammette un G,,+, transitivo, esiste certamente nel caso che n sia dispari, qualche trasformazione infinitesima permutabile con G~+,. p q f f p § 4. Sia ora dato un gruppo, gib. ridotto sotto forma normale, cio~ con tante trasformazioni infinitesime linearmente indipendenti quante sono le variabili su cui opera il gruppo. Noi vogliamo esaminare come si semplifi- cano in questo caso le equazioni di KxLLm(~. Siano '~ ~ (/-----1~ 2,..., n--~-m) r=l le trasformazioni infinitesime del gruppo e sia le trasformazioni infinitesime del gruppo e sia ds~--~ai~dxidx~ (i, k~l, 2,..., n-I-t) l'elemento lineare di uno spazio a , n-f-t ,, dimensioni che ammetta il no- stro gruppo. l'elemento lineare di uno spazio a , n-f-t ,, dimensioni che ammetta il no- stro gruppo. g pp Se noi seriviamo le equazioni di KmLxN(~ noi riconoseiamo faCilmente: 1. ° :Per le ai~ (i, k ~ 1, 2 7... n) queste equazioni assumono proprio la stessa forma, come se noi volessimo cereare gli spazii ad n dimensioni ammessi dal gruppo. g pp 2. o Per le alk(i, k~---n-{-1, n+2~..., n~t) le equazioifi di KIn- LInG diventano semplicemente: ~ ~ aih ~ O. .'~1 E poich~ delle Xz proprio n sono linearmente indipendenti, si ha sem- plicemente : aih --0 (r= 1, 2,..., n) ~Xr e le equazioni di KILLISe dicono soltanto che le aik in discorso sono funzioni soltanto di Xn~,,..., X~+t. e le equazioni di KILLISe dicono soltanto che le aik in discorso sono funzioni soltanto di Xn~,,..., X~+t. 3. ° Per le aik (i ~ n, k > n) le equazioni di KILHSa diventano: ~ -- 0 ~, O x~ r the ~ un sistema di equazioni ehe non eontiene nessuna aik del primo o del secondo tipo. Anzi questo sistema di equazioni si seinde per eiaseun valore di k in t sistemi di equazioni distinti, l'uno relativo alle a6~+,, il secondo alle aim+~ eee. (i~ 17 2,..., n). Annag eli Matematica~ Serle III, tomo VIII. 7 50 Fubi~i: Sugli spazii che ammeltono Le equazioni relative alle ai~ (i, k ~ n) si semplificano, ossia si possono ridurre a un nuniero minore di incognite mediante le equazioni lineari (4)"'~ mentre hello stesso tempo per ciaseuna delle aik da determinarsi si ottengono soltanto ~t equazioni. ossia Queste osservazioni riescono utili specialmente per il caleolo effettivo degli elementi lineari da determinarsi. § 5. Ora not vogliamo dimostrar% servendoci dei risultati finora ot- tenuti~ il seguente teorema fondamentale : Con la sola risoluzione di effuazioni algebriche si possono determi~a~'e tutti i gruppi 7 che si posso~o co~siderare, come 9ruppi di movimenti, tro- vando le loro tras[ormazioni infinitesime; con sole quadrature si posso~o determinate Ie trasformazioni finite di tall gruppi~ e gli elemeuti lineari degli spazii loro corrispondenti. g p p La dimostrazione di questo teorema ~ semplicissima: basra a tale scopo la semptiee ossei'vazione che ogni gruppo eontinu% ehe si possa eonsiderare come gruppo di movimenti~ si pub, con un cangiamento di variabili ridmore sotto forma normale, cio~ si pub far si che possegga tante trasformazioni in- finitesime linearmente indipendenti quante sono le variabili su cut opera ef- fettivamente: in aitre parole si pub ridurre a essere transitivo sulle variabili ehe esso effettivamente trasforma. Ora not sappiamo che con la risoluzione di sole equazioni algebriche si possono trovare tutte 18 possibili composizioni realmente distinte dei gruppi a un numero qualsiasi di parametri. Di pih (LIE, Kap. 27, 29) con la sola risoluzione di equazioni algebriehe si possono trovare tutti i gruppi eontinui transitivi, appena se ne eonosea la eomposi- zione, e non si considerino distinti due gruppi simili. E eosi dimostrata sen- z'altro la prima parte del nostro teorema. Esaminando la forma delle tra- sformazioni infinitesime dei nostri gruppi si potrebbe dimostrare anehe la terza parte del nostro teorema; mail proeedimento pifi breve a tale seopo g il seguente: Si rieordi anzitutto ehe con sole quadrature si possono trovare le equazioni finite del nostro gruppo, perehg il gruppo ~ transitivo (LI~ 1. c.), cib ehe intanto dimostra la seeonda parte del nostro teorema. Atlora distin- guiamo due easi: seeondoehe le dimensioni dello spazio da determinarsi sono in numero uguale o maggiore del numero detle -¢ariabili, su eui il nostro gruppo, ehe col proeedimento preeedente g stato evidentemente ottenuto sotto forma normale, opera effettivamente. Comineiamo a trattare il primo easo: sia eiog uguale il numero n delle dimensioni dello spazio da determinarsi e delle variabili su eui opera (transitivamente) il gruppo. Sia A un punto generieo dello spazio e siano a(,°h valori qualsiasi ehe soddisfano alle equa- un gruppo continuo di movimenti. 5! ossia zioni lineari (4)"' (supposte naturalmente risolubili) (dove alle xi si sostitui- seano le coordinate (x¢ °') del punto A) e, se v.ogliamo restringerei a spazii reali, anche alle solite disuguaglianze, se questo b possibile. Sia ora B un punto qualunque dcllo spazio, in cui si vogliono trovare i valori delle ai~. Siceome il gruppo opera transitivamente nello spazio ambiente esiste almeno una trasformazione T del gruppo ehe trasporta il punto B nel punto A; essa porter~ anche un intorno qualunque di B in un intorno di A; di pih essa si potrb, ottenere con la sola risoluzione di equazioni finite. Sieno x~ 'J le coordinate di B, e siano al),i valori finora ineogniti delle alh nel punto B. La T, stabilendo una corrispondenza biunivoca tra gli intorni di B o di A, stabilisee una corrispondenza biunivoea tra i punti x~"+ d x~ '~ e i punti • y+ d x~ °', corrispondenza determinabile con differenziazioni ; le d x~ °~ ven- gono date come espressioni lineari omogenee helle d x~ '~, a coefficienti eo- stanti. Sostituiamo nella ~o,~-'°' d_,~'°'d x~ ) alled x~; i loro valori cosi determi- nati, otterremo una forma quadratica helle d~ '-')_~ , che deve essere identiea "~ d x~'; d x~; csprimendo questa identith, si hanno senz'altro i valori con z a~i,, delle a~ nel punto qualunque xi" dello spazio ambiente, espressi in funzione delle costanti iniziali aii?2. Passiamo ora al caso ehe il numero n + m delle dimensioni dello spazio sia maggiore del numero n delle variabili (x,, x.~,.., x,,), su cui opera effet- tivamente il gruppo, cosiceh~ nello spazio in diseorso il gruppo non opera pih transitivamente. E sia ds'---~'a~l,,dxidx~ (i~ k=l, 2,..., n+m) i,k ds'---~'a~l,,dxidx~ (i~ k=l, 2,..., n+m) i,k l'elemento lineare dello spazio. Per le a;, (i, k-- n -~- 1,..., n -t- m) nutla vi sarebbe da dire, poich~ noi sappiamo gi~ che esse sono flmzioni (del resto arbitrarie) delle x~,, x,,~,..., x~+,, (§ 4). ossia In ogni modo si osservi che x affinche il nostro spazio ammetta effettivamente il gruppo in discorso, ~ con-- dizione necessaria e sut~ciente che una trasformazione T del gruppo che porta un punto B~x~" di una variet~ V~ invariante in un altro punto A ~ x~' della stessa V~ e quindi stabilisce anehe una corrispondenza biuni- voca ira gli intorni dei due punti~ porti anche la forma ,,,/,,d i,k nella forma (x?') d d x . ,,,/,,d i,k (x?') d d x . ,,,/,,d i,k ,,,/,,d i,k nella forma (x?') d d x . (x?') d d x . 52 Fubini: Sugli spazii che ammettono Allora prendiamo una variefft qualsiasi V,~ a m dimensioni ehe abbia uno e un sol punto eomune con eiaseuna delle variefft V,~ minime invarianti e fissiamo in ciascun punto della Vm dei valori arbitrarii per le aik aneora da de~erminarsi, purch~ natmralmente essi soddisfino alle solite equazioni li- neari: eib ehe eorrisponde all'introduzione di funzioni arbitrarie. Per poi de- terminate i valori al.}~ delle a~.l~ in un altro punto B qualsiasi di S,~+m basra trovare quel punto A, in eui la varietb~ invariante V,~ passante per B incontra la Vm, e in eui le aie rieevono dei valori noti .(o) ~,.~ e applieare quindi ai punti A, B il precedente proeedimento. II nostro teorema ~ cos). dimostrato in generale. g Naturalmente non con ogni speciale seelta delle funzioni e eostanti ar- bitrarie su deseritte si otterr~ un tipo di spazio realmente distinto: anzi per le eonsiderazioni generali del § 1, noi possiamo supporre, senza diminuire la generalith an+~,g ~-- 0 (an+3,~ ~-- O)x,,+,=x.+,~=o (k~l, 2,..., n, n+2,..., n+m) (k =1 = n + 2) =I=, + 3) are+t,n+l === 1 (an+~,n+3)x.+,=eo.+~=o ~ 1 7 eee. an+~,g ~-- 0 (an+3,~ ~-- O)x,,+,=x.+,~=o (k~l, 2,..., n, n+2,..., n+m) (k =1 = n + 2) =I=, + 3) are+t,n+l === 1 (an+~,n+3)x.+,=eo.+~=o ~ 1 7 eee. (an+~,n+3)x.+,=eo.+~=o ~ 1 7 eee. dove le uguaglianze scritte in basso fuori delle parentesf indicano su quali variet~ si possono supporre senz'altro verifieate le equazioni stesse. {} 6. Ora noi ei vogliamo ehiedere qual'~ il significato delle re]azioni lineari (4)"', a cui i valori iniziali delle air devono sempre soddisfare. A. questa domanda risponde subito la seguente eonsiderazione. Se noi ei rife- riamo p. es. ossia al primo dei casi test~ trattati e rieordiamo il metodo di cui noi ei siamo serviti, notiamo tosto the per trovare i valori delle ail, in un punto B in funzione dei valori delle aik in un punto A, ei siamo serviti di una trasformazione T ehe eonducesse il punto B nel punto A. 0ra nel caso di trasformazioni infinitesime linearmente dipendenti osserviamo ehe i punti A, B non individuano la trasformazione T, ma che anzi di tall trasforma- zioni ve ne sara, in generale pill di una: eh~ se S ~ la trasformazione pih generale ehe laseia fisso A, tutte le trasformazioni TS (o S T a seeonda della notazione ehe si usa) eondueono il punto B nel punto A. E poieh5 le (4)"' ei danno le uniehe eondizioni a eui devono soddisfare i valori ini- ziali delle aih~ g ben ehiaro ehe il loro signifieato ~ questo, ehe eio~, qual- un gruppo continuo di movimenti. 53 siasi trasformazione TS venga usata, i valori delle ai~ nel punto B siano sempre quelli the si otterrebbero eonsiderando la T. In altre parole esse si- gnifieano, che ogni trasformazione S del gruppo che lascia fisso il punto A deve trasformare in s~ la forma 2 ai~-~°)d #o, d x~;. Del resto risulta senz'altro ehiaro dal procedimento usato test~ chese questa condizione ~ soddisfatta, a]lora i valori che si ottengono in un punto B sono perfefitamente determi- nati, eil gruppo ~ ammesso da uno spazio, il cui elemento lineare ha dei coefficienti, che nel punto A assumono il valore ~,,~'°'. Possiamo dunque espri- mere sotto forma pitt elegante e coneisa le condizioni necessarie e suffieienti amnch~ un gruppo possa essere considerato come gruppo di movimenti. 6bndizione necessaria e sufficiente affinch~ un gruppo G ridotto o no sotto forma normale si possa conside~:are come gruppo di movimenti ~ che quel suo sottogruppo F che lascia fisso un pu~to A determinato, in cui il gruppo sia regolare, sia tale che esista almeno un sistema di quadriche omo- tetiche in guisa che un punto infinitamente vicino ad A non esca mai per le trasformazioni di F da quella di queste .quadriche, che passa per ess% o~ in altre parole F trasformi in s~ ciascuna quadrica di un sistema di qua- driche omotetiche infinitamente vicine ad A (*). (~) Si noti the cos[ immaginiamo il gruppo operante in uno spazio euclideo. ossia Se il gruppo ~ transitivo baster~ chiaramente che questo avvenga in un solo punto (perch~ allora avviene in tutti); se ~ intransitivo cib dow'~ acca- dere in un pu~to di ciascuna varieth minima invariante. Questa distinzione evidentemente superflua, seil gruppo ~ gik r~dotto a forma normale. p g pp g Se queste condizioni sono soddisfatte, esiste infatti una forma X a~,dx~ °~ dx~ ~, che F trasibrma in s~. Osservazi(me 12 Se uno di questi cosifatti sistemi di quadriche ~ for- mato di elissoidi, to spazio corrispondente si pub supporre reale. Osservazione 2. ~ Le condizioni analitiche~ che traducono queste condizioni geometriche sono date dall'annullarsi di quella matric G che esprime essere le (4)'" compatibili. § 7. ]~ ben evidente ora ehe i gruppi G~ transitivi sun variabili pos- sono essere considerati come gruppi di movimenti (teorema del prof. BiA~cm) perch~ in tal caso il sottogruppo F si riduce all'identitb~; o in altre parole~ compiutamente determinata la trasformazione the porta un punto B in un punto A; e quindi~ dati arbitrariamente i valori delle a,.~ in A riescono sen- z'altro, coi nostri metodi~ determinati i valori delle a,7~ nel punto B. Quest'os- servazione rende evidente a priori il bel teorema del prof. BiAl~Cm. 54 Fubini: Sugli spazii che ammettono Ma questa stessa osservazione si pub senz'altro applicare a ogni gruppo con trasformazioni infinitesime linearmente indipendenti e ne traggiamo il teorema : Ogni gruppo a trasformazioni infinitesime linearmenle indipe~denti si pub co~siderare come gruppo di movimenti. Confi'ontando questo risultato, che ~ immediato corollario del precedente teorema generale, con quello del § 2 otteniamo il seguente teorema: Ogni gruppo G,~ generato dan trasfovmazioni linearmente indipendenti su un numero qualsiasi n q-m di variabili ~ simile a u~ gruppo G',~ sem- piicemente transilivo in uno spazio ctd n dimensioni~ e~ cib ch'~ lo stesso, si pub pensare sottogruppo di un gruppo transitivo sun-[-m q--k lettere e con n -~ m + k parametri (dove k ~ un intero qualsiasi nullo o positivo). Net precedenti paragrafi not abbiamo risoluto due questioni: la prima, di riconoscere se un gruppo contin~to si pub considerate come gruppo di mo- vimenti, la seconda di indicare un mezzo, col quale si possa determinare con sole quadrature, differenziazioni e risoluzione di equazioni finite tutti questi gruppi, e gli spazii che loro corrispondono. g pp g p p § 8. Not aggiungeremo ora una facile osservazlone, che ci condurrb. ossia aun notevole risultato: Nessun gruppo, chesi possa considerare come gruppo di movimenti, pii~ di una volta transilivo. p Infatti se G bun tale ~'ruppo ed S lo spazio corrispondente, due punti di S hanno almeno un invariante: la loro distanza geodetica. Per la stessa ragione: Per la stessa ragione: Per la stessa ragione: Se un gruppo G, si pub considerate come gruppo di movimenti~ quel sub Se un gruppo G, si pub considerate come gruppo di movimenti~ quel sub soltogruppo che lascia fisso u~ punto generico ~ certo intransitivo. f Ne deduciamo : Ne deduciamo : Nessun spazio S,~ aun numero u > 2 di dimensioni pub ammettere ur~ n ~ -~ n -- 2 parametri n~ come gruppo di movimenli, n~ come gruppo reale a 2 sottogruppo del gruppo totale di movimenti Questo teorema per n ~ 3 fu dimostrato dal prof. BiAscm; not lo am- metteremo vero per n ~---m-- 1 lo dimostreremo per n ~m. St% se b possi- bil% Sm uno spazio che ammetta uu G,,,+,,_2 di movimenti. Questo gruppo 2 b certamente transitivo, perch~ se non lo fosse, esso sarebbe per il teorema del § 1 simile aun gruppo di movimenti di uno spazio a meno dim di- un gruppo eontinuo di movimenti. 55 assurdo, pereh~ m~ + 2m -- ~ > m (m2-- 1) Quindi quel mensioni: cib che b suo sottogruppo che lascia fisso un punto generico b proprio un gruppo a m~ + m -- 2 m 2 parametri~ ciob b proprio un G(,n_l~+(,,_l)_ 2 . Questo gruppo per l'osservazione '2 preeedente b intransitivo in G,~ e per il teorema del § 1 b simile a un gruppo di movimenti di uno spazio a , m--1, dimensioni. Cib che b pure assurdo, perchb not abbiamo supposto dimostrato il nostro teorema per ~t~m~ 1. parametri~ ciob b proprio un G(,n_l~+(,,_l)_ 2 . Questo gruppo per l'osservazione '2 preeedente b intransitivo in G,~ e per il teorema del § 1 b simile a un gruppo di movimenti di uno spazio a , m--1, dimensioni. Cib che b pure assurdo, perchb not abbiamo supposto dimostrato il nostro teorema per ~t~m~ 1. La nostra asserzione resta cosl dimostrata in generale. Cosl pure si di- mostra: Se un Sn ammette un G,%n_ ~ (certo immaginario sen > 2), esso ~ 2 a curvalura costante~ ossia ammette anche un G~+,. ossia Anche questo teorema 2 si dimostra col metodo di induzione completa. Ecco p. es. come si dimostra per un $4. Quel sottogruppo G~ di G9 che lascia fisso un punto generico di S~ sar'h intransitivo e avr~t delle V~ per variet~ minime invarianti~ p. es. le ,c4 ~ cost.~ geodeticamente parallele. Esso si potr~ immaginare operante (transitivamente) sulle x~, x~, x~ (§ 1). Questo Gs pub chiaramente portare ogni segmento T di una di queste variet~ in ogni altro segmento della va- riet~ stessa uguale a T. Quindi dovendo esso rieondurre tutto lo $4 in s~ senza mutare le distanze, a due segmenti uguali di una delle x, = cost. cor- rispondono (per mezzo delle geodetiche normali alle x4 ----- cost.) due segmenti tra di loro uguali su ogni x4 =cost. Ma una x4 ~ cost. ammettendo un Gs, ammetterh un G~. Quel gruppo G'o che ope~:a sulle x,~ x~, x~ come questo Gs e lascia invariato x4 trasformerh dunque in sb ogni x4 = cost., senza mutar l'elemento lineare e percib sarh un gruppo di movimenti dello S~, ehe quindi ammetter~ un G,o. § 9. Ritorniamo ora alle condizioni necessarie e sufficienti date al § 6 afflnehb un dato gruppo continuo si possa considerare come un gruppo di movimenti. Queste condizioni si possono anche enunciate cost: Quel sotto- gruppo che lascia fisso un punto A deve essere un sottogruppo di uno spazio a curvatura costante, quando lo si pensi operante sulle rette uscenti da A~ considerate come elementi, o, in altre parole~ deve lasciare fisso un cono qua- drico col vertice in A; di pi?t egli deve laseiare invariale le quadriche in- § 9. Ritorniamo ora alle condizioni necessarie e sufficienti date al § 6 afflnehb un dato gruppo continuo si possa considerare come un gruppo di movimenti. Queste condizioni si possono anche enunciate cost: Quel sotto- gruppo che lascia fisso un punto A deve essere un sottogruppo di uno spazio a curvatura costante, quando lo si pensi operante sulle rette uscenti da A~ considerate come elementi, o, in altre parole~ deve lasciare fisso un cono qua- drico col vertice in A; di pi?t egli deve laseiare invariale le quadriche in- Fubini: Sugli spazii che ammettono 56 finitamente vici~e ad A aventi questo cono per cono assintotico. La prima parte di queste condizioni non sarebbe evidentemente sufficiente~ perch5 p. es. ossia anehe ii G7 delle similitudini dello spazio euc]ideo soddisfa ad esse. Esistono dunque altri gruppi che soddisfano so]tanto a quella prima Condizione e che non sono altro che i gruppi di trasformazioni conformi. Osserverb che]a con- dizione affineh~ una trasformazione infinitesima generi un gruppo di trasfor- mazioni conformi per lo spazio d s ~ ~ ~ av~ d x~ d x:, the esista una funzione k delle xl tale ehe the esista una funzione k delle xl tale ehe X (2 a+k d xl d x~) ----- k 2 alk d xi d x~. Se k ~ costante essa si pub ehiamare una trasformazione simile. Se k ~ costante essa si pub ehiamare una trasformazione simile. Un'altra osservazione ~ la seguente: Almeno per gruppi transitivi val- gono ancora i teoremi seguenti, che si dimostrano come gli analoghi dei pa- ragrafi precedenti per le forme quadratiehe: g p p q Si possono costruire per quadrature tutte le forme di2~erenziali di or- dine qualunque che ammettono un gruppo continuo transitivo di operazioni, e i gruppi corrispondenti. Condizione necessaria e sufficiente affinch~ un gruppo transitivo possa trasformare in s~ una forma differenziale di ordine qualunque n, ~ che quel suo sottogruppo che lascia fisso un punto A trasformi i punti infinitamente vicini ad A in modo che esista un sistema di superficie omotetiche din ~'~° ordine infinitamente vicine ad A~ ciascuna delle quali venga dal detto sotto- gruppo trasformata in s~. Cosi si possono pure generalizzare molti altri dei precedenti risultati. § 10. Rieerchiamo ora effettivamente i gruppi a 1, 2~ 3~ 4 parametri che possono essere realmente considerati come gruppi di movimenti. Osser- viamo chese il numero delle loro trasformazioni dipendenti ~ minore o uguale a 3, essi devono (§ 1) potersi eonsiderare come gruppi di movimenti di uno spazio a non pi~ di tre dimensioni~ e quindi si possono prendere senz'altro dalla Memoria del prof. BiA~cm; si potrebbe per5 anehe~ poich~ il LIE diede tutti i gruppi su due variabili e insegnb un metodo per trovare tutti quelli su tre variabili, esaminare l'un dopo l'altro tutti quelli dei tipi enun- ciati da Lm che non posseggono pih di quattro parametri, e di questi rite- nere soltanto quelli che soddisfano alle condizioni del § 6. Questo metodo sarebbe assai rapido~ e servirebbe a trovare senz'altro direttamente i ri- un gruppo eontinuo di movimenti. 57 sullali della p#t volte eitata Memoria del prof. 8 Annali di Matematiea, Ser'ie III, tomo VIII. ossia +*.3 ; " ,P a'~ a x, a .:c, 1 a c q X, ---- z, £7', + 2- (*~ - xl) ~ -- a', a x, a a 3 a II") X,----- ax, ; X, = a:,3 ; X3 = ax---7 -- x, a.v, ; a 1 ( e -'x,_ ."c'~) a n e -x, a X, = X, ~ -t- g ~ 1 - n"- 3 x---,' 1 --- n ~ a xa I") X,= a a a a ax,; X°=--; X, .... +*.3 ; " ,P a'~ a x, a .:c, 1 a c q X, ---- z, £7', + 2- (*~ - xl) ~ -- a', a x, a a 3 a II") X,----- ax, ; X, = a:,3 ; X3 = ax---7 -- x, a.v, ; a 1 ( e -'x,_ ."c'~) a n e -x, a X, = X, ~ -t- g ~ 1 - n"- 3 x---,' 1 --- n ~ a xa a a II[ °) X,=ax---;; X,=ax~; 3 n sen x, a Xa = cos x, -- eotg x, sen x, ~ q- -sel-l x~ ~ xa ; X, = (X, X,). I1 tipo II °) e II[)° si potrebbero (In un punto di vista generale conside- rate come identici. I1 tipo II °) e II[)° si potrebbero (In un punto di vista generale conside- rate come identici. § 11. Trovinmo ora i gruppi G4 a tvasformazioni infinitesime indi- pendenti. Questo gruppo (§ 1) si deve poter ridurre a un gruppo h'ansitivo su 4 lettere. Per determinate questi gruppi potremmo) seguendo il procedi- mento generale del § 5, rieorrere senz'al)ro ai metodi di LIE. ]Via perb noi possiamo usare di metodi pitt rapidi,in quanto the noi conosciamo tu(ti i G3 transitivi. Potremo dunque prendere un sottogruppo G3 di G4, di cui cer- cheremo la composizione. Da questa pota'emo subito dedm're (per mezzo della penultima tabella) una form% a eui possiamo immaginare ridotte le sue trasfovmazioni infinitesime; la quarta trasfovmazione infinitesima di G~ si de- termina poi in guisa che non sia combinazione linea,'e delle preeedenti, e che il G4 abbia la eomposizione voluta. Sia ora il G, non integrabile (ell'. Lm: Vol. ossia Bix~cm. Si potrebbe anche infine cosh-uire questi gruppi secondo il mio metodo del {} 5; ma questa via sarebbe un po' pih lunga della preeedente. ~loi nafm'almente prenderemo questi gruppi addiritiura dalla Memoria del prof. Br.~cm, perchh il ]ettore pub facilmente da s~ vedere che questi altri metodi condurrebbero appunto ai risultati del prof. BIA~em. Gruppi G, : a X, = a x~ Gruppi G~ a trasformazioni infinitesime naturatmente linearmeute indipendenti. a I°) X'=b x, ~Xq--a x, II °) X, -~ e-oo .... X, = -- 0 x, a x, Gruppi Ga a tra.¢formazioni infinitesime linearmente dipendenti. a a a a ------ x, = --- x~ = x, -, .... x, I °) X, a oc, a x~ v x~ 0 x.- I[ °) a X, ~--- sen x, a a X, = b ~ ~ + co~g x, cos z~ ~; X, = (X, X:) a Xo= a z, a a 1 (e_:oo,--x~) ~ Ig °) x, = a-x~ - ~-;, -- aT, X, - z, ~-:}~ + -g aT.. Gruppi Ga a h'asformazioni infinitesime linearmente indipendenti. ~ v + -+x~ ~-~x.+ 2 ~x~ I") X,=~x~. ; X,=a.ra ; X~-----ax--- ~ ---- I[ °) X,-- a a a +xo a 8 e.=; X':--a.~; *~=-a.---: -aT;x~+hX'ax~ IIi°) X,-- 0 a a x~ a +(x~q_hx,) a 0 a a a a IV °) x,---e', -.:e~:, --2x, e~ ..... ; X.= • X,=e~,-- a x, a x.. a x~ " ~ ' a x, sen.r., 0 a __ cotg x, sen x, ~7~ -~- sen x~ a xa ; V °) X,=ax,; X.=cosz,~z, --- X, = (X, :~q) Fubi,ni: Sugli spazii che ammeltono 58 VI °) VII °) O a a O x,=~-;; x:=~-7, ; x~= o.~+.,~a., O a O X, = o x--~ ; X~ = ~ x--- ; X3-----O.- ~ VI °) O a a O x,=~-;; x:=~-7, ; x~= o.~+.,~a., VI °) O a a O x,=~-;; x:=~-7, ; x~= o.~+.,~a., VII °) O a O X, = o x--~ ; X~ = ~ x--- ; X3-----O.- ~ Gruppi G4 a trasformazioni infinilesime lh, earmenle dipendenti. Gruppi G4 a trasformazioni infinilesime lh, earmenle dipendenti. I") X,= a a a a ax,; X°=--; X, .... ossia III,§ 137); il gruppo de- rivato (X,, X2, X~) dowh avere una delle seguenti eomposizioni: (X, X~) = X, (X, X3) = 2 X~ (X~ X,) = .X~ oppure (~¥, G) = G (~; X~) = X, (G X,) = X,. ( , ) , ( , ) ( ,) oppure (~¥, G) = G (~; X~) = X, (G X,) = X,. 59 un gruippo continuo di movimenti. 59 E in ambi i casi si pub poi scegliere .V4 in modo the sia permutabile con le precedenti trasformazioni. II G3(X,, X~, X~) ~ certo a trasformazioni infinitesime linearmente in- dipendenti e (§ 1) si pub immaginare semplicemente transitivo sutre lettere. :Nel primo di questi easi awemo: :Nel primo di questi easi awemo: O (9 0 0 0 X,= e-'~.----x~ - --2x~e -a',-; X.=--; X.--e ~-. Posto : 't O (~i~-----~I(X,, X2, X3, X.)) avremo, essendo (XI, X~)= 0 (i= 1, 2, 3) e_,~ ~ O ~, x~ 4 ~3 e --x' = O ; e_~, O ~, ~ x, + 2 x: ~ e - ~,, -- "~ e-(~.,+xo) __~ x~ e-x, ~ e -x, --~2~,e -x~-2~3x~e -x~==O 0 ,)7-,i ~4 .t . ~.4 e--X~ ~.r--~l = ~xo. ~x.. D.~'~ 0 &~, ~ 0 Ox~ = 0 (i 1, 2, 3) O (9 0 0 0 X,= e-'~.----x~ - --2x~e -a',-; X.=--; X.--e ~-. O (9 0 0 0 X,= e-'~.----x~ - --2x~e -a',-; X.=--; X.--e ~-. Posto : 't O (~i~-----~I(X,, X2, X3, X.)) avremo, essendo (XI, X~)= 0 (i= 1, 2, 3) Posto : 't O (~i~-----~I(X,, X2, X3, X.)) avremo, essendo (XI, X~)= 0 (i= 1, 2, 3) e_,~ ~ O ~, x~ 4 ~3 e --x' = O ; e_~, O ~, ~ x, + 2 x: ~ e - ~,, -- "~ e-(~.,+xo) __~ x~ e-x, ~ e -x, --~2~,e -x~-2~3x~e -x~==O 0 ,)7-,i ~4 .t . ~.4 e--X~ ~.r--~l = ~xo. ~x.. D.~'~ 0 &~, ~ 0 Ox~ = 0 (i 1, 2, 3) donde, integrando, donde, integrando, 1 ~3 = -- 2 (~ x, -[- fl) ~, = h dove ~, /3, 7, l, sono funzioni qualunque di x,. Con procedimento analogo troviamo nel secondo caso: dove ~, /3, 7, l, sono funzioni qualunque di x,. ossia Con procedimento analogo troviamo nel secondo caso: O ~ O sell x2 X, == ~x, ; X~ = cos x~ ~ ~ cotg x, sen x~ ~ + sen x~ ~x~ x, ---= (x,-:~;). E, posto E, posto 4 D i=l 0 0 ¢, = ¢~=- o .L = ~ ~-x3 + ~ 0.r, 4 D i=l si ottiene dove ?, J/ sono funzioni di x,. 60 Fu b in i: ~uyh spazii ehe ammettono Del resto questi due easi, quando non si distingua reale da immaginario, sono da riputarsi identiei. p Sia era il G, integrabile e sia un G~ a h'asformazioni non permutabili il g'ruppo derivato. Indieando con X,, _X~, X.~, X, le trasformazioni genera- triei di G4 potremo porte (Lm~ lee. eit.) o F) (X, .X;) = (X, ~X~) = 0 (X, .~) = X, (G G) = c G (.X', X,) = X~ (.\~ .xS) = (c -- 1) X, oppure pp II °) (x, x,) = (x, -vO = o (x, x~) = x, (x, xO ---- 2 x,; (.v, ~5) = x~; (x~ x,) = x: + .\:~. pp II °) (x, x,) = (x, -vO = o (x, x~) = x, (x, xO ---- 2 x,; (.v, ~5) = x~; (x~ In ambi-i easi potremo fare In ambi-i easi potremo fare x,=~----;; x,=a~;; .~=--o--~+z~ax. E posto a i ~Xi avremo, nel primo case, p ~, = (c - 1)x,-~- ,~, do ~, ----- x, -t- ~, 1 h -- -'2 -- x, % q- 2 x~. q- ~, 1 5 -= ,3 -- x, + ~3 ~4 = -- zU p ~, = (c - 1)x,-~- ,~, 1 h e nel seeondo -- -'2 -- x, % q- 2 x~. q- ~, 1 ~4 = -- zU ~, ----- x, -t- ~, 5 -= ,3 -- x, + ~3 dove %, D, ~, 14 sono funzioni di x~. dove %, D, ~, 14 sono funzioni di x~. Sia era il gruppo derivato di G~ un G~ e non eontenga il G4 un Ga a trasformazioni permutabili. Allora o il G~ ha la penultima eomposizion% dove si ponga c = 17 oppure avr~ la eomposizione: (NN)=(X,~¥~)=-(X, XO=(X~N)=O (X, X3)=X, (X,X~)=N. (NN)=(X,~¥~)=-(X, XO=(X~N)=O (X, X3)=X, (X,X~)=N. Potremo fare : Potremo fare : b ~ 0 X,=~x--G; X,= ; X,~=- +x, - 0.r~ 0), 0 ,:e~ 61 un gruppo continuo di movimenli. ossia (..x~ ~;) =- a s~ (X, X,) = c X~ e quindi ~, = x, + ~,, oppure I[°) (x, x,) = c x, e quindi : l (& &) =-= (1 + c) x, ( x~ x,) = x,-t-cx, ~, ~- c x, + x~ -+- ~o, ; oppure III°) (X, X,) = X, e quindi : oppure IV °) e quindi : oppure : Vo) ~, : xa -t- ~, G--(I -[- c) z,+ %; ( x, x,) = o 1 (&iv,) = x, 1 14 (x, &) = x, + x, (x, x,) = x, (x, x,) = x, + x, (x, x,) = (G x,) - o, (x~ x,) = G 1 h ~, ~- c x, + x~ -+- ~o, ; oppure III°) (X, X,) = X, e quindi : oppure IV °) e quindi : ~, : xa -t- ~, G--(I -[- c) z,+ %; ( x, x,) = o 1 (&iv,) = x, 1 14 (x, &) = x, + x, (x, x,) = x, (x, x,) = x, + x, ~, ~- c x, + x~ -+- ~o, ; oppure III°) (X, X,) = X, e quindi : : xa t G--(I -[- c) z,+ %; ( x, x,) = o 1 (&iv,) = x, 1 oppure III°) e quindi : oppure IV °) e quindi : ~, : xa -t- ~, 1 14 (x, &) = x, + x, (x, x,) = x, (x, x,) = x, + x, oppure : oppure : Vo) (x, x,) = (G x,) - o, (x~ x,) = G Vo) Fubiui: Sugli spazii che ammettono 62 e quindi : e quindi : e quindi : ~, = ~, ; 5 - x~ -4- ~-~ ; ~ -- ~,~; ~, - oppure : VI °) e quindi : ~,= x,~, (x, x~) = x, (x, x,) = x, ~., = x~ + x~ + ~.~ oppure vw) e quindi (x~ x,) = o h (x~ x,) = x, + xq (i=1, 2 7 3) 1 h e quindi : ~, = ~, ; 5 - x~ -4- ~-~ ; ~ -- ~,~; ~, - oppure : VI °) e quindi : (x, x~) = x, (x, x,) = x, h (x~ x,) = x, + xq e quindi : q ~,= x,~, ~., = x~ + x~ + ~.~ oppure vw) e quindi (x~ x,) = o (i=1, 2 7 3) 1 h dove ~,, ~:, ~3, l, sono funzioni di x4. ossia 61 e, posto e, posto 4 X, ~i a xi avrelrlO : avrelrlO : 1 dove ~,, ~,, 7a, 14 sono funzioni di x,. dove ~,, ~,, 7a, 14 sono funzioni di x,. dove ~,, ~,, 7a, 14 sono funzioni di x,. Contenga ora il G, un Ga~(X,, XS, 3i~) a trasformazioni a due a due permutabili. Potremo fare x,=~-~, x,= a-~, x~=~- G, .,c,= ~ g,a. ~..= Si potrg poi scegliere X~ in guisa the o I °) (x, x,) = ~. (..x~ ~;) =- a s~ e quindi (X, X,) = c X~ (x, x,) = ~. ossia dove ~,, ~:, ~3, l, sono funzioni di x4. {} 12. Veniamo ora a un'altra parte del presente lavoro, dove io voglio esporre un metodo per la rieerea dei sottogruppi finiti diseontinui dei gruppi eontinui e, in modo speeiale, dei gruppi ehe si possono considerate come gruppi di movimenti. Io svolgerb questi metodi, applieandoli dapprima effet- tivamente a due esempii, agli spazii eio~ del tipo ¥II[ °) e IX °) del prof. BtA~cm, eib ehe ei permetter~ anehe di trovare delle notevolissime rappresentazioni geometriehe di tali spazii sulla sfera e sulla pseudosfera. g p p Comineeremo dagli spazii del tipo IX°), ehe ammetton% con le notazioni del prof. BIA~em un gruppo generato dalle senx~ X~ -- ~ ~ eotg x, sen x~ ~r~ + senx~ ~,r3 ~:~ ; 32, = cos x~ ~r, X~ ---- (X, X~) eomposizione : eon la eomposizione : eon la eomposizione : (X, X~) = X, ; (X~ X3~ = X, ; (& X,) = X,. Cosh'uiamo il gruppo aggiunto di questo gruppo. Con le usuali notazioni di Lm, esso si oitiene dali'integrazione del sistema" de', ),, e' -- (1) dt de'~ = )~3 e', -- )., e'3 (2) dt d e'3 -- ~., e'~ -- ?,~ e', . (3) dt (1) (2) (3) d e'3 -- ~., e'~ -- ?,~ e', . dt un gruppo conlimw di movimenti. 63 63 Donde de', de', de'3 0 e', -~ -1- e', -d-i- @ e'3 ~-[ = de', de', de'3 0 e', -~ -1- e', -d-i- @ e'3 ~-[ = ossia e'~ -~- e'g -[- ~'~ ~ cost. (4) (4) Questo gruppo aggiunto non ~ pereib altro che il gruppo delle rotazioni di una sfera in si~ siessa; e poich~ se T ~ una trasfi)rmazione qualunque (]el gruppo inizMe Questo gruppo aggiunto non ~ pereib altro che il gruppo delle rotazioni di una sfera in si~ siessa; e poich~ se T ~ una trasfi)rmazione qualunque (]el gruppo inizMe T--'TT=T, T--'TT=T, ne traggiamo che alla trasformazione )., t, ~ t, ),3 t del gruppo iniziale corri- sponde una rotazione della sfera attorno al diametro che ha i coseni di di- rezioni proporzionali a ),, t, ),,. t, ).3 l; cib chesi conferma col calcolo seguente. ossia Derivando ]a (1) rispetto a t, otteniamo per le (2) e (3): d~ e ', d t, + (~'; + )4) e:, -- ~,, ,(~,., e', + ~ e ~) ~ 0 donde : d3e '' de', ( de', de'31 d t~ 4 (~,~ + ),~) -dT -- ~' ~'' -f[ + )'~ --fi/= 0 e poich~ per le (1), (2), (3) si ha 3 d e'i si avrh 3 d e'i si avrh si avrh d 3 e't , ~ O. ' e' Indieando con At, Bi, Ci dello Identiea equazione si lrova per e~, ~, costanti avremo dunque e'i-- Ai@ Bisen Ht-}- Cicos Ht (i=1, 2, 3) (5) (5) dove Servendoci delle (1), (2 ~, (3) e del fatto ehe per t-----0 e'i~ei (i=l, 2, 3) Servendoci delle (1), (2 ~, (3) e del fatto ehe per t-----0 e'i~ei (i=l, 2, 3) Servendoci delle (1), (2 ~, (3) e del fatto ehe per t-----0 e'i~ei (i=l, 2, 3) (1), (2 ~, (3) e del fatto ehe pe e'i~ei (i=l, 2, 3) potremo determinare le Ai, Bi, Ci e otterremo infin% scrivendo ?.,, )~,, ),~, H potremo determinare le Ai, Bi, Ci e otterremo infin% scrivendo ?.,, )~,, ),~, H Fubini: Sugli spazii che ammettono 64 in luogo di ),, t, L t, ),3 t, H t e, = e, I,H, + cos [t [ -- tt"-"] nt- e.~ ~-~7- @ i[ sen H H. ~ i tt) (6) -Jr- e~ \ H~ H sen It-- H---wF, cos / (6) o le eguaglianze ehe se ne ottengono rotando. Queste eguaglianze rappvesen- tano appunto una rotazione attorno alla retta, i cui eoseni di direzione sono proporzionali a )~,, ),~, ),3. Per trovarne l'ampiezza ~ si osservi che per note formole di Geometria analitiea ),, ).~ ),3 )q ),.. ),3 )., ).~ ),3 • ----- cos e~ e~ e3 e', e'~ e'3 e, e~ e3 donde sostituendo e sviluppando otteniamo: a=H. Abbiamo cos~ trovato, con metodo evidentemente applicabile a casi pih generati, un gruppo di sostituzioni lineari isomorfo al gruppo dato. ossia E il no- stro risultato si pub esporre cos]: I1 gruppo in discorso ~ oloedricamente isomorfo al gruppo dei movimenli di una sfera in s~ : l'isomorfismo si stabilisce~ facendo corrispondere alla trasformazione )~, X, + L X~ + ~3 323 una rolazione di ampiezza ~ X~-~- ),~ + ?,~ attorno a quel punto di una sfera (col centro nell'origine) le cui coordhmte sono proporzionali a ),,, ;~, 2~. ( g ) p p ),,, ; , I gruppi non sono perb simili, perehb mentre uno opera su tre variabili, l'altro opera in realt'X soltanto su due. I gruppi non sono perb simili, perehb mentre uno opera su tre variabili, l'altro opera in realt'X soltanto su due. Ma ora osserviamo ehe dati due gruppi oloedrieamente isomorfi, non sempre dall'esistenza di sottogruppi finiti diseontinui per l'uno si pub eon- chiudere l'esistenza di sottogruppi finiti diseontinui per l'altro. Cosl p. es. il gruppo detle rotazioni attorno a un asse e il gruppo delle traslazioni in una direzione sono oloedrieamente isomorfi, mentre perb per l'uno esistono sotto- gruppi diseontinui finiti e per l'altro non ne esistono affatto. Cib ehe si deve alle polidromie, eee., ehe si possono presentare am- pliando il campo in eui opera un dato gruppo. 1~ ben ehiaro perb ehe a un sottogruppo finito discontinuo di un gruppo, eorrisponderh un sottogruppo un gruppo continue di movimentL ~5 isomorfo nel gruppo aggiunto, in generale. E quindi dovremo prima ricercare i sottogruppi discontinui finiti del gruppo aggiunto. Questo problema ~ sen- z'altro risoluto nel nostro case: i sottogruppi cercati net gruppo aggiunto non sono che i gruppi dei poliedri regolari. Eil nosh~o calcolo preeedente deft- nisce senza ambig, dt~ i sottogruppi corrispondenti del gruppo iniziale, perch~ d~ nel mode pih perspicuo quale trasformazione del nostro gruppo si deve far corrispondere a una trasformazione del gruppo aggiunto. Ma per peter affermare che le trasformazioni del gruppo inizia]e, corrispondenti alle tra- sformazioni del gruppo di un po]iedro regolare contenuto nel gruppo aggiunt0, formino effettivamente un gruppo, bisogna, come risu]ta chiaramente dalle considerazioni precedenti, fissare bene il significato delle coordinate. A tal fine serve ]'importante osservazione che due gruppi oloedricamente isomorfi non simili si possono spesso rendere simili, mutando le variabili su cui essi operano. ossia Nel case nostro il gruppo aggiunto, invece di essere considerate come un gruppo operante sui punti della sfera, sar~t pensato come gruppo operante sugli elementi della sfera, cio~ sulla sfera immaginata come luogo degli in- finiti enti che si definiscono dando un punto della sfera stessa e una direzione uscente da esso tangente alla sfera. I due gruppi risultano altora senz'altro simili, e con opportuna see]ta delle coordinate di un elemento della sfera, essi riescono identici. Si prendano come coordinate di un elemento della sfera la colatitudine e la longitadine ~, ~o del punto corrispondente e la derivata d--~' dove d s ~ la lunghezza dell'elemento, d ~ l'incremento di ~ muo- vendoei lunge di esso. Le trasformazioni infinilesime del gruppo aggiunto sono e~-~--e.~; e,~es--e3~-~e; e~3-~--e~-~e ~. e3 = cos ~, e~ ~ sen ~ sen $, e, ~ sen ~ cos ? avremo ehe l'ultima, rappresentando una rotazione attorno all'asse delle ,, e3 ,, sark eguale a avremo ehe l'ultima, rappresentando una rotazione attorno all'asse delle ,, e3 ,, sark eguale a X•=--. La soconda, essendo una rotazione attorno sen ~ sen ~0 e quindi sar'~ della forma all'asse e2, lascia invariato e~--e.~=k -- cos ~ sen ~ ~ -{- sen6cos~ .' 9 9 Annali di Matematica, Serie IH, tome VIII. Fubini: Sugli spazii che ammettono 66 dove // ~ una funzione da determinarsi. Ma: poichb dove // ~ una funzione da determinarsi. Ma: poichb 0 D O~------d sen 0 sen ~ ~e~ q- sen ~ cos ? ~e, 0 D O~------d sen 0 sen ~ ~e~ q- sen ~ cos ? ossia ~e, 0--0 = -- sen 0 ~e~ -}- cos 0 sen 50 Uee~ +" cos ~ c 0--0 = -- sen 0 ~e~ -}- cos 0 sen 50 Uee~ +" cos ~ cos ~ si trova senz'altro e quindi Analogamente : 1 sen 0 a X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0" 0 0 X, ----- sen 50 ~- A- cos 50 cotg 0 ~ • si trova senz'altro 1 sen 0 a X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0" 0 0 X, ----- sen 50 ~- A- cos 50 cotg 0 ~ • e quindi a X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0" 0 0 X, ----- sen 50 ~- A- cos 50 cotg 0 ~ • a X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0" Analogamente : Ampliamo ora il gruppo X,~ X~, X~ immaginandolo su ~(') dO ~(,) O~. Avremo: operante anche X~--O? - -- ; X~ =_ sen • cotg 0 ~ -- cos 50 ~ -J- 50(') sen ~f ~ q- [~(o cos 50 cotg 0 -- 0(') sen ? ] + sen ~/jj ~ ~(1/ L x (x x 3 x, = (x, x.3. O Laseiando era il termine in 0,~' perchb noi vogliamo soltanto oceu- parci di come il gruppo trasforma ~o), e osservando che ds'= dO'q-sen~d~ 2, donde ~(') x,'i -- 0 ~:~ ( d 0(,) -- sen 0 , sostltuendo alla ~(') la nuova variabile ~k =d x/1 -~-~',)' il gruppo assume l'aspetto il gruppo assume l'aspetto 0 sen ~ 0 X2 = sen 50 cotg 0 -- cos ~ 0-~ q- sen 0 0 endo dunque x, = xo. X3 ~ Ponendo dunque questo gruppo assume proprio la forma data dal prof. BIANcnI. questo gruppo assume proprio la forma data dal prof. BIANcnI. un gruppo continuo di movlmenti. 67 Abbiamo dunque : Le /ormule precedenti stabiliscono una rappresentazione del nostro spazio sulla sfera euclidea pensata come luogo di elementi~ tale che ai movimenti dello spazio in s~ corrispondono rnovlmenti della sfera in s~ stessa. p p f Se noi vogliamo che]a rappresentazione sia biunivoca su tutta la sfera senza eceezion% resta senz'altro fissata la corrispondenza ira i punti dello spazio e ]e ]oro coordinate. ossia E possiamo dire: I gruppi finiti discontinui degli spazii in discovso sono quelli~ che, ~zella corrispondenza precedentemente fissata~ corrispondono ai gruppi dei poliedri re qolari. I1 fatto che qui vediamo, ciog ehe i gruppi finiti discontinui dei nostri spazii sono oloedricamenle isomorfi a gruppi finiti discontinui di movimenti di uno spazio ellittico ~ un fatto generale, che si ripele sempre appena il gruppo totale dei movimeJ~ti non contenga trasformazioni infinitesime ecce- zionali. Infatti a un gruppo finito discontinuo di mo,dmenti corrisponde un gruppo finito discontinuo nel gruppo aggiunto, che g in tal easo oloedrica- mente isomorfo al gruppo primitivo. E questo gruppo~ essendo un gruppo finito di operazioni lineari laseia sempre invariata una forma quadratica de- finita ehe ~ p. es. la somma delia forma e~-1-e~ + el e delle sue trasformate. ~el easo precedente questa forma ~ proprio ,, e} + e~.-1-el ,. p q p p } Da queste osservazioni scaturisce senz'altro un metodo per ricercare in ogni caso cosiffatto se esistono nel gruppo considerato sottogruppi finiti di- scontinui. I1 nostro metodo consiste nel formare il !lruppo aggiunto, e di de- terminare quali delle sue trasformazioni lascino fissa una forma quadratica definita, cib che si compie con operazioni algebriche : considerata poi questa forma quadratica posta uguale a zero come assoluto di uno spazio ellittico, vedere ~uali sottogruppi discontinui finiti di questo spazio appartengono pure al gruppo aggiunto. In conclusione anche nel case pitt generale la ricerea si riduee alla determinazione dei gruppi finiti diseontinui di uno spazio el- littico. Applieheremo ora questo proeedimento, un po' modifieato, al tipo VIII ° del prof. B~A~cnl. Comincieremo dal costruirne il gruppo aggiunto. Esso viene definito dalle de', 4- ).,e' -- d t ' ~ ).2 e', = O d e'~ e' -- 9~ ' ~-- dt ~'2),i 3 2 3ei 0 de'3 dt de 3 dt 68 Fubini: Sugli spazii che ammetlono Yediamo se qualche sua trasformazione lascia fissa una conica. ossia E, poich~ pet" le prccedenti equazioni, q ale's e' de't 1 de'2 -- 0 e'l --d- [- -{- 3 d t 2 e'2 d t e' " 4 e't 3 -- e ~ = cost., ale's e' de't 1 de'2 -- 0 e'l --d- [- -{- 3 d t 2 e'2 d t e' " 4 e't 3 -- e ~ = cost., ossia ossia tutte le trasformazioni del gruppo lasciano fissa la conica rcale e ~ _ '~ 4e'i 3 e~=O. Allora senz'altro deduciamo che il nostro gruppo ~ oloedricamente iso- morfo ai movimenti di una pseudosfera in s~ perch~ questa conica ~ reale, e~ analogamente a quanta s'~ ratio prima noi avremmo: g q p II noslro spazio si pub riferire biunivocameste alla pseudosfera pensala come luogo dei suoi o~ ~ elehzenti in modo che ai movimenti della ioseudosfera in s~ stessa corrispondano movimesti dello spazio is s}. Questa corrispon- denza definisce per noi senz'alti'o la corrispondenza tra un punto di S~ e le sue coordinate. E allora senz'altro poieh5 eccetto casi bana!i, la pseudosfera non ammette gruppi finiti di movimenfi in s~, ma ammette bensi gruppi in- finiti discontinui, avremo: Gli spazii in discorso non ammetlono (tranne qualche caso banale) gruppi finiti discontinui di movimenti : essi ammettono perb i~finiti gruppi infiniti discontinui di moviment% isomorfi oloedricamente ai gruppi di PoI:~cAR~. Per gli altri tipi di S~, che ammettono un G3 transitivo di movimenti 5 ben facile riconoscere l'impossibilith di gruppi discontinui finiti. Basra in- fatti riconoscere l'aperiodicit~ di una trasformazione qualsiasi del gruppo ag- o'Junto. P. es. nel tipo Vl ° il gruppo aggiunto ~ definito da d e~i t 1 dt -]- ?. e3-- ;% e ~ = O de'o, e' dt d e'3 dr ----0. dond% se ?,3 =[= O, dond% se ?,3 =[= O, )~4 ~Lt ) e~ t e't -- ~3 @3 - e3 n t. e, . -- ~" e3 (1 -- d ~Jt) -{- e2 e h~t e%- ~3 t ea~es un gruppo continuo di movimenti. 69 ehe non pub essere periodica nel campo reale, poich~, essendo ~=',=0, e~ ' ='i= 1. ossia 8e invece ),~ = O, si avr'a: e'~ ----- -- ~.l e3 t + e~ e', = -- h X~ ea t + e, e"3 ~ ea pure aperiodica se e'~ ----- -- ~.l e3 t + e~ e', = -- h X~ ea t + e, e"3 ~ ea z,=]=0, o x,=',=0. pure aperiodica se pure aperiodica se z,=]=0, o x,=',=0. z,=]=0, o x,=',=0. La discussione ~ analoga per gli altri tipi. Invece ora di studiare quegli &che ammettono un G~, not osserveremo che questi G4 contengono una trasformazione infinitesima eccezionale e di. remo poehe parole sui gruppi G,. che eontengono k trasformazioni Y,... Y~ infinitesime eccezionali. Per trovarne i sottogruppi finiti discontinui si trovino prima tutti i cosiffatti sottogruppi eventtia]i del gruppo aggiunto. Si deter- minano in un modo qua]unque le trasformazioni eorrispondenti del gruppo iniziale, cercando pot, se ~ possibile, di aggiungervi delle trasformazioni di quel sottogruppo di G,. che viene generato dalle Y,... Yk~ che siano perio- diehe e che insieme a]le preeedenti generino effettivamente un gruppo della specie voluta. p § 13. Daremo ora un'app]icazione dei nostri metodi alla determina- zione degli spazii che ammettono un Gt oppure un G~ o un Ga o un G4, oppure un gruppo a pih di 4 parametri con sole 4 trasformazioni infinite- sime indipendenti. :Per i primi quattro cast il problema si risolve facilmente: per il quinto easo inveee si vedr~ ehe oeeorrono nuo~¢i e partieolari artifici, senza i quali i ealeoli diverrebbero estremamente lunghi e ben diffieilmente condurrebbero in rondo. A questo caso, ~ riservata una prossima Memoria. Per trovare gli spazii the ammettono un G, basta (cir. Bl~t~cm (A) pag. 8) supporre i eoefficienti dell'elemento lineare indipendenti da x,. Per trovare gli spazii che ammettono un G,, basta, nel primo caso, ammettere i coetficienti dell'elemento lineare indipendenti da x,, x~. lgel secondo caso posto ds~-~aikdxidxh (i~1, 2,... n) Fubini: Sugli s pazii che ammettono 70 le formule di KILLI~ ei danno subito ehe le formule di KILLI~ ei danno subito ehe a~ ----- x, a,~ + c,~ (k ='= 2) a,~ = x~ ai, + 2 c,~ x, + c., dove le c;k sono arbitrarie, puretlb non dipendano da xi, x~. Spazii cite ammettono un G3 a trasformazioni infinitesime indipendenti. Qui ricorderemo i risultati generali del § 4. ossia Per trovare gli spazii cor- rispondenti a uno di questi tipi noi dovremo scindere il caleolo in due patti: uno relativo alle aik per i, k~-1, 2~ 3 e l'altro relativo alle air, per i> 3, k ~ 3. Quanto alle alh dove i> 3, k> 3 noi sappiamo gib. ehe esse posseno essere funzioni arbitrarie indipendenti dalle x,~ x,, ix3. Esamineremo ora uno dopo l'altro i varii tipi di G~, transitivi helle x,, x,, x~ gi~t trovati. p p , ,, ,, g 1. ° tipo}. Per ]e aia dove i~ k-= 1, 2, 3 otteniamo, iniegrando le equa- zioni di KILT.~% .T, dove le ci~ sono indipendenti da x,, x,, x~. Se lo spazio in diseorso ~ un S~, potremo supporre aa, = 1, a, = a~ = a~ ~ 0 e le cik funzioni di x4. In ge- nerale avremo per le aiR, dove i> 3, k ~ 3 Ctit 0 a,~ __ 1 0 a,3 1 O x, ~ x3 0 x, 0 x, equazioni~ che si integrano senz'altro. 2. ° tipo). Per le aik dove i, k = 1~ 2~ 3 aw'emo per le equazioni di KmLis% cite subito si integrano, 2. ° tipo). Per le aik dove i, k = 1~ 2~ 3 aw'emo per le equazioni di KmLis% cite subito si integrano, a,, = c,, a,~ = c,, e x' a,z = cis e hx' a~ = c~ e :x, ; a,a = C~3 eO+h)x, as3 -~ ca3 e ~hx' a,, = c,, a,~ = c,, e x' a,z = cis e hx' a~ = c~ e :x, ; a,a = C~3 eO+h)x, as3 -~ ca3 e ~hx' as3 -~ ca3 e ~hx' X x3 dove le cik sono indipendenti da x,,. ~, • X x3 dove le cik sono indipendenti da x,,. ~, • X x3 dove le cik sono indipendenti da x,,. ~, • Se lo spazio ~ un $4 si pub fare a4, =-1 ai4 =0 (i- 1~ 2, 3). In generale per le a~n (i> 3~ k ~ 3) avremo: p Se lo spazio ~ un $4 si pub fare a4, =-1 ai4 =0 (i- 1~ 2, 3). In generale per le a~n (i> 3~ k ~ 3) avremo: 0 x~ ~ x~ ~x~°i~ ~ ~ ai~ = 0 ~x~a;~ + a~ ~ --[- a~ h ~ = 0 the subito si integrano. the subito si integrano. ossia un gruppo continuo di movimenti. 71 71 3. 0 tipo), kvremo per le aik (i, k == 1, 2, 3) 0 aik 0 aik . Ox~ = ~-~ = O, 3. 0 tipo), kvremo per le aik (i, k == 1, 2, 3) 0 aik 0 aik . Ox~ = ~-~ = O, at3 x~ ~ x~ 0 x~ a22 6923 0 a83 -- ~ x~ a~ + h a~3 + a~ = O x'--T -- 2 a~3 + 2 h a~ . Derivando la seconda di queste ultime equazioni rispetto a x, e portan- dovi il valore di ~al3 dato dalla terza e tra la seconda e la terza formula x~ cost ottenuta eliminando al3, si ottiene: 0 ~ al2 ~ ~t2 0x~+h-yE-x +a,, =0. E, se = +-+-i/3 sono le radiei di z'+ h z-{-1------0 si avrh: a,, : ¢/e ~'~, cos fl x, -]- Z e:X' sen fl x, E, se = +-+-i/3 sono le radiei di z'+ h z-{-1------0 si avrh: E, se = +-+-i/3 sono le radiei di z'+ h z-{-1------0 si avrh: a,, : ¢/e ~'~, cos fl x, -]- Z e:X' sen fl x, a,, : ¢/e ~'~, cos fl x, -]- Z e:X' sen fl x, dove q,, Z non dipendono da x,, x~, x3. Si ha quindi: dove q,, Z non dipendono da x,, x~, x3. Si ha quindi: q,, p Si ha quindi: ai~ ~ a2~ ~a~ a,3 ----- -- "0 ,~ 0 x, ~ 0 ,z~ Sostituendo nell'ultima per le 0 a~2 D a23 0---~[ ' ~ i loro valori, si ha: Sostituendo nell'ultima per le 0 a~2 D a23 0---~[ ' ~ i loro valori, si ha: Sostituendo nell'ultima per le 0 a~2 D a23 0---~[ ' ~ i loro valori, si ha: ~ a~3 Da23 = 2 h a33 x' ~ ÷ 4 a~3 ÷ h ~ x---7 Derivando ed eliminando tra l'equazione cosl ot/enuta e la la a33 si trova: precedente Derivando ed eliminando tra l'equazione cosl ot/enuta e la la a33 si trova: precedente ~a a~a 0 ~ a~a 0 a2a ~ x------~ -k- a h O x----~ + ( 4 + 2 h') -U-~ -[- 4 h a..~ = O. ossia analoga equazione si troverebbe per a2~ e si avrebbe: a~2 ---- ~ e -hx' + ~ e --hx' cos 2fl x, 27 z e-~X' sen 2/3 x, 1 ~ a2~ a~3 = 2 ~xi a33--------- ~x, ~+~vx~ 1+ • Un'analoga equazione si troverebbe per a2~ e si avrebbe: n'analoga equazione si troverebbe per a2~ e si avrebbe: a~2 ---- ~ e -hx' + ~ e --hx' cos 2fl x, 27 z e-~X' sen 2/3 x, 1 ~ a2~ a~3 = 2 ~xi a33--------- ~x, ~+~vx~ 1+ • a~2 ---- ~ e -hx' + ~ e --hx' cos 2fl x, 27 z e-~X' sen 2/3 x, 1 ~ a2~ a~3 = 2 ~xi a33--------- ~x, ~+~vx~ 1+ • 72 Fubini: Sugli spazii che ammettono Si ha poi a~,----- Z, dove ~,, ~, Z sono funzioni indipendenti da x,, x~, x~. Se 1o spazio ~ un S,, potremo fare a, = 1, ai, = 0 (i= 1, 2, 3). In generale per le ai~ (i> 3, k-1 3) otteniamo : Si ha poi a~,----- Z, dove ~,, ~, Z sono funzioni indipendenti da x,, x~, x~. Se 1o spazio ~ un S,, potremo fare a, = 1, ai, = 0 (i= 1, 2, 3). In generale per le ai~ (i> 3, k-1 3) otteniamo : ai~ ~ alh ~ alk ai, ~ xa ~ (x, -~ h xa) __~ 0 Dah ~ 0 ~ ai, ~aia ~ x---[ ~[ + ai~ -= 0 ~ x----[ + h a~ ----- O ossia ossia ehe si integrano senza diffieolth. ehe si integrano senza diffieolth. g 4. 0 tipo). Per le alk (i, k---- 1, 2, 3) le equazioni e la loro integrazione si trova gi~ nelle pag. 67-68 di (A). Baster~ immaginarvi le a, b, c, d fun- zioni indipendenti da x,, x~, x3. ossia p Per le ai~ (i> 3, k~ 3) troviamo le: p Per le ai~ (i> 3, k~ 3) troviamo le: aik ~ aii ~ ai, D aia x---7 ----- 0 ~ x-----[ = ~ x----[ ~ ~ x----'7 + a i, ----- 0 0_o0 3 ~ aik .3 V ah ~ (e-x') ¢ti, ~ (x~ e -~) ~ (2 x, e -x.) 0 x~ ~ xh ~ x~ ai~ ~ xk = 0_o0 3 ~ aik .3 V ah ~ (e-x') ¢ti, ~ (x~ e -~) ~ (2 x, e -x.) 0 x~ ~ xh ~ x~ ai~ ~ xk = che si integrano senz'altro. che si integrano senz'altro. che si integrano senz'altro. g 5. ° tipo). Yalgono per questo considerazioni perfettamente analoghe a quelle del tip() preeedente. 5. ° tipo). Yalgono per questo considerazioni perfettamente analoghe a quelle del tip() preeedente. 6. 0 tipo). Per le a~ (i, k = 1, 2, 3) si trova, integrando le equazioni di KmLI~% e indieando con Z~ delle funzioni indipendenti da x,, x~, x3 ehe: a,,~---Z,,; a~=Z~..; a,z-----Z,3; a,~-----x~z~-t- Z,~; a~3-----x~z,,d-Z,~ Per le aik (i> 3, k L 3) si ha: aik ~ aik ~ ah ~ ai2 D ala a,,~---Z,,; a~=Z~..; a,z-----Z,3; a,~-----x~z~-t- Z,~; a~3-----x~z,,d-Z,~ Per le aik (i> 3, k L 3) si ha: Per le aik (i> 3, k L 3) si ha: aik ~ aik ~ ah ~ ai2 D ala ehe tosto si integrano. g 7. o tipo). Basta supporre le ai~ indipendenti da x,, x~ x~, x,. g 7. o tipo). Basta supporre le ai~ indipendenti da x,, x~ x~, x,. Spazii che ammettono un G3 a trasformazioni infinitesime linearmente dipendenti. In questo caso si dovrh scindere lo studio per le ai~ in due altri casi, uno relativo alle aih (i, k~--2), l'altro per le ai~, (i> 2, k~ 2). Spazii che ammettono un G3 a trasformazioni infinitesime linearmente dipendenti. In questo caso si dovrh scindere lo studio per le ai~ in due altri casi, uno relativo alle aih (i, k~--2), l'altro per le ai~, (i> 2, k~ 2). un gruppo continuo di movimenti. 73 1. ° tipo). Tutte le aik sono indipendenti da x,, x,. Per le air, (i, k= 1, 2) si trova dalle formole di KmH~a the: dove c,, ~ indipendente da xl, x~. ossia ,, p , Per le alh (i~>2, k-1 2) si ha: p Per le alh (i~>2, k-1 2) si ha: aik ? aik --0 0 x, D x... X~ Xt che si integrano senz'altro. che si integrano senz'altro. g 2. ° tipo). Per le a~, (i, k-----1, 2) si hanno le: g 2. ° tipo). Per le a~, (i, k-----1, 2) si hanno le: m, --0 a,~(; ~a~ .__2a~cotgx, a~=a,,sen-'x~ the si integrano tosto. g Per le aik (i:~ 2, k L 2) le formule di KILLI~e danno, poieh~ le ao~ sono indipendenti da x~, che: ai~ = ai~ = O. 3. 0 tip()). La discussione ~ perfettamente analoga alla precedente. 3. 0 tip()). La discussione ~ perfettamente analoga alla precedente Spazii che ammettono un gruppo G4 a trasformazioni infinitesime di- Tendenti. La ricerca di questi spazii in generale si fa come preeedente- mente : noi, col solo fine di una maggiore brevith, studieremo soltanto gli S,, accontentandoci di aver fatto rilevare dagli esempii precedenti il metodo generale. 1. ° tipo). Potremo chiaramente porte, quando ci si restringa agli $4~ d s ~ = d xI + ~, aik cl xi d xk i,k (i, k = 1, 2, 3). Integrando le equazioni di KILLIN% si trova a.-----c,t; a~=c~; a23=x, c22; a33=x]c~+c.; a,~+a,3=0 a.-----c,t; a~=c~; a23=x, c22; a33=x]c~+c.; a,~+a,3=0 dove le ci~ sono indipendenti da x,, x., x~. dove le ci~ sono indipendenti da x,, x., x~. 2. ° tipo). Posto, come sopra d s ~ = d x~ -t- Z a~k d z, d x~ %k (i, k=l~ 2~ 3) 10 10 Annali di 2¢Iatematica. Serie III, tomo VIII. Annali di 2¢Iatematica. Serie III, tomo VIII. 74 Fubini: Sugli spazii che ammettono otteniamo dalle e(~uazioni di KXLLI~O, otteniamo dalle e(~uazioni di KXLLI~O, otteniamo dalle e(~uazioni di KXLLI~O, otteniamo dalle e(~uazioni di KXLLI~O, Cttl = Cjj ~ (tas ~ Cs~ ~ ~,~3 ----- n e x, c3a ~ at.. = aja == 0 a,, = e'~, [(1 - n9 c,, + n' c3~] dove ]e elk sono indipendenti da x,, ",c,, x,. 3. ° tipo). Posto ds*~dx]+~aikdxidxk (i, k=], 2, 3) i,k si trova, con ii solito procediment% che: si trova, con ii solito procediment% che: al~ ~ ct~ ; aa3 =: Caa ; aj~ ~-- a~a = 0 ; a~a = n C~3 COS Xt a~ : n ~ c3a cos' ~ + c,, sen ~ x~. ossia Spazii che ummettono un gruppo G, non integrabile transitivo a trasfor- .mazioni infinitesime.linearmente indipendenti. Prima di trattare questo caso particolar% faremo alcune consideraz]oni generali sugli spazii che ammettono un Ga, a trasformazioni infinitesime in- dipendenti. Osserveremo cio~ che in ognuno dei gruppi G4 cosiffatti, prece- dentemente determinati~ entrano degli elementi arbitrarii. E not potremmo va]ercene per condurre in due maniere i caleoli. Il primo modo consiste nel dare a questi elementi arbitrarii valori opportuni della massima semplicith~ ricordando il teorema di Lm che due gruppi semplicemente transitivi sullo stesso numero di variabili e isomorfi sono anche simili. I1 secondo metodo consisterebbe nel prefissare all'elemento lineare da determinarsi una forma saputa , a priori , possibil% laseiando quindi agli elementi arbitrarii the en- trano nel gruppo tutta l'arbitrariet~ compatibile con questa forma. I1 primo metodo ~ pih opportuno per 1o studio degli spazii a un numero qua]unque di dimensioni, il seeondo per lo studio degli $4. Diamo ora un esempio del primo metodo, p Prendiamo p. es. il gruppo G4 generato dalle diamo p. es. il gruppo G4 generato dalle X,=b--~; X~--O-~v; X~=--~x--~+X~x,; O X,_[(~_ 1)x, + ,.,,] U £ + [--x,~+ cz, + ~,] ~x--- + + [z~ + ~] ~ x~ -- 1-7 ~x," O X,_[(~_ 1)x, + ,.,,] U £ + [--x,~+ cz, + ~,] ~x--- + + [z~ + ~] ~ x~ -- 1-7 ~x," un gruppo continuo di movimenti. 75 Secondo i principii test~ esposti potremo porre ~,=7,=?~=¢), l~=:l col che si ha X~ = (c-- 1) x, ~ -[- c x~ ~ -Jr x~ ~ x~ ~ ~x~ " Per le ai~ (i> 4, k> 4) basra supporre le funzioni indipendenti dalle x,, x~, x~, x~. Per le a~ (i, k= l, 2, 3, 4) troviamo per le formule di K~LL~O, d as3 ~aih ?Crib --- -- 0 x, -= 2 a~ ; ~ x~ ~ x~ a(3 0=',=3) D x, a~_2c; a~; --=0; ~a'L--2(cx4 -- 1) a,,; ~x, -- ~x4 ~x, --- (x --~1) a,, -4- c a,2, ecc., ecc., 0 x4 --- (x --~1) a,, -4- c a,2, ecc., ecc., 0 x4 the si integrano senz'altro. Per le a;k (i> 4, k ~-~ 4) si hanno le: DCti___._~k --~ Oaik 0 ; --- ~ aia. ossia @ ai, 0 x3 _ 0 (c -- 1)x, ~aih ~x~ ~ X a ~ 2c c a i~ ~ -j- a i,3 -~w k = 0 DCti___._~k --~ Oaik 0 ; --- ~ aia. @ ai, 0 x3 _ 0 (c -- 1)x, ~aih ~x~ ~ X a ~ 2c c a i~ ~ -j- a i,3 -~w k = 0 the pure si integrano senza difflcoltk. Con questo stesso metodo si possono chiaramente studiare tutti gli altri tipi di G4. Noi, col solo scopo di non allungare questa )lemoria coll formule, che poi non utilizzeremmo, ci restrin- geremo agli $4 e useremo naturalmente del secondo metodo. Cominciamo ora a trovare gli $4 con un G~ transitivo non integrabile del primo tip% avver- tendo che la prima parte dell() studio ci servir~ anche per i casi ulteriori. Sic(.ome il sottogruppo formato dalle X,, X.,, X3 ha le x4 == cost. come va- riet~ invarianfi potremo porte: ds~-dx~-~aikdxidxk (i, k-l, 2, 3). 4 ~ f ~ una trasf0rmazione infitfitesima che quelIo spa- i=l 4 ~ f ~ una trasf0rmazione infitfitesima che quelIo spa- i=l Fubini: 5'ucjli spazii the ammettono 76 zio ammette allora~ serivendo ]e condizioni di K~LLI~ relative ad aik =-a~, a,:~ a~3~ a, otteniamo: ~7:~ = 0 Ox4 (~) (~) (i = l, 2, 3). @ @ Indicando al solito con Ai~ il il valore eertamente non nullo del complemento algebrico di alk diviso per determinante [alh[ otterremo dalle ([3) Indicando al solito con Ai~ il il valore eertamente non nullo del complemento algebrico di alk diviso per determinante [alh[ otterremo dalle ([3) ~4 ~ :~ A~k-- (i= 1 2, 3). (I) 094 k=l ~ '~l~ ~ " (I) esso Nei easi in eui~ come nel nostr% ~4 pub essere soltanto funzione di x~ ;~ quindi per la (~) eostante~ e pereib per le (I) abbiamo ehe: = ~3 p Ox--~--~ ~x, oX~,~----0. Dunque le ~i non possono contenere x,; eosiech~ la nostra seelta dell'elemento Iineare fa si ehe tutte le funzioni arbitrarie (della x,) che compariseono in X, sono semplici eostanti. Queste considera- zioni va]gono anche per molti dei casi seguenti e noi non le ripeteremo. Intanto avremo che le a, fl~ 7 sono effettive costanti. E se noi seriviamo ]e formole di KIL~,IsG per le ai~, (i, k-~-l, 2, 3) relative a X,, X~ X, ot- teniamo le formule pag. 67 eli B. ossia E ei basterk dall'esame di X4 ricavare le a, b~ c, d, e, f ehe nel nostro easo sono funzioni di x4. Osserviamo intanto che nella X4 potremo (dividendo le v., /3, 7 per una costante certo non nulla) supporre 14-----1. Scrivendo le equazioni di KmLI~( b otteniamo dei polinomii in x,, x~ da uguagliarsi a zero. Annullando i coefflcienti dei singoli termini (che saranno soltanto funzioni di x 0 raggiungeremo il nostro scopo. L'equa- zione di K~Lm~(~ relativa ad a,~ ci dh cos'l: d x4 d x4 L'equazione di KIT,Lira per a33 ci dS, indicando con un apice la deri- vazione rispetto x4: L'equazione di KIT,Lira per a33 ci dS, indicando con un apice la deri- vazione rispetto x4: b' =a"- 7 -k 2b~--~.(cq-2d) e' q- 2 d' ---= 6 (bT--e.) d' = 2 (b 7 - - e ~.) e'--(c-~- 2 d) y-- 2e[3--~ ~. un gruppo continuo di movimenti. 77 L'equazione di KILLI~o relativa ad a~2 ci d~ infine: f' ---- 4 (e 7 - f Lea m b, c, d, e~ f si ottengono dunque integrando un sistema lineare di equazioni differenziali ordinarie del prim'ordine. q p 2. ° tipo).,Prefisso come sopra all'elemento lineare la forma 1~2,3 dx] + Z ai~dxidxk si trova che ~=~, 6,=~ sono costanti. Per Ie ait,(i~ k~l, 2~ 3) tro- viamo, poich~ lo spazio deve ammettere il G3 ~(X,, X~, X3) le equazioni di pag. 69 (A) dove soltanto le eostanti di integrazione saranno funzioni op- portune di x4. Se ~ ~ 0, queste funzioni saranno proprio costanti effettive ; se ~.3~ 0~ notiamo che mutando x4 in Kx4, dove K ~ una costante oppor- tuna~ potremo fare ~3-----~4~ mentre, sostituendo al nostro spazio uno spazio simil% potremo ancora porre a,4-----1. Si ha cosi e le aik (i-----1~ 2~ 3) si troveranno ancora date dalle formule a pag. 69 di (A)~ dove le a, b, c... si intendano ancora effettive costanti~ ma al posto di x~ si sostituisca x~--x4. Spazii che ammettono un G4 a trasformazioni linearmente indipendenti, integrabile e il cui gruppo derivato ~ un G3 a trasformazioni infinitesime non permutabili. 1. o tipo). Anehe qui potremo porre (i, k~l, 2, 3). ds'~dx~A-~aihdxidXh (i, k~l, 2, 3). ossia Avremo per l'osservazione fatta che ~,, ,%~ y3, 14 sono costanti effeitive che indicheremo con l,, l~, 13, l~ eosicchh si potrh porte: 1 Essendovi le X,, X~, X~ il nostro spazio sar~ del tipo: ds' = dx]--~ ~ dx~-{- 2 ~ dx, dx,-~- Z dx~ +2 (x, ~ + #) dx, d x~ -~- 2 (x, Z -}- Z) d x~ d x3 -J- (x~ Z -[- 2 z, ), --[- ~) d x; , dove le ~ ~, 7., 3, ),, u sono funzioni di x,. dove le ~ ~, 7., 3, ),, u sono funzioni di x,. 78 Fubini: Sugli spazii the ammettono Scrivendo le formule di KILLll~(3 relative a X~ abbiamo: Scrivendo le formule di KILLll~(3 relative a X~ abbiamo: [(c - 1) x, + z,] Y-kT, ~ "~'~ + "~' (c -- a) ~ ~ x, + ,~h, (c -- 1) U~ ~ :~' + 0 (c x: -- l~ x~) ~ (c x, -- l~ x,) "q- (/is - - X3 ~Xa= 1 ~aik Sostituendovi i valori trovati delle aih~ e integrando otteniamo: Z ~ P:~ e~cl'x~ ----- e a.(~-~)x, [ ~ 2 p,~ l~ e z,x, -I--p,,-[- 1] p,~ e ~,'~, ] )==e~,(~÷,)., I z, 1 ' 1) p,~ e z(~-~)~' ~p~3 (si noti che c =,= ----= e~t'~' I 2 l, et,(~_,)~, + l~ p~ e a'(~-')x' + p~3 I ------ ecZx~ I c~--lli pt~ e(C_i)Z,x4__l 3 p~3 e l'x~ li ls t c -- i p'~ ed'~' + p,3 dove le p;k sono costanti effettive. dove le p;k sono costanti effettive. dove le p;k sono costanti effettive. 2. ° ripe). Anehe qui si pone d s ~ = d x~ + ): aih d xi d x~. ik E se ne deduce che le li sono effettive costanti. Per l'esistenza di X,, X~, X3 si ha che ds ~ ~ del tipo gi~ citato al 1. ° case. p g Scrivendo le equazioni di KmLI~G relative alla X4- l~ X,- 13 -¥2 e in- tegrando, troviamo : Z ~P~ ea'x' ----- e ~z,x, (l~ p~2 e '~z.~' + 2 l, p~3 e t''~' + P3.0 q~ = e~Z'x' I-- (l, + 13) p..~ e z''~' -- 1, p.~3 x4 + p,~ ] 3' = e :z,', I p,3 -- l, (l, 2 r. ossia 13) p~ e ~l.x' -- (1, -~- 13) p23 e z'x' q- l, p,~ e l,x, -- --/~ p33 x4 -- l, 14 p~3 x4 e ~x' ,! ~ e'Z'~' I (l, -q- l~)~p~ e 'Z,`~, -- 2 (1, -t- 13)p,~ e 1.~ -}- 2 p~ l~ x, (1, H- 13) e Z.È~ -1- + 1", p~ x ~, -- 2 l~ l). z, + p. i Z ~P~ ea'x' dove le Pik sono eostanti. un gruppo continuo di movimenti. 79 Spazii che ammeltono un G~ integrabile a trasformazioni infinitesime in- dipendenti, di eui il gruppo derivato ~ un G~. p g pp Per il primo dei due casi di questo tipo, troviamo col solito proeedi- mento delle pagine precedenti e con notazioni analoghe :. ds'= dx~ + ?dx~ + 2kdx, dx, + z dx~ + 2(x,~-~.2) dx, dxa+ + 2 (x, >c -ki) dx~dx~ + (xl z + 2 x, X + t,)dx] dove dove dove = e z'~' (-- h p~, e Z~' + ~,,) ---- - - 2p,, l~ eZ, '~, q- l~p~.~ e ~hx~ + p,, ~, = e'-Z. ~, (1, l, p,, x, + p~,) = e<~, 12 l, l, p,, x~ + l~ l] zlp. + p,, I = e~'°°' I P,, + l, 1, p,, x, -- 1, 1~ p,, e z.°°, -- l, p,a e z,'~, + 1, I, p.~, e z,~, -- -- 1, la 1, p,, x~ e z,~, ] . = e z'~' (-- h p~, e Z~' + ~,,) ---- - - 2p,, l~ eZ, '~, q- l~p~.~ e ~hx~ + p,, ~, = e'-Z. ~, (1, l, p,, x, + p~,) = e<~, 12 l, l, p,, x~ + l~ l] zlp. + p,, I = e~'°°' I P,, + l, 1, p,, x, -- 1, 1~ p,, e z.°°, -- l, p,a e z,'~, + 1, I, p.~, e z,~, -- -- 1, la 1, p,, x~ e z,~, ] . Studiamo il secondo tip() di tali gruppi. Studiamo il secondo tip() di tali gruppi. Esso eontiene un G~ a trasformazioni tipo 2. ° , dove si faccia h = 0. infinitesime indipendenti del tipo 2. ° , dove si faccia h = 0. ossia Sarh percib : d s' -- d x~ + ~ d x; ~ 2 ~ e*' d x, d x~ ~ S e '*, d x~ -~ ~ d x~ .-9 + 2 Z dx, dx3 -4 2). e'~,dx~dx3 7 + 2 Z dx, dx3 -4 2). e'~,dx~dx3 7 dove le ?, ~,... sono funzioni di x4. dove le ?, ~,... sono funzioni di x4. Nell'espressione trovata per ]a 2/4 f saranno al solito ]e ~i vere costanti~ % come si vede percib tost% potremo porr% indicando con a~ ~ l, costanti 7 0 0 ~ 1 X, = ~ U~ + [~ e-~. ~ -t- x ~ . . . . ~x~ ~x3 h ~x, Integrando le relative equazioni di KmLI~% e indicando con pil, delle costanti~ abbiamo : fz ~" ]Oaa e ~l'x~ fz ~" ]Oaa e ~l'x~ Fubini: Sugli spazii cite ammeUono 80 ( ) ~------ 2~ p,~- T Se ~ ~ 0, valgono le stesse formule, dove perb al termine--e"l'x' (ehe eom- parisee in ~0, ~, Z) si deve sostituire il termine 14 X4 • Annali di Matematioa, Serie II[, tomo VIII. 14 X4 • Spazii che ammetlono un G, che contengono un G3 a trasformazioni in- finitesime permutabili e le cui trasformazioni infinitesime sono linearmente indi2endenti. In tutti i tipi gik enumerati al § 11 i coeflleienti dell'elemento lineare corrispondente saranno funzioni della sola x~. E noi potvemo porte: d s' = d xl -]-- ~ ai~ d xi d xt, (i, k = 1, 2, 3). -$~P., Se ne hg. ehela %, ~, ~, l~ sono eostanti. g Le equazioni di Kmrm~(~ diventano: g Le equazioni di Kmrm~(~ diventano: 2 aik UY~ + ~' 0 x, Z a,~ + ~ az~ ~ = 0 (i, l = 1, 2, 3). Sostituendovi per le ~i i loro valori 7 integrando, indieando con hia delle costanti, troviamo : I °) a,, = h,, e a,x, a~2 = h2~ e ~l'~x' a33 == h~3 e ~lxx~ a,~ = h,, e l,(i4aix, a.~ -=- h,s e l,(~+c)x' a,s ~ h,s e l'('+°)w' a.~ = (l, h,, x, -1- h,~) e t,('+'c)x* a,~--= e,h~x, (l h,, x, + h,~) a~ = e a-*~, (l~ h,, x~ + 2 h,~ l, x, + ~) un gruppo conlinuo di movimenti. 81 III °) IV °) V ° ) 1 a 3 l, h,, ° ÷ (h,, + h.,~) l, x, + h,~ a,, ---= l, h,~ x~ .{- 2 h,, 14 x, -[- h.,, ; II . a33 = -~ h,, xl --~ l~ h,~. x] -4- 14 (h,, + h.~) xi + 2 l, h,~ x, + h~ a.22 ~- h~, e?t.~, ; a,~ = e:l. ~, [ h,..~ 1,, x, -4- h,, ] ; a,, = e<~, (li h:~ x~ -4- 2 l, h,, x, A- h,,) a,~ ~--e ~t,°~, h,3 --~ ~ x] -4- ~2- li h,2 x4 --}- (h,, + h23) l, x, a~---- e *~,', h. II ¥ -t-- l~ h,, :r~ + (h,, -4- h:~) l~ z~ + 2 l, x, h,3 + h~ a,,~h,; a~----h,~; a,,----h,.; a.,3=14h.~2x~Th~; a,3 = l, h,~ X4 "-1 t- hia III °) 1 a 3 l, h,, ° ÷ (h,, + h.,~) l, x, + h,~ VI °) ] [ , a~3 ~ e ~,x, [ l, h,.. x, -[- h,s [ a23 ---- e ~t':e~ [ 14 h..a x, -[- h23 [ 11
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Heterogeneous and rapid ice loss over the Patagonian Ice Fields revealed by CryoSat-2 swath radar altimetry
Remote sensing of environment
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Document Version: V i d Document Version: Version created as part of publication process; publisher's layout; not normally made publicly available Document Version: Version created as part of publication process; publisher's layout; not normally made publicly a Published In: Remote Sensing of Environment Edinburgh Research Explorer Heterogeneous and rapid ice loss over the Patagonian Ice Fields revealed by CryoSat-2 swath radar altimetry Edinburgh Research Explorer Citation for published version: Foresta, L, Gourmelen, N, Weissgerber, F, Nienow, P, Williams, J, Shepherd, A, Drinkwater, MR & Plummer, S 2018, 'Heterogeneous and rapid ice loss over the Patagonian Ice Fields revealed by CryoSat-2 swath radar altimetry', Remote Sensing of Environment. https://doi.org/10.1016/j.rse.2018.03.041 Citation for published version: Digital Object Identifier (DOI): 10.1016/j.rse.2018.03.041 Link: Link to publication record in Edinburgh Research Explorer Document Version: Version created as part of publication process; publisher's layout; not normally made publicly available Published In: Remote Sensing of Environment Heterogeneous and rapid ice loss over the Patagonian Ice Fields revealed by CryoSat-2 swath radar altimetry L. Forestaa,⁎, N. Gourmelena,b, F. Weissgerbera, P. Nienowa, J.J. Williamsa, A. Shepherdc, M.R. Drinkwaterd, S. Plummerd a School of GeoSciences, University of Edinburgh, UK b Université de Strasbourg, CNRS, IPGS UMR 7516, F-67000 Strasbourg, France c Centre for Polar Observation and Modelling, School of Earth and Environment, University of Leeds, UK d European Space Agency, ESA-ESTEC, Keplerlaan 1, 2201 AZ Noordwijk, Netherlands A R T I C L E I N F O The Northern and Southern Patagonian Ice Fields (NPI and SPI) in South America are the largest bodies of ice in the Southern hemisphere outside of Antarctica and the largest contributors to eustatic sea level rise (SLR) in the world, per unit area. Here we exploit swath processed CryoSat-2 interferometric data to produce maps of surface elevation change at sub-kilometer spatial resolution over the Ice Fields for six glaciological years between April 2011 and March 2017. Mass balance is calculated independently for nine sub-regions, including six individual glaciers larger than 300 km2. Overall, between 2011 and 2017 the Patagonian Ice Fields have lost mass at a combined rate of 21.29 ± 1.98 Gt a−1, contributing 0.059 ± 0.005 mm a−1 to SLR. We observe widespread thinning on the Ice Fields, particularly north of 49° S. However the pattern of surface elevation change is highly heterogeneous, partly reflecting the importance of dynamic processes on the Ice Fields. The Jorge Montt glacier (SPI), whose tidewater terminus is approaching floatation, retreated ~2.5 km during our study period and lost mass at the rate of 2.20 ± 0.38 Gt a−1 (4.64 ± 0.80 mwe a−1). In contrast with the general pattern of retreat and mass loss, Pio XI, the largest glacier in South America, is advancing and gaining mass at 0.67 ± 0.29 Gt a−1 rate. Keywords: CryoSat-2 Radar altimetry Swath processing Glacier elevation change Patagonian ice fields https://doi.org/10.1016/j.rse.2018.03.041 Received 24 November 2017; Received in revised form 22 March 2018; Accepted 29 March 2018 0034-4257/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). ⁎ Corresponding author. E-mail address: luca.foresta@ed.ac.uk (L. Foresta). Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 1. Introduction comparatively high amounts of shortwave energy (Lenaerts et al., 2014). Thus, the glaciers west and east of the ice divide are thought to be more sensitive to changes in precipitation and air temperature re- spectively (Rivera and Casassa, 1999; Warren and Sugden, 1993). The Northern and Southern Patagonian Ice Fields are the two largest ice bodies in the Southern Hemisphere excluding Antarctica, with areas of about 4200 and 13,000 km2 and volumes of about 1200 and 4300 km3 (Carrivick et al., 2016), respectively, and elevation ranging from sea level to about 3900 m. They experience relatively warm and wet climatic conditions (Sagredo and Lowell, 2012) and lie on top of the narrow Andean mountain range, which forms an efficient barrier to the predominantly westerly winds and moisture rich air transported inland from the Pacific Ocean (e.g. Garreaud et al., 2013). The moun- tain and associated ice divide separates areas with contrasting climatic conditions. On the western side, orographic uplift of moist air produces extreme annual precipitation of up to 10 m a−1 (e.g. Lenaerts et al., 2014; Sakakibara and Sugiyama, 2014 and references within) as well as extensive and persistent cloud coverage with associated low shortwave and high longwave energy fluxes (Lenaerts et al., 2014). To the east of the divide, the Ice Fields are in the rain shadow and receive The two Ice Fields have been experiencing long-term thinning and retreat. Between the end of the Little Ice Age (LIA, ~1870) and 2011, their area shrank by ~12.5% on average (Davies and Glasser, 2012), associated with a combined mass loss of about 1.70 ± 0.25 Gt a−1 (Glasser et al., 2011). However, geodetic studies based on gravimetry (Chen et al., 2007; Ivins et al., 2011; Jacob et al., 2012; Gardner et al., 2013) and comparisons of Digital Elevation Models (DEM; Rignot et al., 2003; Willis et al., 2012a, 2012b; Jaber, 2016) estimated that between 1975 and 2012 the rate of mass loss of the Ice Fields has been in the range of 15 to 35 Gt a−1, one order of magnitude more compared to the long term trend. 1. Introduction L2swath data are then used to compute rates of surface elevation change for six glaciological years between April 2011 and April 2017 using a plane-fit algorithm (e.g. McMillan et al., 2014b). One glaciological year is defined as the period between 1st April in year n and 31st March in year n + 1. CS2's acquisition dates vary spatially for different pixels due to the satellite's orbital path as well as to the local topography, so that the temporal resolution at the pixel scale is non-uniform and longer than monthly. However, seasonality biases are avoided given the regular flight path followed by CS2, which ensures that data within each pixel are acquired at the same epochs (within a few days) in each glaciological year. L2swath data are gridded at 500 m × 500 m spatial resolution and for each pixel, we model eleva- tion z(x,y,t) using a linear relationship in space and time: (Glasser et al., 2011 and references within), increasing to 15.4% in the first decade of the 21st century (Jacob et al., 2012; Gardner et al., 2013), second only to glaciers in Alaska and the Canadian Arctic, and larger than high mountain Asia (Brun et al., 2017) which all extend over areas ~5–8 times larger. Currently, the Patagonian Ice Fields are the largest contributor to sea level rise per unit area in the world (Gardner et al., 2013; Carrivick et al., 2016). Velocities of glaciers draining the Ice Fields (up to 10 km a−1) are amongst the fastest in the world (Sakakibara and Sugiyama, 2014; Mouginot and Rignot, 2015) and substantial ice flow acceleration has been observed, coincident with rapid frontal retreat, for a number of tidewater and lacustrine glaciers (Sakakibara and Sugiyama, 2014). These observations implicate the importance of the role of dynamics and tidewater glacier calving in the rapid wastage of the Ice Fields. In fact, > 80% of them terminate in proglacial lakes (mostly across the NPI and east of the SPI) or fjords (western side of the SPI) (Sugiyama et al., 2016 and references within). Since 2010, the European Space Agency (ESA) radar altimetry mission CryoSat-2 (CS2) (Drinkwater et al., 2005; Wingham et al., 2006) has been acquiring topography data over land ice. Radar in- struments are particularly suited to land ice applications since they can penetrate through clouds and do not depend on sunlight. 1. Introduction We iteratively fit the model to the data using 3σ clipping until there are no more outliers. The formal uncertainty εḣ on each pixel's rate of elevation change ḣ is extracted from the model covariance matrix M: where x, y and t are measured easting, northing, and acquisition time, respectively, and c0, c1, ḣ, c2 are the model coefficients. The time-de- pendent coefficient ḣ retrieved from the model fit is the linear rate of surface elevation change for each given pixel. Each observation is as- signed a weight according to the sample power as in Gourmelen et al. (2017a). We iteratively fit the model to the data using 3σ clipping until there are no more outliers. The formal uncertainty εḣ on each pixel's rate of elevation change ḣ is extracted from the model covariance matrix M: = = − − P cov p G cov z G ( ) ( )[ ]T 1 1 Despite their important contribution to ice mass loss and global SLR, studies quantifying mass changes of the Patagonian Ice Fields are lim- ited in number and do not cover the most recent period. This paper focuses on quantifying the mass balance of the NPI and SPI during six glaciological years between April 2011 and March 2017. To this aim, we exploit swath processed CS2 SARin data to generate maps of surface elevation change rates at sub-kilometer spatial resolution, and convert them into estimates of glacier volume and mass change. For a number of large catchments on the SPI, such estimates are derived at the basin scale. Additionally, the dense L2swath elevation field enables the pro- duction of time series of elevation change for different sub-regions of the Ice Fields exhibiting contrasting patterns of change. where p is the vector of coefficients [c0 c1 ḣ c2] of the model para- meters, z are the input elevations and G = [x y t 1] is the model matrix. We simplify the data covariance matrix cov(z) to a variance matrix whose diagonal values are the squared elevation differences between the observed and modelled estimates (z-z′)2. The square root of the diagonal elements of P represents the standard deviations of the model parameters p. Due to the complex topography (see Discussion), the ḣ maps do not have complete coverage. 1. Introduction We use the relation between elevation and elevation change to model estimates for the gaps in the maps of surface elevation change rates (i.e. hypsometric averaging, see e.g. Moholdt et al., 2010; Nilsson et al., 2015a; Foresta et al., 2016), using the SRTM DEM for the elevation field. Polynomial models of order 1 to 3 are fitted to the data and used to generate elevation change rates for the in- dividual pixels without an estimate. In order to avoid over-fitting the 1. Introduction During the last 50 years, the Patagonian Ice Fields contributed an estimated 10% to the total mass loss from glaciers and ice caps, ex- cluding those at the periphery of the Greenland and Antarctic ice sheets https://doi.org/10.1016/j.rse.2018.03.041 Received 24 November 2017; Received in revised form 22 March 2018; Accepted 29 March 2018 0034-4257/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). Please cite this article as: Foresta, L., Rem ote Sensing of Environm ent (2018), https://doi.org/10.1016/j.rse.2018.03.041 e this article as: Foresta, L., Rem ote Sensing of Environm ent (2018), https://doi.org/10.1016/j.rse.2018.03.041 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. this technique (Hawley et al., 2009; Gray et al., 2013; Foresta et al., 2016; Gourmelen et al., 2017a) can generate up to two orders of magnitude more data than conventional Point-Of-Closest-Approach (POCA) processing and, importantly, provides more homogeneous spatial coverage over relatively small glaciated regions with consider- able topography (Foresta et al., 2016; Gourmelen et al., 2017a). The L2swath processing scheme is similar to Foresta et al. (2016) and Gourmelen et al. (2017a), but we use a different procedure to discard noisy waveform samples before performing the phase unwrapping. This procedure, first developed for InSAR images (Weissgerber, 2016) and updated for CS2 SARIn data (Weissgerber et al., 2018), was shown to further increase the density of the L2swath elevation field and to im- prove the spatial coverage of the Jakobshavn glacier, Greenland (Weissgerber and Gourmelen, 2017) (Appendix A). The L2swath algo- rithm makes use of an external DEM to improve the precision of ele- vation measurements in the presence of slopes larger than 0.54°, where an entire waveform may be affected by a phase shift. Without this correction, observations may be wrong by several tens of meters in elevation and a few kilometres in geo-location (Gourmelen et al., 2017a). It is not straightforward to predict the accuracy needed for the DEM (Gourmelen et al., 2017a). However given the magnitude of the geolocation and elevation shifts, the DEM need not be extremely ac- curate. We used the SRTM C band DEM (Farr et al., 2007) acquired in 2000 as a reference for elevation, after including a correction to ac- count for the elevation change occurred between 2000 and 2011 (Appendix B). 1. Introduction Radar alti- metry data have been previously exploited to map elevation change over ice caps (Rinne et al., 2011a, 2011b), but the technique has not been applied widely due to the limitation caused by the large radar footprint. CS2's state-of-the-art radar altimeter uses Synthetic Aperture Radar (SAR) along-track to reduce the footprint size as well as inter- ferometry across-track to accurately locate the position of the surface reflection (SARIn mode; Wingham et al., 2006). Additionally, its orbit inclination of 92° and repeat cycle of 369 days provides an inter-track spacing of ~5 km on average over the Patagonian Ice Fields. Finally, CS2's relatively short wavelength (2.2 cm; Ku band) restricts the pe- netration of the radar pulse in the snowpack, compared to, e.g., sensors working in C or X bands. These characteristics make CS2 better suited for monitoring changes in glacier areas with frequent cloud cover and considerable slopes. CS2 SARIn data have successfully mapped topo- graphic changes over Arctic ice caps (McMillan et al., 2014a; Gray et al., 2015). Additionally, swath processing (Hawley et al., 2009) of CS2 SARIn data has been applied to generate high resolution DEMs of ice caps and selected areas of the Greenland and Antarctic ice sheets (Gray et al., 2013; Ignéczi et al., 2016; Gourmelen et al., 2017a) and to produce sub-kilometer maps of surface elevation change (Christie et al., 2016; Foresta et al., 2016; Gourmelen et al., 2017a; Gourmelen et al., 2017b), with a wide range of applications such as the identification of supraglacial lakes in NE Greenland (Ignéczi et al., 2016), subglacial lakes in West Antarctica and regions of subsidence in Iceland (Smith et al., 2017; Gourmelen et al., 2017a) as well as quantifying channe- lized basal melt under the Dotson ice shelf in West Antarctica (Gourmelen et al., 2017b) and volume and mass change of individual ice caps in Iceland (Foresta et al., 2016). = + + + z x y t c x c y ht c ( , , ) ̇ 0 1 2 where x, y and t are measured easting, northing, and acquisition time, respectively, and c0, c1, ḣ, c2 are the model coefficients. The time-de- pendent coefficient ḣ retrieved from the model fit is the linear rate of surface elevation change for each given pixel. Each observation is as- signed a weight according to the sample power as in Gourmelen et al. (2017a). 2. Data and methods We exploit swath processed CS2 SARIn baseline C data (L2swath) as 2 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Combining data from different glaciers is needed if coverage is insufficient to provide a representative figure of elevation change in each and is jus- tified providing that they show a similar pattern of change. data, the F-test is used to evaluate if the improvement of the additional model parameters on the fit is statistically significant at the 99% con- fidence level. The median rate of elevation change is then computed in each 50 m elevation band k and multiplied to the area Ak, extracted from the DEM, to produce elevation dependent volume change V̇ k. The sum of these contributions represents the total rate of volume change. Uncertainties are calculated by error propagation using the same method as in Foresta et al. (2016), summarized in Appendix C. The observed median rates of elevation change for these nine sub- regions are shown as a function of cumulative surface aerial extent (10% steps; Fig.1, side panels) and as a function of elevation (50 m steps; Fig.2, side panels). Widespread thinning is occurring in the northern part of the SPI across all elevations, with average rates of 2 m a−1 on the plateau up to and above 1400 m elevation (SPI-G1) and in excess of 10 m a−1 at the terminus margins of Jorge Montt (tide- water), Viedma and Upsala (both lacustrine) glaciers. Most glaciers in the south/southwest are close to balance (SPI-G2), with the exception of Grey and Tyndall on the eastern side. Pio XI glacier is thickening at rates of ~2 and 1 m a−1 below 1000 m and between 1000 and 1500 m elevation, respectively, and thinning by about 1 m a−1 above 1500 m altitude. Similar to the northern part of the SPI, the NPI is experiencing widespread thinning of up to 8 m a−1 with the exception of the ice divide close to the eastern margin of the ice field (Figs. 1 and 2). Hypsometric averaging is applied in each sub-region (Fig. 2, red lines) to generate maps of modelled elevation change rates for the Ice Fields (Fig. 2), from which mass change is computed (Table 1). The poly- nomial models (Fig. 2, red lines) compare well with the observed median rates of elevation change (Fig. 2, black lines with dots). 2. Data and methods A few exceptions are visible over the Tyndall and Upsala glaciers (at low and high elevation respectively) as well as over the Pio XI glacier below 1000 m and above 2500 m elevation. Model misfits have marginal im- pact on the glacier mass change when glacier area is negligible or data coverage is high (Fig. 2). For example the rate of mass loss of glacier Tyndall, assuming no elevation change below 350 m elevation, is re- duced by about 9% (or 0.054 Gt a−1), which is well within its asso- ciated uncertainty (Table 1). Similarly for Pio XI glacier, the thinning predicted by the model above 2500 m elevation reduces the glacier net mass gain by only 6% (or 0.04 Gt a−1). However, at low elevation where the area of Pio XI glacier is not negligible and where there are no observations to constrain the elevation change (Figs. 1 and 2), the impact of the misfit on the glacier mass balance may be significant (see Discussion). Between April 2011 and April 2017, the NPI and SPI have been losing mass at rates of −6.79 ± 1.16 and −14.50 ± 1.60 Gt a−1, respectively, contributing 0.059 ± 0.005 mm a−1 to eu- static SLR. About 35% of the SPI mass loss is concentrated on glaciers in the SPI-G1 group (−5.07 ± 0.79 Gt a−1), which represent ~28% of the SPI surface. The Upsala glacier is the single largest contributor to the mass loss (−2.68 ± 0.40 Gt a−1) and is also the glacier with the second highest rate of loss per unit area (Table 1) after Jorge Montt. Both glaciers have retreated between 2011 and 2017, by about 0.5 and 2.5 km, respectively. Pio XI is the only glacier in the Patagonian Ice Fields with positive mass balance (0.67 ± 0.29 Gt a−1). Its southern tidewater and northern lacustrine termini have both advanced, re- spectively by about 500 m and 800 m during our study period. Using a dual density scenario, the rates of mass change in the nine sub-regions are lower by 11–19% compared to using the density of glacial ice at all elevations and the total rate of mass loss of the Ice Fields is 17.89 ± 2.03 Gt a−1 (Table 2). For most basins, dynamic processes are dominating the mass loss and hence the ice density scenario is the preferred option. 2. Data and methods However, in a few sectors the dual density scenario may be more accurate. For example, over the Pio XI glacier (SPI), where surface thickening is suspected (Malz et al., 2018), the mass change is 0.67 ± 0.29 Gt a−1 and 0.57 ± 0.25 Gt a−1 for the single and dual density scenarios, respectively. Ti i f b d l i l ti h (Fi 3) h This interpolation scheme is applied independently for the NPI and for different sub-regions of the SPI displaying highly contrasting pat- terns of change at similar elevations (Fig. 2). Finally, we assume that all changes relate to the gain or loss of ice of density ρice = 900 kg m−3 when converting the rate of volume change to mass balance. This simplification is based on the assumption that at least part of the ob- served changes are due to dynamics (see Discussion) and ignores pos- sible differences in snow pack density below and above the firn line. To explore mass loss related to material with lower density, we calculate mass balance estimates using a dual density scenario. In this case the densities of glacial ice and firn are used when converting volumetric changes occurring, respectively, in the ablation and accumulation areas. We assign ρfirn = 600 kg m−3 (Malz et al., 2018 and references within). We used Equilibrium Line Altitude (ELA) values as reported in De Angelis (2014) and Barcaza et al. (2009), respectively, for the gla- ciers on the SPI and NPI. For each group of glaciers (SPI-G1, SPI-G2, NPI), we computed an average ELA from all glaciers with surface area larger than 100 km2 in the given catchment. Glacier outlines over the Ice Fields record their extent in 2000–2001 (RGI Consortium, 2017) and the Upsala and Jorge Montt glaciers (SPI) have retreated considerably since then and, for the latter, even during our study period. Their front positions in 2017 were manually digitized using Landsat8 scenes (Appendix D, Table D1) and their mass loss between 2011 and 2017 is calculated against their updated fronts. Area changes between 2011 and 2017 are not included in our estimates of mass loss. The only exception is Jorge Montt (SPI), which retreated considerably in this time period and for which we provide an additional estimate of mass loss due to area change. 2. Data and methods The front outline of the Jorge Montt and Upsala, as well as of Pio XI (SPI), was additionally digitized for a number of years between 2005 and 2017 (Appendix D, Table D1). This data is used for context in the Discussion (subsections Jorge Montt, Upsala and Pio XI) and is not em- ployed for calculating mass balance. Finally, we produce time series of mean observed glacier elevation change with the same methodology as Gray et al. (2015) and Foresta et al. (2016). The time series are generated at the catchment scale for each of the nine sub-regions with 90 (Pio XI, SPI-G2, SPI) or 120 days time step (Fig. 3). 3. Results Swath processing of CryoSat-2 SARIn data provides 6.7 and 26.6 mil- lion valid observations of ice topography over the NPI and SPI, respec- tively, with the rate of elevation change for a single pixel being constrained by ~1700 elevations (median) over a period of 5.6 years (median) between April 2011 and March 2017. For comparison conventional CS2 POCA de- livers about 17,000 and 55,000 observations over the NPI and SPI re- spectively. Fig. 1 displays the maps of observed rates of elevation change over the Ice Fields. On the SPI, different catchments show distinct patterns of change over the study period (Fig. 1). Given such heterogeneity, we apply the hypsometric averaging model independently for six large glaciers on the SPI, namely Jorge Montt, Viedma, Upsala, Pio XI, Grey and Tyndall. The spatial coverage of ḣ estimates, at 500 m posting, ranges between 61 and 73% of total catchment areas (Table 1), with the exception of Grey and Tyndall (~52%). We combine data from the rest of the SPI in two groups of neighbouring glaciers, labelled SPI-G1 and SPI-G2. The former includes all glaciers north of Pio XI and Viedma excluding Jorge Montt, while the latter is composed of all the glaciers west and south of Upsala excluding Grey and Tyndall (Fig. 1). Over the NPI, all glaciers are analysed together. Time series of mean observed glacier elevation change (Fig. 3) show negative trends for all sub-regions with the exception of Pio XI, which shows increasing elevation. Most sub-regions display a seasonal oscil- lation on the order of 1–3 m. The amplitude is highest (4 m) for the Grey Glacier, while it is less discernible for glaciers with the strongest mass losses per unit area (Jorge Montt and Upsala), possibly reflecting the importance of dynamic thinning also during the accumulation period. 3 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based h elevations. The insets show observed median rates of elevation change (black lines with dots) against cumulative glacier surface area (10% steps), to Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice elevations. The insets show observed median rates of elevation change (black lines with dots) aga uncertainty envelope (grey shade). 3. Results Elevation (non-linear) is also shown for clarity. Fields between April 2011 and March 2017 base ainst cumulative glacier surface area (10% steps) Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March elevations. The insets show observed median rates of elevation change (black lines with dots) against cumulative glacier surface area (1 uncertainty envelope (grey shade). Elevation (non-linear) is also shown for clarity. served rates of surface elevation change of the Northern and Southern Patagonian Ice Fi . The insets show observed median rates of elevation change (black lines with dots) agai ty envelope (grey shade). Elevation (non-linear) is also shown for clarity. Fig. 1. Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based on CS2 L2swath elevations. The insets show observed median rates of elevation change (black lines with dots) against cumulative glacier surface area (10% steps), together with the uncertainty envelope (grey shade). Elevation (non-linear) is also shown for clarity. Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. n change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based ian rates of elevation change (black lines with dots) against elevation (50 m bands), together with the pol vation change (not shown for clarity). The normalized histograms of the distribution of glacier area a inuous line and shaded patch, respectively). Maps of modelled rates of surface elevation change of the Northern and Southern Patagonian Ice Fields be h elevations. The insets show observed median rates of elevation change (black lines with dots) against elev red line) fitted to the original rates of elevation change (not shown for clarity). The normalized histogr e versus elevation are shown in grey (continuous line and shaded patch, respectively). modelled rates of surface elevation change of the Northern and Southern Patagonian Ice ons. The insets show observed median rates of elevation change (black lines with dots) ag ) fitted to the original rates of elevation change (not shown for clarity). The normalize elevation are shown in grey (continuous line and shaded patch, respectively). Maps of modelled rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based th elevations. 4.1. Spatial coverage Furthermore, using a single sensor and frequent repeat measurements is advantageous as it limits penetration biases associated both with using multiple sensor types (e.g. optical vs radar or radar with varying wavelengths) and seasonal variations in surface mass density, thereby limiting the impact on surface elevation change estimates (Jaber et al., 2013; Jaber, 2016; Willis et al., 2012b; Malz et al., 2018). Given the similarity of the Patagonian Ice Fields to mountain glaciers, swath alti- metry may also provide one additional tool for monitoring elevation change over these complex areas (Paul et al., 2015). 4.1. Spatial coverage The Patagonian Ice Fields are a challenging region for radar altimetry. The topography is similar to mountain glaciers, with elevation ranging from sea level to above 2000 m over distances of < 30 km. Furthermore, the flow of most glaciers on the Ice Fields is almost perpendicular to CS2's approxi- mately north-south flight direction so that elevations change abruptly (> 1000 m) over short distances (400 m) along the flight track (Fig. 4), in- creasing the occurrence of loss-of-lock in the altimetric record and leading to gaps in the collected data (Dehecq et al., 2013). Over the Southern Pata- gonian ice field, conventional CS2 POCA altimetry provides about 30% spatial coverage at 500 m posting. Although swath altimetry is affected by loss-of-lock as much as POCA, enhanced spatial coverage is achieved because a swath of heights, rather than one single elevation, is acquired when the on board tracker correctly sets the range window. Swath altimetry thereby provides 61–73% surface coverage over the large (A > 400 km2) glaciers in the northern part of the SPI and between 47% and 54% in other areas (Table 1). The only exception is SPI-G2 (39%), where the ice field is narrower and where there are no observations over a number of glaciers with rela- tively small surface area (Fig. 1 and Fig. 2, inset). Despite the limited extent, their mass loss may be non-negligible. For example HPS12 (south of Pio XI) has an area of 165 km2 and lost 0.63 Gt a−1 between 2000 and 2011/12 (Willis et al., 2012b). In comparison, work based on high resolution radar TanDEM-X DEMs have almost complete coverage at higher spatial resolution (Jaber et al., 2013; Jaber, 2016; Malz et al., 2018), although this data does not allow yet to generating time series of elevation change. Compared to TanDEM-X DEMs, optical ASTER DEMs achieve similarly high spatial Fig. 3. Time series of cumulative mean observed elevation change for the nine sub-regions (Table 1), including the SPI as a whole (grey), in order of in- creasingly negative specific mass balance (top to bottom). coverage for decadal periods, decreasing to 57–73% for the entirety of the Ice Fields over shorter time periods comparable to that in this paper (Willis et al., 2012b). Despite CS2 L2swath's lower spatial coverage, we still capture in detail the various patterns of change. Table 2 Estimates of mass balance Ṁ [Gt a−1] for the NPI and individual sub-regions of the SPI based on CS2 L2swath data at 500 m spatial resolution using two dif- ferent density scenarios (see text). ELA [m] Single density Ṁ [Gt a−1] Dual density Ṁ [Gt a−1] Abs Diff Ṁ [Gt a−1] NPI 1005 −6.79 ± 1.16 −5.67 ± 1.26 1.13 Jorge Montt 930 −2.20 ± 0.38 −1.96 ± 0.41 0.25 Upsala 1170 −2.68 ± 0.40 −2.29 ± 0.46 0.39 Viedma 1260 −2.27 ± 0.36 −1.90 ± 0.40 0.38 SPI G1 1077 −5.07 ± 0.79 −4.17 ± 0.90 0.90 SPI G2 1096 −1.66 ± 1.16 −1.35 ± 1.03 0.31 Tyndall 940 −0.60 ± 0.14 −0.53 ± 0.14 0.07 Grey 980 −0.69 ± 0.23 −0.59 ± 0.20 0.09 Pio XI 930 +0.67 ± 0.29 +0.57 ± 0.25 0.10 NPI + SPI −21.29 ± 1.98 −17.89 ± 2.03 3.40 3. Results The insets show observed median rates of elevation change (black lines with dots) against elevation (50 m bands), together with the poly (red line) fitted to the original rates of elevation change (not shown for clarity). The normalized histograms of the distribution of glacier area an ge versus elevation are shown in grey (continuous line and shaded patch, respectively). ge of the Northern and Southern Patagonian Ice tes of elevation change (black lines with dots) ag h ( h f l i ) Th li Fig. 2. Maps of modelled rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based on CS2 L2swath elevations. The insets show observed median rates of elevation change (black lines with dots) against elevation (50 m bands), together with the polynomial model (red line) fitted to the original rates of elevation change (not shown for clarity). The normalized histograms of the distribution of glacier area and data coverage versus elevation are shown in grey (continuous line and shaded patch, respectively). 5 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Fig. 3. Time series of cumulative mean observed elevation change for the ni ub-regions (Table 1), including the SPI as a whole (grey), in order of i creasingly negative specific mass balance (top to bottom). Table 1 Estimates of mass balance Ṁ [Gt a−1] and specific mass balance [mwe a−1] as well as area A [km2] and spatial coverage [%] of the maps of surface elevation change rates ḣ for the NPI and individual sub-regions of the SPI based on CS2 L2swath data at 500 m spatial resolution. Frontal retreat of Jorge Montt (SPI) amounts to an additional ~0.07 Gt a−1 (see Discussion). A [km2] ḣ Coverage [%] Ṁ [Gt a−1] mḃ [mwe a−1] NPI 4046.4 45.7 −6.79 ± 1.16 −1.68 ± 0.29 Jorge Montt 474.4 68.0 −2.20 ± 0.38 −4.64 ± 0.80 Upsala 863.1 61.3 −2.68 ± 0.40 −3.11 ± 0.46 Viedma 992.3 72.7 −2.27 ± 0.36 −2.29 ± 0.36 SPI G1 3570.1 47.4 −5.07 ± 0.79 −1.42 ± 0.22 SPI G2 4829.5 39.1 −1.66 ± 1.16 −0.34 ± 0.24 Tyndall 332 49.9 −0.60 ± 0.14 −1.81 ± 0.42 Grey 333.3 54.0 −0.69 ± 0.23 −2.07 ± 0.69 Pio XI 1242.6 65.0 +0.67 ± 0.29 +0.54 ± 0.23 SPI total 12,637.2 49.9 −14.50 ± 1.60 −1.15 ± 0.13 NPI + SPI 16,683.6 −21.29 ± 1.98 −1.28 ± 0.12 A [km2] ḣ Coverage [%] Ṁ [Gt a−1] mḃ [mwe a−1] 4.2. Rates of mass change Spatial patterns in the rates of surface elevation change (Figs. 1 and 2) are comparable with those observed over the period 2000–2011/16 (Willis et al., 2012a, 2012b; Jaber et al., 2013; Jaber, 2016; Malz et al., 2018). The 6 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. NPI and the northern part of the SPI (SPI-G1, Jorge Montt, Viedma and Upsala) are thinning very rapidly and account for 89% of the mass loss of the Patagonian Ice Fields between 2011 and 2017 (Table 1). The rate of mass loss of the Patagonian Ice Fields has increased in recent decades (Fig. 5a), with our estimated mass loss rate (21.29 ± 1.98 Gt a−1) being 46% higher than between 1944 and 1996 (Aniya, 1999), 42% higher than between 1975 and 2000 (Rignot et al., 2003) and 24% higher than between 2000 and 2012/14 (Jaber, 2016). However, for the period 2000–2011/12 Willis et al. (2012b) estimate a total rate loss of 24.39 ± 1.20 Gt a−1, comparable to those based on gravimetry data (Chen et al., 2007; Ivins et al., 2011; Jacob et al., 2012; Gardner et al., 2013) but 30% more negative than that of Jaber (2016) (Fig. 5a). GRACE-based estimates rely on model predicted corrections for postglacial rebound and land water storage, which are a large source of uncertainty to the estimated rates of mass loss in Pa- tagonia (e.g. Chen et al., 2007; Jacob et al., 2012). Additionally, mass loss from glaciers and ice fields in the vicinity (Möller and Schneider, 2008; Melkonian et al., 2013; Falaschi et al., 2017) may impact on the estimate since gravimetry methods are always sensitive to mass leakage effects from neighbouring areas (e.g. Sørensen et al., 2017). The disagreement between Willis et al. (2012b) and Jaber (2016) may be related to the 2 m elevation correction applied to the SRTM data by Willis et al. (2012b) in order to account for potential radar penetration through the glacier surface. How- ever, analysis of the SRTM mean backscattering coefficient suggests wet surface conditions on the Ice Fields at the time of the SRTM acquisition (Jaber, 2016), which would be expected to prevent the radar signal from penetrating through the surface (Nilsson et al., 2015b). Additionally, Dussaillant et al. (2018) find that radar penetration over the NPI occurs only above 2900 m elevation, i.e. < 0.75% of the ice field's area. 4.4. Jorge Montt glacier Jorge Montt, a tidewater glacier at the northernmost tip of the SPI, has been retreating since 1898 when it reached its LIA maximum extent (Rivera et al., 2012b). Its recession has been linked to fjord water depth, with periods of stable front positions corresponding to shallow depths and underwater pinning points (Rivera et al., 2012b). Additionally, water temperatures at depth (> 100 m) have been shown to be as high as 8 °C in summer 2012 only 1 km from the glacier front (Moffat, 2014), which may further destabilise the glacier through submarine melting (Straneo and Heimbach, 2013). By 2011 Jorge Montt had retreated almost 20 km w.r.t 1898, with the highest rates of recession between 1990 and 1997 (993 m a−1) occurring when water depths beneath the glacier increased sharply to ~300 m (Rivera et al., 2012b). The recent retreat history of Jorge Montt's reveals a slowdown to about 100–300 m a−1 in the early 2000s, followed by increased retreat after 2009 initiated at a location where bathymetry data reveals the deepest trough in the fjord (Rivera et al., 2012b; Moffat, 2014; Fig. 7). Between 2010 and 2011, Jorge Montt retreated almost 1 km (Rivera et al., 2012b; Rivera et al., 2012b) and calved at a rate of 2.4 km3 a−1, when the terminus was floating (Rivera et al., 2012a). Manual delineation of the glacier front between 2011 and 2017 using Landsat optical data (Fig. 7) indicates that Jorge Montt retreated by an additional ~2.5 km, likely through enhanced calving following retreat into deeper water (Rivera et al., 2012a). Given an average glacier freeboard height of 22 m above sea level at the terminus (Rivera et al., 2012a, 2012b) and width of 1.15 km, the glacier frontal retreat amounts to a mass loss rate of 0.07 Gt a−1 (~3% of the catchment's loss due to thinning, Table 1) between 2011 and 2017. This value is however likely underestimated since the slope of the glacier surface, and thus upglacier thickening, has not been considered. Due to the un- certainty associated with this calculation, and that at least part of the glacier terminus was floating in 2010/11 (Rivera et al., 2012a) and likely during our observational period, we report this loss separately in Table 1 and do not include it in our total estimate of glacier contribution to sea level rise. 4.2. Rates of mass change In absolute value, this correction has a larger impact on the SPI than on the NPI (Willis et al., 2012b), where the estimates from Willis et al. (2012b) and Jaber (2016) differ by only ~10%. Separating the contributions of the Ice Fields (Fig. 5b) shows the dif- ference in the progressive increase in mass loss between the NPI and SPI. Between 2011 and 2017 the NPI's rate of loss (6.79 ± 1.16 Gt a−1) is ~70% more negative compared to the previous decade (about 4 Gt a−1, Willis et al., 2012a, 2012b; Jaber, 2016; Dussaillant et al., 2018), which in turn was ~37% higher than between 1975 and 2000 (Rignot et al., 2003) (Fig. 5b). Compared to the latter, Rivera et al. (2007) record higher rates of mass loss in a similar time period (1979–2001) (Fig. 5b), but their estimate is based on data mostly lying in the ablation zone of the NPI. In contrast, the g. 4. (left panel) Example ascending and descending CS2 sub-satellite tracks (red) displayed over the SRTM topography for the SPI. Glacier outlines from R otted in black. (right panel) Along-track topography (black) for the same sub-satellite tracks (red). For reference, the SPI (light blue) is shown in the ba 7 Fig. 4. (left panel) Example ascending and descending CS2 sub-satellite tracks (red) displayed over the SRTM topography for the SPI. Glacier outlines from RGI v6 are plotted in black. (right panel) Along-track topography (black) for the same sub-satellite tracks (red). For reference, the SPI (light blue) is shown in the background. Fig. 4. (left panel) Example ascending and descending CS2 sub-satellite tracks (red) displayed over the SRTM topography for the SPI. Glacier outlines from RGI v6 are plotted in black. (right panel) Along-track topography (black) for the same sub-satellite tracks (red). For reference, the SPI (light blue) is shown in the background. 7 mass loss over the SPI varies by just 8 10% between these three periods 4 4 Jorge Montt glacier Fig. 5. Estimates of mass balance Ṁ [Gt a−1] for the Patagonian Ice Fields combined (a) and separate (b) as published in the literature as well as calculated in this study. Note that the time line is not linear. The estimate from Gardner et al. (2013) is for the whole of the Southern Andes. In (b) different shades are used for visual clarity. L. Foresta et al. 4.4. Jorge Montt glacier Between 2011 and 2017, Jorge Montt shows the highest mass loss per unit area, 4 times above the average for the SPI as a whole (Table 1). Its absolute rate of mass loss (2.20 ± 0.38 Gt a−1) is comparable to what reported by Jaber (2016) for the period 2011–2014 (2.59 Gt a−1, uncertainty not re- ported), which increased by 50% compared to the 1.72 Gt a−1 (uncertainty not reported) rate of mass loss between 2000 and 2011 (Jaber, 2016). 4.3. Glacier dynamics We observe a sharp transition around 49° S (Figs. 1–2, dashed green line) from intense thinning in the north to a large area facing limited mass loss (SPI-G2), which spans about 4800 km2 or about 40% of the total surface of the SPI (Table 2). This pattern is in agreement with earlier observations between 2000 and 2012/16 (Willis et al., 2012b; Jaber et al., 2013; Jaber, 2016; Malz et al., 2018), therefore suggesting constant behaviour over decadal time scales. The topography of the northern and southern parts of the SPI has different characteristics, with a greater proportion of the northerly ice field lying at lower altitudes. In fact, about 72% of SPI-G1's surface lies below 1500 m elevation, ~12% more than SPI-G2's at the same altitude (Fig. 1, insets SPI-G1 and SPI-G2). The Ice Field also narrows and steepens south of 49° S and even at low elevations the southern SPI shows only moderate thinning (Fig. 6). Glaciers Grey and Tyndall, at the south- eastern tip, are the exception to this pattern. However the latter lies almost entirely below 1500 m altitude (Fig. 1, inset Tyndall) and both glaciers re- ceive scarce precipitation due to their location east of the ice divide. The NPI has similar area-altitude distribution as SPI-G1 (Fig. 1, inset SPI-G1 and NPI) and the two areas show comparable mass losses per unit area (Table 1). However, the northern part of the SPI contains some of the fastest flowing glaciers on the Ice Fields, including Jorge Montt and Upsala (Sakakibara and Sugiyama, 2014; Mouginot and Rignot, 2015) which accelerated significantly (> 500 m a−1) in the period 1984–2000 (Jorge Montt) and 2000–2010 (Upsala), coincident with rapid frontal retreat (Sakakibara and Sugiyama, 2014). These observations confirm the im- portance of dynamics in impacting the overall mass balance of the northern part of the SPI (e.g. Sakakibara and Sugiyama, 2014; Mouginot and Rignot, 2015), where three of the largest glaciers (Jorge Montt, Upsala and Viedma) are thinning very rapidly (Table 1 and Fig. 1–3). 4.2. Rates of mass change Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. i i f b l ̇ [ 1] f h i i ld bi d Fig. 5. Estimates of mass balance Ṁ [Gt a−1] for the Patagonian Ice Fields combined (a) and separate (b) as published in the litera Note that the time line is not linear. The estimate from Gardner et al. (2013) is for the whole of the Southern Andes. In (b) diffe Fig. 5. Estimates of mass balance Ṁ [Gt a−1] for the Patagonian Ice Fields combined (a) and separate (b) as published in the literature as well as calculated in this study. Note that the time line is not linear. The estimate from Gardner et al. (2013) is for the whole of the Southern Andes. In (b) different shades are used for visual clarity. mass loss over the SPI varies by just 8–10% between these three periods (Rignot et al., 2003; Jaber, 2016; Malz et al., 2018) (Fig. 5b) although its rate of loss is still more than twice that of the NPI. Estimates from Jaber (2016) and Malz et al. (2018), both based on comparing TanDEM-X data to the SRTM DEM, differ by 1.29 Gt a−1 although they agree within their uncertainties. The difference may be due to the 4 years longer time period analysed by Malz et al. (2018), who report positive elevation changes in the southernmost part of the SPI between 2011/12 and 2015/16. We observe only a slightly positive trend in elevation change in this time period, fol- lowed by a marked drop after 2015/16 (Fig. 3, SPI-G2). However, our time series for SPI-G2 is representative of an area roughly twice as large than that analysed by Malz et al. (2018) over multiple time periods. Finally, the es- timated SPI's rate loss of 34.83 ± 3.96 Gt a−1 between 1995 and 2000 (Rignot et al., 2003), which is even more negative than any estimate for both Ice Fields combined (Fig. 5a), appears out of line with other values. 4.5. Upsala glacier 20–50% (Sakakibara et al., 2013; Mouginot and Rignot, 2015) and thin- ning at a maximum rate of ~40 m a−1 near the terminus (Sakakibara et al., 2013). The rapid retreat may have been caused by the glacier front reaching an area where the lake depth exceeds 560 m (Sugiyama et al., 2016). In early 2013 the glacier's velocity at the front was 2.9 m d−1 (Moragues et al., 2018), ~33% lower compared to 2008 and more similar to values recorded in the early 2000s (Sakakibara et al., 2013). Moragues et al. (2018) report a doubling in maximum velocity between 2013 and 2014. However this increase is unlikely to have been sustained in time. In fact, between 2011 and 2017, the glacier front has been comparatively stable (Fig. 8), with a retreat rate of ~85 m a−1 similar to the period 2000–2008 (Sakakibara et al., 2013). Coincident with a more stable front position, the average thinning rate within 16 km of the terminus decreased by a factor two from 13.4 m a−1 between 2006 and 2010 (Sakakibara et al., 2013) to 6.2 m a−1 between 2011 and 2017 (Fig. 8). Bertacchi Glacier, a tributary of Upsala, shows a similar pattern with current rates of elevation change decreasing to 8.5 m a−1 (Fig. 8) from ~15 m a−1 be- tween 2008 and 2011 (Sakakibara et al., 2013). We observe maximum thinning rates of ~12 m a−1 5 km from the terminus of Upsala glacier, comparable to estimates at the front in the early 1990s (Naruse et al., 1997). Despite these reduced thinning rates, Upsala remains the glacier with the second most negative specific mass balance in the Patagonian Ice Fields after Jorge Montt, and is the largest single contributor to net mass loss amongst individual glaciers (Table 1 and Figs. 1–3). south and Lake Greve to the north. From 1945 to present, the tidewater terminus advanced ~13 km and is currently at its Neoglacial maximum (Wilson et al., 2016; Fig. 9 and Fig. E1). Looped supraglacial moraines were used to identify up to six surge events since 1926 (Rivera et al., 1997a; Wilson et al., 2016), two of which were concurrent with front retreat, possibly due to enhanced calving at the tidewater terminus (Wilson et al., 2016; Fig. E1). 4.5. Upsala glacier Upsala, a freshwater calving glacier located on the north-eastern side of the SPI and draining into Argentino Lake, has also been retreating since the late 1970s (Naruse et al., 1997). Between 2008 and 2011, retreat rates quadrupled w.r.t the previous 8 years and the glacier retreated by almost 3 km (Sakakibara et al., 2013), while simultaneously speeding up by 8 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Fig. 6. Median observed rates of surface elevation change for the two Ice Fields every tenth of a degree of latitude and for different elevation bands at [0-80 800-1000 m], [1000–1200 m], [1200–1500 m], [1500–1800 m] and [1800–2200 m]. The Pio XI glacier is not included in this analysis due to its anomalou unique behaviour (see Discussion). Note that the scale on the x-axis varies to display the strong thinning at lower elevations. Fig. 6. Median observed rates of surface elevation change for the two Ice Fields every tenth of a degree of latitude and for different elevation bands at [0-800 m], [800-1000 m], [1000–1200 m], [1200–1500 m], [1500–1800 m] and [1800–2200 m]. The Pio XI glacier is not included in this analysis due to its anomalous and unique behaviour (see Discussion). Note that the scale on the x-axis varies to display the strong thinning at lower elevations. Fig. 7. Frontal retreat of Jorge Montt Glacier (SPI). The glacier retreated almost 2 km between 2000 and 2011 and receded a further ~2.5 km in our study period. Water depth at the glacier front was 400 m in 2013. Bathymetry data after Piret et al. (2017). Fig. 7. Frontal retreat of Jorge Montt Glacier (SPI). The glacier retreated almost 2 km between 2000 and 2011 and receded a further ~2.5 km in our study period. Water depth at the glacier front was 400 m in 2013. Bathymetry data after Piret et al. (2017). Fig. 7. Frontal retreat of Jorge Montt Glacier (SPI). The glacier retreated almost 2 km between 2000 and 2011 and receded a further ~2.5 km in our study period. Water depth at the glacier front was 400 m in 2013. Bathymetry data after Piret et al. (2017). 9 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. 4.5. Upsala glacier The glacier has been thickening in the ab- lation area since the late 1970s, with the highest rates recorded at the termini, while the picture is more complicated in the accumulation area where data is sparse (Fig. E1). Between 2011 and 2017, we observe thickening at almost all elevations, by about 2.33 m a−1 and 0.57 m a−1 (median value) in the ablation and accumulation areas respectively (Fig. 1; Pio XI inset) with thinning at the highest elevations above 1500 m altitude; findings which match those described in Jaber (2016). There is however no coverage in our data close to the two termini. Assuming a thickening trend at the two fronts, which was sustained for the last four decades (Rignot et al., 2003; Willis et al., 2012b; Jaber et al., 2013; Jaber, 2016; Wilson et al., 2016), we estimate that the Pio XI glacier is gaining mass at a rate of 0.67 ± 0.29 Gt a−1 between 2011 and 2017 (Table 1 and Fig. E1). However the rate is likely underestimated since the hypsometric averaging model for Pio XI glacier predicts less thickening compared to the ob- servations below 1000 m elevation (Fig. 2, inset). The mass gain is likely a result of complex dynamics associated with both surge mechanisms and terminus calving processes, since Pio XI is the only advancing glacier within the Patagonian Ice Fields and air temperature has increased over the last 50 years (Rasmussen et al., 2007). 5. Conclusions The tidewater (south) and lacustrine (north) termini advanced about 500 m and 8 retreated ~2.5 km between 2011 and 2017, likely by enhanced calving in deeper fjord waters. The only exception to the overall pattern of thinning and retreat is the Pio XI glacier, which continues to advance at both its tidewater and lacustrine termini. The glacier, which is currently at its Neoglacial maximum, is estimated to have gained mass at a rate of 0.67 ± 0.29 Gt a−1 during our study period. spatial and temporal resolution are essential. CS2 swath altimetry provides an important tool for monitoring these rapidly changing areas and quan- tifying their ongoing mass loss. Appendix A. Filtering CS2 SARIn waveform samples Selecting waveform samples based on fixed thresholds on coherence and power is an empirical approach which has been applied successfully to infer glacial topography and higher products based on it such as topography changes (Gray et al., 2013; Christie et al., 2016; Ignéczi et al., 2016; Foresta et al., 2016; Gourmelen et al., 2017a). However, in this paper we use a different procedure to discard noisy waveform samples before performing the phase unwrapping. This method relies entirely on the phase difference field and consists in identifying, within each waveform, groups of consecutive samples which can be modelled by a straight line. The original phase difference is divided into overlapping segments, their length being set to 64 samples and the overlap half of the length. The slope of the phase difference is then calculated independently in each segment. Instead of applying a linear regression, the algorithm applies a Fourier transform on the normalized complex coherence e(i ðɸ), where ðɸ is the phase difference field. Compared to linear regression, this approach is both more efficient computationally as well as independent on phase wrapping. The Fourier transform enables to testing a large number of possible slopes and the one with the highest correlation with the input data is selected. The signal is oversampled to take into account that the slope of CS- 2's phase difference can represent non-integer frequencies. Thus, each overlapping section has two possible slopes. A correlation is applied again to the data in each overlapping section, this time using only its two estimated slope values. Sections whose correlation is below a set threshold (for this work, 0.95) are considered noisy and discarded. Finally, the remaining segments are used to unwrap the phase difference. With this procedure, no smoothing is applied to the phase difference and no threshold is set on the power or coherence. Acknowledgments Between April 2011 and March 2017, the Ice Fields lost mass at a combined rate of 21.29 ± 1.98 Gt a−1 (equivalent to 0.059 ± 0.005 mm a−1 eustatic sea level rise), an increase of 24% and 42% when compared to the periods 2000–2012/14 and 1975–2000, respectively. We find that the NPI (−6.79 ± 1.16 Gt a−1), which is responsible for a third of the total loss, is losing mass 70% faster compared to the first decade of the 21st century. Given the ongoing and rapid wastage of the Patagonian Ice Fields, and their important contribution to the global budget of mass loss from glaciers and ice caps, continuous observations with excellent This work was performed under the European Space Agency's Support to Science Element CryoSat+ Mountain Glacier (contract 4000114224/ 15/I-SBo). L.F. acknowledges a Young Scientist Training grant from the European Space Agency's Dragon3 program. J.J.W. acknowledges a NERC PhD Studentship. The authors wish to thank ESA for providing free access to CryoSat-2 full bit rate data, NASA for providing free access to the SRTM DEM and USGS for providing free access to Landsat data. The authors are grateful to the Editor-in-Chief, Crystal Schaaf, and to two anonymous re- viewers, whose comments have significantly improved the manuscript. 5. Conclusions Pio XI, the largest glacier on the SPI and in South America, is the only glacier of the Patagonian Ice Fields to have experienced a net large ad- vance since 1926 and the only known surge-type glacier on the SPI (Rivera et al., 1997a; Wilson et al., 2016). Published data of frontal changes, ice flow velocity at the termini, elevation change and mass balance, sum- marized in Fig. E1, reveal a complex history (Appendix E). Between 1951 and 1963, the glacier's westward and southward advance dammed a proglacial river originating from Greve glacier to the north, forming the current Lake Greve for at least the second time since 1926 (Rivera et al., 1997a). Since then, the glacier has been terminating in Ejre Fjord to the CryoSat-2 swath radar altimetry is employed successfully to map ele- vation change over the Patagonian Ice Fields at sub-kilometer spatial re- solution. Despite the challenging topography, similar to that of mountain glaciers, the technique can be used to observe changes over individual glaciers or catchments with an area as small as 300 km2. The northern part of the SPI displays a high degree of complexity, although most of the area is thinning at all elevations, with Jorge Montt, Viedma and Upsala glaciers losing mass at rates higher than 2 Gt a−1. Jorge Montt additionally Fig 8 Front location of Upsala glacier (SPI) The glacier receded about 500 m between 2011 and 2017 Thinning rates from this study are also Fig. 8. Front location of Upsala glacier (SPI). The glacier receded about 500 m between 2011 and 2017. Thinning rates from this study are also shown in the range −10 to 0 m a−1. Fig. 8. Front location of Upsala glacier (SPI). The glacier receded about 500 m between 2011 and 2017. Thinning rates from this study are also shown in the range −10 to 0 m a−1. 10 L. Foresta et al. Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx Fig. 9. Front location of Pio XI glacier (SPI). The tidewater (south) and lacustrine (north) termini advanced about 500 m and 800 m, respectively, between 2011 and 2017. Fig. 9. Front location of Pio XI glacier (SPI). Appendix B. SRTM DEM correction A number of freely available DEMs covering Southern Patagonia exist, namely: the SRTM (i) C and (ii) X band DEMs (Farr et al., 2007), the (iii) ASTER (Advanced Spaceborne Thermal Emission and Reflection Radiometer) GDEM2 (Tachikawa et al., 2011) and (iv) the ALOS (Advanced Land 11 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Observing Satellite) AW3D30 v1.1 (Tadono et al., 2014; Takaku et al., 2014; Tadono et al., 2016; Takaku et al., 2016). The latter is the most recent, but has large gaps over the SPI, which are filled using the SRTM C band DEM. Version 1 of the ASTER GDEM was known to be affected by large artefacts (Arefiand Reinartz, 2011) and, despite large overall improvements, version 2 still has high frequency noise, particularly over glacial terrain (Meyer et al., 2011). Visual comparison between the SRTM C band and ASTER GDEM2 DEMs shows evident noise in the latter, with differences at times on the order of tens of meters between neighbouring pixels. Finally, the SRTM X band DEM does not have complete coverage and gaps over the Ice Fields are significant. Therefore, we used the SRTM C band DEM as a reference for elevation, which fully covers the Patagonian Ice Fields, resampled at 300 m posting and referenced to the WGS84 vertical datum. The down-sampling of the DEM is mostly dictated by achieving a satisfactory performance in computing time while keeping the spatial resolution somewhat comparable to that of an individual elevation based on CryoSat-2 interferometric data (300 m in the along-track direction). We use linear interpolation when querying the DEM. The SRTM DEM is based on data acquired in February 2000, and a few areas at the margins of the SPI have thinned by at least 80 m since then (Willis et al., 2012b; Jaber et al., 2013; Jaber, 2016). This magnitude is comparable to the elevation offset caused by a 2π shift on CS2's phase (Data and methods, this paper; Fig. 3 in Gourmelen et al., 2017a, 2017b). Thus, over areas which experienced intense thinning rates, the swath algorithm may select an incorrect 2π multiple which best matches current observations to the glacier topography from 2000. In order to avoid that, the SRTM DEM needs to be registered to the beginning of our study period. Appendix B. SRTM DEM correction To this purpose, we applied a first order correction of the SRTM DEM based on a visual inspection of results in Willis et al. (2012b) and assuming constant rates of elevation change between the SRTM and CS2 periods. This approach was sufficient to improve the phase unwrapping, leading to further pixels meeting the quality criteria for robust rates of surface elevation change. We refer to this corrected DEM simply as the SRTM DEM in this study. The correction of the SRTM DEM only affects the terminus areas of Jorge Montt and Upsala glaciers (SPI) since there are no CS2 swath altimetry observations over smaller glaciers on the Ice Fields which experienced similar thinning rates in the period 2000–2011/12 (e.g. HPS12, SPI; Willis et al., 2012b; Jaber et al., 2013; Jaber, 2016). Appendix C. Error budget The errors on the mass balance estimates are calculated as in Foresta et al. (2016). The uncertainties εḣ on the observed rates of elevation change for the individual pixels, extracted from the model covariance matrix (see Data and methods), are propagated when applying the hypsometric averaging method: = ∑ = E k ε m N k ( ) ( ) ( .) h m N k h ̇ 1 ( ) ̇ 2 where Eḣ(k) is the elevation change error in elevation band k and N(k) is the number of valid observations in the elevation band. A two-term decreasing exponential is used to interpolate values for elevation bands with no observations (e.g. at low elevation for bands with limited spatial extent). We multiply the area extent A(k) of the elevation band to the related Eḣ(k) and sum all contributions to estimate the total uncertainty on the rate of volume change: where Eḣ(k) is the elevation change error in elevation band k and N(k) is the number of valid observations in the elevation band. A two-term decreasing exponential is used to interpolate values for elevation bands with no observations (e.g. at low elevation for bands with limited spatial extent). We multiply the area extent A(k) of the elevation band to the related Eḣ(k) and sum all contributions to estimate the total uncertainty on the rate of volume change: ∑ = ∗ E E k A k ( ) ( ). V k h ̇ ̇ With this method, the volume change uncertainty is only related to that area of the ice cap where there are valid rates of surface elevation change, but does not account for incomplete data coverage. The volume change uncertainty is therefore rescaled according to the data coverage (Table 1). This procedure generates a rather conservative (i.e. larger) error estimate since it assumes that the lack in data coverage has a direct impact on the total error estimate, which does not hold if the sampling is sufficiently uniform. Finally, we include an error on the density: = − E ρ ρ 1 2 ( ) ρ ice firn when converting volume to mass change (e.g. Nilsson et al., 2015a). when converting volume to mass change (e.g. Nilsson et al., 2015a). Appendix D. Landsat scenes Appendix D. Landsat scenes Table D1 List of Landsat scenes used to manually delineate the front positions of glaciers Jorge Montt, Upsala and Pio XI (SPI). 1 andsat scenes used to manually delineate the front positions of glaciers Jorge Montt, Upsala and Pio XI (SPI). y p g g , p ( ) Jorge Montt 2005 LT52310942005050COA00 2009 LE72320942009156EDC00 2010 LE72310942010088COA00 2011 LE72320942011050EDC00 2013 LE72320942013087ASN00 2014 LC82310942014075LGN00 2015 LC82320942015021LGN00 2016 LC82320942016072LGN00 2017 LC82320942017106LGN00 Upsala 2011 LE72310952011123EDC00 2017 LC82310952017035LGN00 Pio XI 2011 LE07_L1TP_232094_20110219_20161210_01_T1 2014 LC08_L1TP_231094_20140401_20170424_01_T1 2017 LC08_L1TP_231094_20170204_20170216_01_T1 12 Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx L. Foresta et al. Appendix E. Pio XI glacier (SPI): summary of published data Fig. E1 summarises published data of frontal changes, ice flow velocity at the fronts, elevation change and mass balance (Rivera et al., 1997a; Rivera et al., 1997b; Rivera and Casassa, 1999; Rignot et al., 2003; Lopez et al., 2010; Willis et al., 2012b; Jaber et al., 2013; Sakakibara and Sugiyama, 2014; Wilson et al., 2016). Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; (c) mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et al., 2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. Appendix E. Pio XI glacier (SPI): summary of published data Fig. E1 summarises published data of frontal changes, ice flow velocity at the fronts, elevation change and mass balance (Rivera et al., 1997 Rivera et al., 1997b; Rivera and Casassa, 1999; Rignot et al., 2003; Lopez et al., 2010; Willis et al., 2012b; Jaber et al., 2013; Sakakibara an Sugiyama, 2014; Wilson et al., 2016). Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; ( mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et a 2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. Appendix E. Pio XI glacier (SPI): summary of published data Appendix E. Pio XI glacier (SPI): summary of published data Appendix E. Appendix D. 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Pio XI glacier (SPI): summary of published data Fig. E1 summarises published data of frontal changes, ice flow velocity at the fronts, elevation change and mass balance (Rivera et al., 1997a; Rivera et al., 1997b; Rivera and Casassa, 1999; Rignot et al., 2003; Lopez et al., 2010; Willis et al., 2012b; Jaber et al., 2013; Sakakibara and Sugiyama, 2014; Wilson et al., 2016). Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; (c mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et al 2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; (c) mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et al., 2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. 13 13 L. Foresta et al. Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx References Rem ote Sensing of Environm ent xxx (xxxx) xxx–xxx Rivera, A., Lange, H., Aravena, J.C., Casassa, G., 1997b. The 20th-century advance of Glaciar Pio XI, Chilean Patagonia. Ann. Glaciol. 24, 66–71. p Takaku, J., Tadono, T., Tsutsui, K., 2014. Generation of high resolution global DSM from ALOS PRISM. International archives of the photogrammetry. Remote. Sens. Spat. Inf. Sci. 40 (4), 243–248. http://dx.doi.org/10.5194/isprsarchives-XL-4-243-2014. Rivera, A., Lange, H., Aravena, J.C., Casassa, G., 1997b. 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Frequency of osteoradionecrosis of the lower jaw after radiotherapy of oral cancer patients correlated with dosimetric parameters and other risk factors
Head & face medicine
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RESEARCH Open Access Lang et al. Head & Face Medicine (2022) 18:7 https://doi.org/10.1186/s13005-022-00311-8 Lang et al. Head & Face Medicine (2022) 18:7 https://doi.org/10.1186/s13005-022-00311-8 * Correspondence: kristin.lang@med.uni-heidelberg.de Co espo de ce: st . a g@ ed.u e de be g.de 1Department of Radiation Oncology, University Hospital of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany 2Heidelberg Institute of Radiation Oncology (HIRO), 69120 Heidelberg, Germany y Full list of author information is available at the end of the article Abstract Objectives: Osteoradionecrosis (ORN) of the lower jaw is a serious late complication after radiotherapy in patients with oral cavity cancer. The aim of this study is to generate more insight into which patient- and treatment-related factors are associated with the development of ORN in oral cavity cancer patients undergoing postoperative radiotherapy. Material and methods: Retrospective evaluation and comparison of 44 patients with ORN (event group 1) matched according to 45 patients without ORN (control group 2) who received postoperative radiotherapy of oral cavity squamous cell carcinoma at our institution between 2012 and 2020. Dosimetric factors that favor the occurrence of ORN should be detected. The cumulative occurrence rate of ORN was calculated according to the Kaplan–Meier method and analyzed by Cox regression and log-rank test. Results: The median time to develop ORN was 18 months (3–93 months) after radiotherapy. Dental status before radiotherapy (RT) treatment (HR 4.5; 1.8–11.5) and dosimetric parameters including Dmean > 45 Gy (HR 2.4; 1.0–5.7), Dmax > 60 Gy (HR 1.3; 1.1–2.8) and planning target volume (PTV) proportion > 40% intersection with the lower jaw (HR 1.1; 1.0–1.1) were significantly associated with ORN. Conclusion: The results of this retrospective study reveal that oral cavity cancer patients who underwent pre-RT dental surgery as well as dosimetric parameters using Dmax > 60 Gy, higher mean doses > 45 Gy and more than 40% PTV intersection with the lower jaw bone are independent risk factors for ORN. These findings can assist in the management of patients undergoing RT for head and neck cancer regarding ORN prevention. Frequency of osteoradionecrosis of the lower jaw after radiotherapy of oral cancer patients correlated with dosimetric parameters and other risk factors Kristin Lang1,2*, Thomas Held1,2, Eva Meixner1,2, Eric Tonndorf-Martini1,2, Oliver Ristow3, Julius Moratin3, Nina Bougatf1,2, Christian Freudlsperger3, Jürgen Debus1,2,4,5 and Sebastian Adeberg1,2,4,5 © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://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 7 (2022) 18:7 Lang et al. Head & Face Medicine Lang et al. Head & Face Medicine (2022) 18:7 Clinical relevance: Poor oral hygiene and desolate dental status as well as high radiation doses to the mandibular bone significantly increase the risk of developing osteoradionecrosis. Before irradiating a patient with oral cavity cancer, an appointment with the dentist should be made and teeth sanitized if necessary. Likewise, maximum radiation doses to the lower jaw should be minimized. Keywords: Desolate dental status, Osteoradionecrosis, Radiotherapy, Mandibular bone To ensure comparability, and in particular to be able to detect risk factors, patients were divided into two equal groups: those who developed ORN (44 patients, group 1) and those who did not develop ORN (45 patients, group 2). We collected basic patient and treatment data from the National Tumor Center Heidelberg Cancer Registry and imported them into our HIRO Research Database for this study [16]. Inclusion criteria were as follows: histologically squamous cell carcinoma, follow- up duration of at least 3 months after completion of RT, no interruptions during RT, regular follow-up examina- tions including computed tomography (CT) examina- tions every 3 months in the first 2 years after RT as standard clinical practice for all head and neck cancer patients, every 6 months in years 3 and 4 after RT and once a year in years 5 and 6, as well as regular head and neck examinations at the Department of Oral and Max- illofacial Surgery and the availability of sufficient RT treatment plan data to evaluate the dose to the lower jaw bone. Exclusion criteria were patients with follow-up of less than 3 months as well as patients with other hist- ology and patients with cancer outside the oral cavity. Treatment features f Before treatment, patients generally underwent pre-RT dental evaluation and pre-RT dental treatment (from at- tempts to preserve teeth to extraction of teeth not worth preserving), as deemed appropriate by the oral and max- illofacial surgeons, based on risk assessment. The pre- RT dental treatment because of poor dental status was done 2 weeks before the start of radiation. A desolate dental status was defined in our sample as follows: peri- odontal disease, carious changes in more than 5 teeth, attacked gums or exposed tooth necks. In our sample, patients with caries deep into tooth pulpa were involved. There was no special grading system used for degree of carious changes. For periodontal status the oral and maxillofacial surgeons used periodontal risk assessment (RPA), which includes number of teeth and implants, number of missing teeth, percentage of alveolar bone loss and number of periodontal pockets with probing depths > 5 mm. Introduction Oral cancer is sensitive to radiation and is standard treatment either in definitive intention or in the case of pathologic risk factors (positive resection margin, posi- tive lymph nodes, locally advanced disease) as postopera- tive intention. Osteoradionecrosis (ORN) is a feared late complication of radiotherapy (RT) of oral cancer pa- tients which affects mostly the lower jaw between 2 and 22% [1–3]. The incidence of ORN is about 4–8% [4–6]. ORN is defined as exposed irradiated bone that fails to heal over a period of 3 months without any evidence of persisting or recurrent tumor [7, 8]. Radiological evi- dence of bone necrosis within the target volume is also important for diagnosis and classification [9]. Analysis of epidemiological studies of ORN does not offer accurate data about incidence and prevalence of ORN in the jaws because of inconsistencies in the length of follow-up be- tween studies and limited data from prospective studies. Different treatment-, tumor- and patient-related risk fac- tors of ORN have been reported: size and site of the tumor, age, total RT dose, treatment technique (3D-con- formal therapy (3D-CRT) vs. intensity-modulated radio- therapy (IMRT)), dose volume histogram (DVH) parameters, dose per fraction, injury or dental extrac- tions, alcohol and tobacco abuse, tumor size or stage, as- sociation of the tumor with bone and dental hygiene [10–14]. The international standard radiation technique for patients treated for oral cancer is IMRT, primarily aimed at reducing dosage at the major salivary glands in the oral cavity. Therefore, it is not uncommon to achieve high-dose gradients across the lower jaw bone [15]. All patients treated with RT in the oral cavity underwent pre-RT dental evaluation and pre-RT care if necessary according to current uniform policies. The aim of this study is to generate more insight into which patient- and treatment-related factors are associated with the de- velopment of ORN in oral cavity cancer patients under- going postoperative RT and to predict which patients are at higher risk of developing ORN. Material and methods Data from 89 patients who received postoperative RT to the oral cavity between 2012 and 2020 at the University Hospital of Heidelberg were reviewed for this retrospect- ive analysis. Patient data were homogenized by selecting the same tumor stage and only patients with oral cancer. Patients were treated either with IMRT or 3D-CRT. CT simulation was performed in patients immobilized using a thermoplastic mask. Target volume definition was performed in accordance with current guidelines Page 3 of 7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine Page 3 of 7 [17, 18]. Our target definition was based on preoperative and postoperative CT and magnetic resonance imaging (MRI) scans and included the primary tumor region initial gross tumor volume (iGTV) according to the International Commission on Radiation Units and Measurements definition [19]. The planning target volume (PTV) was created by expansion of the clin- ical target volume (CTV) with a 3–5 mm uniform margin. The lymphatic CTV encompasses the patho- logic lymph nodes (pN+) as well as any adjacent re- gions at risk of tumor spread. The lower jaw was contoured for each plan, and DVHs were retrospect- ively reviewed as RT dosimetric parameters. The max- imum dose to the lower jaw (Dmax), mean dose to the lower jaw (Dmean) and PTV correlation to lower jaw were reviewed. The main treatment features are listed in Table 1. Definit Variou curren The m fails to persist surger Treatm Toxici focus mon T criteri Huma asymp interv medic Table 1 Characteristics of 44 patients with osteoradionecrosis (group 1) an postoperative radiotherapy Group 1 (%) Median age at RT in years (range) 70.5 (39–94) Gender Male 35 (79.5) Female 9 (20.5) T-stage 1–2 28 (63.6) 3–4 21 (47.7) Desolate dental status (caries, periodontal disease) 36 (81.8) Need for pre-RT dental treatment 31 (70.5) Tooth protection during RT 41 (93.2) Systemic therapy CHT 22 (50.0) IT 8 (18.2) none 14 (31.8) Smoking history yes 29 (65.9) no 15 (34.1) RT technique 3D-CRT 7 (15.9) IMRT 37 (84.1) Dmean to mandible < 45 Gy 25 (56.8) Definition of ORN [17, 18]. Our target definition was based on preoperative and postoperative CT and magnetic resonance imaging (MRI) scans and included the primary tumor region initial gross tumor volume (iGTV) according to the International Commission on Radiation Units and Measurements definition [19]. The planning target volume (PTV) was created by expansion of the clin- ical target volume (CTV) with a 3–5 mm uniform margin. The lymphatic CTV encompasses the patho- logic lymph nodes (pN+) as well as any adjacent re- gions at risk of tumor spread. The lower jaw was contoured for each plan, and DVHs were retrospect- ively reviewed as RT dosimetric parameters. The max- imum dose to the lower jaw (Dmax), mean dose to the lower jaw (Dmean) and PTV correlation to lower jaw were reviewed. The main treatment features are listed in Table 1. Various definitions of ORN have been proposed, but no current accepted standard of classification or grading exists. The most prevalent definition is exposed bone after RT that fails to heal over a period of 6 months without evidence of persisting or recurrent tumor and required drug therapy or surgery with radical mandibulectomy [20]. Treatment toxicity Toxicity was evaluated in this retrospective analysis with focus on ORN and was described according to the Com- mon Terminology Criteria for Adverse Events (CTCAE) criteria (version 5.0, U.S. Department of Health and Human Services, Washington, DC, USA): grade 1: asymptomatic; clinical or diagnostic observations only; intervention not indicated; grade 2: symptomatic; medical intervention indicated (e.g., topical agents); Table 1 Characteristics of 44 patients with osteoradionecrosis (group 1) and 45 patients in control group (group 2) after postoperative radiotherapy Group 1 (%) Group 2 (%) p-value Median age at RT in years (range) 70.5 (39–94) 71 (42–88) 0.886 Gender Male 35 (79.5) 37 (82.2) 0.124 Female 9 (20.5) 8 (17.8) T-stage 1–2 28 (63.6) 32 (71.8) 0.086 3–4 21 (47.7) 13 (28.9) Desolate dental status (caries, periodontal disease) 36 (81.8) 17 (37.7) 0.228 Need for pre-RT dental treatment 31 (70.5) 12 (26.7) 0.001 Tooth protection during RT 41 (93.2) 40 (88.9) 0.303 Systemic therapy CHT 22 (50.0) 28 (62.2) 0.475 IT 8 (18.2) 1 (2.2) none 14 (31.8) 16 (35.5) Smoking history yes 29 (65.9) 19 (42.2) 0.453 no 15 (34.1) 26 (57.7) RT technique 3D-CRT 7 (15.9) 2 (4.4) 0.519 IMRT 37 (84.1) 43 (95.6) Dmean to mandible < 45 Gy 25 (56.8) 41 (91.1) 0.023 > 45 Gy 19 (43.2) 4 (8.9) Dmax of mandible < 50 Gy 2 (4.5) 10 (22.2) 0.033 51–60 Gy 22 (50.0) 19 (42.2) > 60 Gy 20 (45.5) 16 (35.5) PTV dimension (ccm) (range) 804 (68–4838) 796 (170–1389) 0.028 ristics of 44 patients with osteoradionecrosis (group 1) and 45 patients in control group (group 2) after th Lang et al. Head & Face Medicine (2022) 18:7 Page 4 of 7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine was no correlation between dental intervention before RT and occurrence of ORN detected. limitations in the performance of instrumental activ- ities of daily living (ADLs); grade 3: severe symptoms; limitations in the performance of self-care ADLs; elective operative intervention indicated; grade 4: life- threatening consequences; urgent intervention indi- cated; and grade 5: death [21]. This retrospective study included all patients who had medical interven- tion for ORN. Group 1 p The median patient age at the time of RT was 71 years (range 39–94 years); 35 patients (79.5%) were male and 9 (20.5%) were female. Pre-RT dental treatment was ne- cessary in 31 patients (70.5%), with half of them having 3 or more teeth removed. At the start of radiation treat- ment, significantly more current smokers (53%) had den- tal extraction compared with former smokers (28%) or never smokers (34%) (p = 0.003). Systemic therapy was used simultaneously with RT in 30 patients. The major- ity of patients were treated with IMRT (84.1%), and only 7 patients received 3D-CRT. Statistical analysis Univariate and multivariate Cox proportional hazard models were used to assess patient- and treatment-related factors associated with the development of ORN. Factors with statistical significance in the univariate analysis were included in the multivariate analysis. The cumulative inci- dence was estimated using the Kaplan–Meier method, and the log-rank test was used to compare cumulative in- cidence curves. The follow-up time was calculated from the last date of RT until the most recent follow-up visit at our institution or the date of death. The time to ORN de- velopment was calculated from the last date of RT until the date of ORN occurrence. The Mann–Whitney U test was used to compare patients in terms of the presence of ORN, RT technique and DVH parameters. Cut-off points for DVH parameters were selected. All statistical analyses were performed using IBM SPSS software version 24. P- values of 0.05 or less were considered statistically significant. Dmean to the lower jaw was 40.5 Gy (range 21–62 Gy), Dmax was 59 Gy (49–75 Gy), and the mean PTV dimen- sion was 804 ccm (range 68–4838 ccm), respectively. Group 2 The median patient age at the time of RT was 71 years (range 42–88 years); 37 patients (82.2%) were male and 8 (17.8%) were female. Pre-RT dental treatment before radiotherapy was performed in 12 patients (26.7%). Sys- temic therapy was used simultaneously with RT in 29 patients. The majority of patients were treated with IMRT (95.6%), and only 1 patient received 3D-CRT. Dmean to the lower jaw was 26.0 Gy (range 12.0–67.0 Gy), Dmax was 56.0 Gy (32.0–77.0 Gy), and the mean PTV dimension was 796 ccm (range 170–1389 ccm), respectively. Results p y All tested dosimetric variables were mutually related and associated with the risk of ORN. Patients with ORN had a significantly higher Dmean, Dmax and PTV than the control group: 40.5 Gy vs. 26.0 Gy (p = 0.043), 59.0 Gy vs. 56.0 Gy (p = 0.033) and 804 ccm vs. 796 ccm (p = 0.028). Statistically significant differences in univariate analyses were noted for Dmean > 45 Gy, Dmax > 60 Gy, PTV touch- ing > 40% lower jaw as well as the need for pre-RT den- tal therapy due to potential focus teeth. In the multivariate analysis pre-RT dental surgery (HR 4.5; 1.8–11.5) and dosimetric parameters including Dmean > 45 Gy (HR 2.4; 1.0–5.7), Dmax > 60 Gy (HR 1.3; 1.1–2.8) and PTV > 40% touching lower jaw (HR 1.1; 1.0–1.1) were also significantly associated with ORN. Results of univariable and multivariable analysis are shown in Table 2. Results are shown according to presence of ORN (group 1: with ORN; group 2: without ORN). Patient and treat- ment characteristics are summarized in Table 1. In both groups there were five patients with desolate dental sta- tus included who declined pre-RT dental treatment as preferred. An example for development of osteoradione- crosis is shown in Fig. 1. The follow-up time for the whole cohort was 28 months, and the median time to develop ORN was 18 months (3–93 months). In total, 78% of 44 ORN sites were located in the body of the jaw, and 22% were lo- cated in the angle or ramus. CT scans were evaluated for all ORN sites, and cortical erosion was observed in 73% and loss of spongiosa trabeculation in 27%. Of the 44 patients with ORN, 20 (45.5%) had CTCAE grade 2, 17 (38.6%) had grade 3 and 7 (15.9%) had grade 4. All patients received initial anti-infective therapy with chlor- hexidine rinsing (0.2% chlorhexidine solution; GlaxoS- mithKline Consumer Healthcare GmbH & Co. KG) and antibiotic treatment, followed by surgical intervention in the infection-free interval. In the time between comple- tion of RT and detection of ORN, no patient had any other dental intervention. For the whole cohort there Discussion ORN of the lower jaw is known to be a chronic late com- plication of RT in patients with oral cavity cancer, and many studies have reported its incidence. Incidences in several recent studies range between 4 and 8% [4–6, 15], and there has been a decrease over the past few decades Page 5 of 7 (2022) 18:7 Lang et al. Head & Face Medicine Fig. 1 The images a–d show the planned CT in one patient with oral cavity cancer undergoing postoperative radiotherapy, including PTV that was planned to receive a total dose of 66.0 Gy (single dose 2.0 Gy) in 33 fractions. To the top right image b shows the ORN of the lower jaw (colored in green) in the high-dose region with maximum dose 68.7 Gy. Therefore, we matched the follow-up CT with the planned CT and dose distribution, and then volumetric and dosimetric evaluations were performed Fig. 1 The images a–d show the planned CT in one patient with oral cavity cancer undergoing postoperative radiotherapy, including PTV that was planned to receive a total dose of 66.0 Gy (single dose 2.0 Gy) in 33 fractions. To the top right image b shows the ORN of the lower jaw (colored in green) in the high-dose region with maximum dose 68.7 Gy. Therefore, we matched the follow-up CT with the planned CT and dose distribution, and then volumetric and dosimetric evaluations were performed 24]. Nabil et al. reported that a total dose above 60 Gy was a significant parameter for ORN [12]. since more conformal techniques (IMRT) have become available, as demonstrated by Studer et al. [15] and Ben- David et al. [10]. Also convincing are recent studies which clarify dosimetric prognostic factors for the incidence and severity of ORN. In our analysis, dosimetric parameters showed signifi- cant correlation with the development of ORN as seen with significant values in group 1: patients treated with Dmax to lower jaw higher than 60 Gy (45.5% vs. 35.5%, p = 0.033), Dmean more than 45 Gy (43.25 vs. 8.9%, p = 0.023) and PTV intersection of more than 40% of the lower jaw bone. All patients with ORN had hot spots in the region of ORN. Discussion Dosimetric evaluation of Dmean in our study may reflect a general damaging mechanism in the lower jaw, and from this study, Dmean seems an ap- propriate parameter to consider in dose planning and be included without a threshold. Doses for the lower jaw Lee et al. and Niewald et al. reported on the dose–ef- fect relationship in patients with oral cavity cancer. The frequency of ORN in their analyses was 6.6% [22] and 8.6% [23] in a median time of 2 years [22, 23]; in our analysis the follow-up duration was significantly lower for some patients. Higher radiation doses were signifi- cantly associated with the development of ORN [22, 23]. Lee et al. found that a cumulative total dose of > 54 Gy was a significant factor in the development of ORN [22, Lang et al. Head & Face Medicine (202 Page 6 of 7 (2022) 18:7 Table 2 Univariable and multivariate analysis of risk factors for ORN HR 95% CI p-value Univariate analysis Gender (male vs. female) 1.837 0.847–2.984 0.124 Age 1.002 0.974–1.031 0.886 T-stage 1.320 0.962–1.813 0.086 Poor dental status (caries, periodontal disease) 1.618 0.740–3.537 0.228 Pre-RT dental treatment 4.822 2.055–11.316 0.001 Smoking history 1.279 0.672–2.435 0.453 Dmean to mandible 1.950 1.770–2.170 0.043 Dmax to mandible 1.492 1.257–2.943 0.033 Concomitant systemic therapy 1.163 0.769–1.759 0.475 PTV dimension 1.001 1.000–1.003 0.028 Multivariate analysis Pre-RT dental treatment 4.567 1.817–11.477 0.001 Dmean to mandible 2.421 1.023–5.728 0.044 Dmax to mandible 1.334 1.135–2.827 0.018 PTV dimension 1.001 1.000–1.003 0.046 Further prospective studies are needed to further dis- sect the dental status and evaluate the infectivity of the focus teeth and the relationship of dental treatment and time interval with radiation onset. However, even if we assume that radiation contributes to the development of ORN at this dose constraint, there might be other con- tributory factors. The final goal would be to optimize radiation treatment plans with adaptive treatment plan- ning and offer patients appropriate counseling for dental management to decrease the risk of ORN. bone should be kept as low as possible to reduce the risk of ORN in oral cavity cancer patients. Our general dose constraint (Dmax) for the lower jaw is 72 Gy or as low as achievable, although we do not compromise tumor coverage to achieve this value. The median time to develop ORN in our study was 18 months. Discussion This is in line with other studies [6, 8, 12, 14, 25]. In a recent study of our institution there was a rate of ORN in 5.8% of patients detected [25]. An appropriate follow-up time was ensured in this retrospective study, and we include cases (group 1) and controls (group 2) for better correlation with factors leading to ORN in pa- tients undergoing postoperative radiotherapy. Therefore, all patients had routine follow-up at our department as well as at the Department of Oral and Maxillofacial Sur- gery in Heidelberg. Our retrospective study showed that poor dental status which entails pre-RT dental treatment was significantly correlated with the development of ORN (70.5% vs. 26.7%). This is in line with previous studies [26]. Acknowledgements Not applicable. Authors’ contributions Kristin Lang: First author, search on the electronic databases, triaged by title, abstract and full text, acquisition, analysis or interpretation of data and statistics. Thomas Held, Eva Meixner, Eric Tonndorf-Martini, Oliver Ristow, Julius Moratin, Nina Bougatf, Christian Freudlsperger, Jürgen Debus, Sebastian Adeberg: Critical revision of the article. All authors read and approved the final version of this manuscript. Conclusion The results of this retrospective study reveal that oral cavity cancer patients who underwent pre-RT dental surgery as well as dosimetric parameters using Dmax > 60 Gy, Dmean > 45 Gy and more than 40% of the PTV touching the lower jaw bone are independent risk factors for the development of ORN. These findings can assist in the management of patients undergoing RT for head and neck cancer regarding ORN prevention. A larger study with focus on dental status is planned. The limitation of this study is its retrospective na- ture, which led to a shortage of necessary data on some single cases. However, we were able to retrieve follow-up data covering a lengthy time period for all patients. The power of this study is that we were able to show—in a dedicated sample of oral cavity cancer patients undergoing radiotherapy—risk factors for de- velopment of ORN: high risk was associated with poor dental status before beginning RT and with dosimetric parameters using Dmax > 60 Gy, higher mean doses > 45 Gy and more than 40% of the PTV touching the lower jaw bone. Acknowledgements Not applicable. Acknowledgements Kristin Lang: First author, search on the electronic databases, triaged by title, abstract and full text, acquisition, analysis or interpretation of data and statistics. Thomas Held, Eva Meixner, Eric Tonndorf-Martini, Oliver Ristow, Julius Moratin, Nina Bougatf, Christian Freudlsperger, Jürgen Debus, Sebastian Adeberg: Critical revision of the article. All authors read and approved the final version of this manuscript. References 1. Store G, Boysen M. Mandibular osteoradionecrosis: clinical behaviour and diagnostic aspects. 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Ann N Y Acad Sci. 2011;1218(1):47–54. https://doi.org/10.1111/j.1749-6632.2010.05771.x. 14. Chen JA, Wang CC, Wong YK, Wang CP, Jiang RS, Lin JC, et al. Osteoradionecrosis of mandible bone in patients with oral cancer-- associated factors and treatment outcomes. Head Neck. 2016;38(5):762–8. https://doi.org/10.1002/hed.23949. Funding h 10. Ben-David MA, Diamante M, Radawski JD, Vineberg KA, Stroup C, Murdoch- Kinch CA, et al. Lack of osteoradionecrosis of the mandible after intensity- modulated radiotherapy for head and neck cancer: likely contributions of both dental care and improved dose distributions. Int J Radiat Oncol Biol Phys. 2007;68(2):396–402. https://doi.org/10.1016/j.ijrobp.2006.11.059. g This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Open Access funding enabled and organized by Projekt DEAL. Author details 1 1Department of Radiation Oncology, University Hospital of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany. 2Heidelberg Institute of Radiation Oncology (HIRO), 69120 Heidelberg, Germany. 3Department of Oral and Maxillofacial Surgery, University Hospital Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany. 4Heidelberg Ion Therapy Center (HIT), Im Neuenheimer Feld 450, 69120 Heidelberg, Germany. 5Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. 1Department of Radiation Oncology, University Hospital of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany. 2Heidelberg Institute of Radiation Oncology (HIRO), 69120 Heidelberg, Germany. 3Department of Oral and Maxillofacial Surgery, University Hospital Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany. 4Heidelberg Ion Therapy Center (HIT), 17. Gregoire V, et al. Delineation of the primary tumour clinical target volumes (CTV-P) in laryngeal, hypopharyngeal, oropharyngeal and oral cavity squamous cell carcinoma: AIRO, CACA, DAHANCA, EORTC, GEORCC, GORTEC, HKNPCSG, HNCIG, IAG-KHT, LPRHHT, NCIC CTG, NCRI, NRG oncology, PHNS, SBRT, SOMERA, SRO, SSHNO, TROG consensus guidelines. Radiother Oncol. 2018; 126(1):3–24. https://doi.org/10.1016/j.radonc.2017.10.016. Im Neuenheimer Feld 450, 69120 Heidelberg, Germany. 5Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. 18. Lapeyre M, Toledano I, Bourry N, Bailly C, Cachin F. Target volume delineation for head and neck cancer intensity-modulated radiotherapy. Cancer Radiother. 2011;15(6–7):466–72. https://doi.org/10.1016/j.canrad.2011.07.239. Received: 21 October 2021 Accepted: 15 February 2022 Received: 21 October 2021 Accepted: 15 February 2022 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Single Bout Exercise in Children with Juvenile Idiopathic Arthritis: Impact on Inflammatory Markers
Mediators of inflammation
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Rochette Emmanuelle ,1,2,3,4 Duché Pascale,3,4 Hourdé Christophe,5 Evrard Bertrand,6,7 Pereira Bruno,8 Echaubard Stéphane,1 and Merlin Etienne1,2,6 1Pédiatrie Générale Multidisciplinaire, Hôpital Estaing, CHU Clermont-Ferrand, 63000 Clermont-Ferrand, France 2Université Clermont Auvergne, INSERM, CIC 1405, Unité CRECHE, 63000 Clermont-Ferrand, France 3 3Laboratoire des Adaptations Métaboliques en conditions Physiologiques et Physiopathologiques (AME2P), Université Clermont Auvergne, EA 3533, Clermont-Ferrand, France 5Laboratoire Interuniversitaire de Biologie de la Motricité (E A7424), Université Savoie Mont Blanc, 73376 Le Bourget-du-Lac, France Laboratoire Interuniversitaire de Biologie de la Motricité (E A7424), Université Savoie Mont Blanc, 73376 Le Bourget-du-Lac, France 5Laboratoire Interuniversitaire de Biologie de la Motricité (E A7424), Université Savoie Mont Blanc, 73376 Le Bourget-du-Lac, France 6Université Clermont Auvergne, INRA, UMR 1019 UNH, ECREIN, 63000 Clermont-Ferrand, France 7 6Université Clermont Auvergne, INRA, UMR 1019 UNH, ECREIN, 63000 Clermont-Ferrand, France 7 7Service d’Immunologie, CHU Clermont-Ferrand, 63000 Clermont-Ferrand, France CHU Clermont-Ferrand, Délégation de la Recherche Clinique et Innovations, 63000 Clermont-Ferrand, France Correspondence should be addressed to Rochette Emmanuelle; e_rochette@chu-clermontferrand.fr Received 28 February 2018; Accepted 27 May 2018; Published 13 June 2018 Received 28 February 2018; Accepted 27 May 2018; Published 13 June 2018 Academic Editor: Elena Dozio Academic Editor: Elena Dozio Copyright © 2018 Rochette Emmanuelle et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. In a context of inflammatory disease such as juvenile idiopathic arthritis (JIA), we do not know what impact physical activity may have on a deregulated immune system. The objective is to measure the impact of a single bout of exercise on plasma inflammatory markers such as calprotectin, IL-6, sIL-6R, sgp130, and the hypothalamic-pituitary-adrenal axis in children with juvenile idiopathic arthritis. Methods. Twelve children with JIA performed a nonexercise control day and a consecutive day that included a 20 min exercise bout at 70% of max-HR at 08:30 am. Venous blood samples were taken at 08:30, 08:50, 09:30, 10:30 am, and 12:00 pm to measure plasma concentrations of calprotectin, IL-6, sIL-6R, sgp130, cortisol, and ACTH. Pain was evaluated at 08:30, 08:50 am, and 06:00 pm. Results. There was a transient twofold increase in postexercise self- evaluated pain (p = 0 03) that disappeared in the evening. A single bout of exercise resulted in a 1.7-fold increase in plasma calprotectin (p < 0 001) but not IL-6 and its soluble receptors. Calprotectin levels returned to baseline within 3 hours after cessation of exercise. Conclusion. Hindawi Mediators of Inflammation Volume 2018, Article ID 9365745, 6 pages https://doi.org/10.1155/2018/9365745 Hindawi Mediators of Inflammation Volume 2018, Article ID 9365745, 6 pages https://doi.org/10.1155/2018/9365745 Rochette Emmanuelle ,1,2,3,4 Duché Pascale,3,4 Hourdé Christophe,5 Evrard Bertrand,6,7 Pereira Bruno,8 Echaubard Stéphane,1 and Merlin Etienne1,2,6 Acute exercise in children with JIA induced slightly musculoskeletal leg pain and transient increased plasma calprotectin levels but not IL-6 levels. Trial registration in ClinicalTrials.gov, reference number NCT 02502539, registered on 29 May 2015. 1. Introduction secreted by activated monocytes and phagocytes and induce the expression of proinflammatory cytokines (IL- 1β, IL-6, or TNF) [2]. In JIA, the MRP8/14 complex, also called calprotectin, is a reliable marker of subclinical disease activity [3, 4]. Juvenile idiopathic arthritis (JIA), the most common inflammatory pathology in children, is a chronic disease characterized by persistent joint inflammation due to immune system disruption [1]. Some of the proteins that per- petuate inflammatory mechanisms in JIA are calgranulins (myeloid-related protein (MRP)-8 and MRP-14). They are It has emerged that exercise may have direct effects not only on metabolic and fitness parameters but also on the pathogenesis of autoimmune diseases as it attenuates chronic Mediators of Inflammation 2 10 min at 4°C and stored at −80°C until analysis, while all other blood samples were analyzed immediately. low-grade systemic inflammation [5]. After acute exercise of varying intensities and lengths, plasma calprotectin levels in healthy adults increase at a rate of between 3.4-fold (VO2 max test) and 96.3-fold (marathon) [6]. Although IL-6 is known as a potent proinflammatory signal (involved in the pathology of JIA), the transient rise in circulating IL-6 during exercise appears to have anti-inflammatory effects [7]. In addition, IL-6 released from contracting muscle acts in a hormone-like manner to mobilize extracellular substrates or inhibits low-level TNF-α production. Likewise, physical activity is known to impact the hypothalamic-pituitary- adrenal (HPA) axis in healthy children [8-10] and modulate the anti-inflammatory effect of cortisol. However, the dis- turbed HPA axis in JIA leads to impaired secretion of adre- nocorticotropic hormone (ACTH) and cortisol [11, 12]. Even though physical activity is increasingly recognized as a useful intervention in patients with rheumatisms [13], to our knowledge, there is no data on the effect of acute bouts of physical activity on IL-6 and its receptors, calprotectin and HPA axis in children with inflammatory disease. p y y Plasma ACTH levels were measured by chemilumi- nescent immunometric assay (Immulite 2000, Siemens Healthcare Diagnostics; analytical sensitivity = 5 pg/mL). Serum cortisol levels were measured by chemiluminescence on an ADVIA Centaur XP Analyzer (Siemens Healthcare Diagnostics; analytical sensitivity= 27.6 nmol/L). Plasma MRP8/14 levels were measured using commercially avail- able ELISA kits (Bühlmann Laboratories, Schönenbuch, Switzerland; analytical sensitivity =400 ng/mL). Plasma highly sensitive C-reactive protein (hs-CRP) levels were measured by nephelometry on a Dimension Vista® ana- lyzer (Siemens Healthcare Diagnostics; analytical sensitivi- ty = 0.16mg/L). 1. Introduction Plasma IL-6 levels were measured using commercially available ELISA kits (EH2IL65, Thermo Fisher Scientific, USA; analytical sensitivity < 1 pg/mL). Plasma sgp130 levels were measured using commercially available ELISA kits (EHIL6ST, Thermo Fisher Scientific, USA; analytical sensitivity = 4 pg/mL). Plasma sIL6-R levels were measured using commercially available ELISA kits (KHR0061, Thermo Fisher Scientific, USA; analytical sensiti- vity =<8 pg/mL). All analyses were made on duplicates. Here, we set out to explore the impact of an acute bout exercise on pain and on the secretion of molecules involved in the inflammatory process in JIA. y pg y p Statistical analyses were performed using Stata software version 13 (StataCorp, College Station, TX, USA). Tests were two-sided, with a type I error set at α = 0 05. Contin- uous data were expressed as mean ±standard deviation (SD) or median (interquartile range) according to statistical distribution (assumption of normality assessed by using the Shapiro-Wilk test). To take into account between- and within-patient variability due to several measures for the same subject, random effects models for correlated data were performed rather than the usual statistical tests which would be inappropriate due to unverified assumption of indepen- dence. Time-point evaluations, sessions, and their interac- tions were considered fixed effects whereas a patient was a random effect (slope and intercept). The normality of resid- uals from these models was studied as described above. When appropriate, the data were log-transformed to achieve normality of the dependent endpoint. 2. Methods Twelve children aged 8 to 16 years with nonsystemic JIA according to the criteria of the International League of Asso- ciations for Rheumatology were enrolled in the study and were followed at the pediatric unit of Clermont-Ferrand Uni- versity Hospital in France. Patients were excluded if they had a physician-diagnosed infection, had been treated by IL-6 blockade in the last 6 months, and had received oral corticosteroids within the last 3 months. Treatments were continued during the study. The study was approved by the governing ethics commit- tee (Comité de Protection des Personnes Sud-Est VI, refer- ence number: AU1190). All participants and their parents have given consent. Experimental design consisted of two consecutive days (day 1: control, day 2: exercise day). 3. Results Pain was self-evaluated by the patient on a visual analog scale at 08:30, 08:50 am, and 06:00 pm during the 2 days. Baseline patient characteristics are reported in Table 1. Mean age was 12.3± 2.8 years, BMI was 19.7 ±2.9 kg/m2, and dis- ease duration was 29.4 ± 18.4 months. Mean fasting hs-CRP was 0.33 ± 0.4 mg/L. The exercise test consisted of a 20 min bout, between 08:30 am and 08:50 am, at 70% of the maximal theoretical heart rate (max-HR) performed on a cycle ergometer (Ergo-metrics 800S, Ergoline, Bitz, Germany) during day 2. Theoretical max-HR was defined as 220-age. Exercise bout induced a transient but significant increase in pain postexercise that resolved in few hours (Figure 1). This musculoskeletal pain involved muscles and joints, mainly in the legs. Venous blood samples were taken via an indwelling cath- eter at 08:30, 08:50, 09:30, 10:30 am, and 12:00 pm, into EDTA K2 Vacutainer® tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for analysis of ACTH, IL-6, sIL-6R, sgp130, and calprotectin, into Vacutainer Lithium Heparin Tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for anal- ysis of hs-CRP and into Vacutainer SST II Advance Tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for analysis of cortisol. Samples collected for calprotectin, IL-6, sIL-6R, and sgp130 were immediately centrifuged at 3000g for Exercise induced a 1.7-fold increase in calprotectin level immediately after the exercise (p < 0 001) that returned to normal within three hours after the end of the exercise bout (Figure 2). However, there was no effect of exercise on IL-6, sIL-6R, and sgp130 levels (Figure 3). Area under the curve (AUC) for plasmatic ACTH and serum cortisol decreased, respectively, from 11.8% (from 3228 ± 128 to 2848 ± 124; p = 0 58) to 17.4% (from 43,455 Mediators of Inflammation 3 Table 1: Baseline demographics, clinical characteristics, and baseline laboratory findings. 3. Results Age (years) Sex BMI (kg/m2) Z score JIA subtype Disease duration (months) Concomitant DMARDs hs-CRP (mg/L)ǂ Heart rate during exercise: min–max (bpm) 11 F 14.8 −1.4 Psoriatic 16.8 MTX 0.24 141–149 10 M 18.1 +0.6 ERA 14.1 Sulfasalazine <0.16 144–150 8 F 21.7 +1.8 pJIA RF− 4.9 MTX 1.4 142–152 9 M 15.5 −0.4 ERA 35.6 MTX <0.16 143–152 14 F 19.7 +0.2 pJIA RF− 9.0 MTX <0.16 140–149 14 M 20.1 +0.3 Psoriatic 40.1 Infliximab + leflunomide 0.33 141–149 16 F 21.1 +0.2 ERA 25.8 NSAIDs 0.53 138–147 9 M 21.3 +1.7 oJIA 55.2 NSAIDs <0.16 140–151 15 F 23.1 +0.9 ERA 20.7 NSAIDs <0.16 140–149 13 F 19.4 +0.2 pJIA RF+ 23.0 MTX + etanercept <0.16 138–150 12 F 16.9 −0.5 Undif 67 None <0.16 139–151 16 F 24.4 +1.0 Undif 20.5 Etanercept 0.22 138–147 JIA: juvenile idiopathic arthritis; oJIA: oligoarticular JIA; pJIA RF−: rheumatoid factor-negative (RF−) polyarticular JIA; pJIA RF+: rheumatoid factor-positive (RF+) polyarticular JIA; ERA: enthesitis-related arthritis; Psoriatic: psoriatic JIA; Undif: undifferentiated; DMARDs: disease-modifying antirheumatic drugs; MTX: methotrexate; NSAIDs: nonsteroidal anti-inflammatory drugs; hs-CRP: high-sensitivity C-reactive protein; bpm: beats per minute. ǂNormal value < 3.0 mg/L. Values of <0.16 are below the minimum detectable limit of the kit. Table 1: Baseline demographics, clinical characteristics, and baseline laboratory findings. JIA: juvenile idiopathic arthritis; oJIA: oligoarticular JIA; pJIA RF−: rheumatoid factor-negative (RF−) polyarticular JIA; pJIA RF+: rheumatoid factor-positive (RF+) polyarticular JIA; ERA: enthesitis-related arthritis; Psoriatic: psoriatic JIA; Undif: undifferentiated; DMARDs: disease-modifying antirheumatic drugs; MTX: methotrexate; NSAIDs: nonsteroidal anti-inflammatory drugs; hs-CRP: high-sensitivity C-reactive protein; bpm: beats per minute. ǂNormal value < 3.0 mg/L. Values of <0.16 are below the minimum detectable limit of the kit. 8:30 8:50 Ex ⁎⁎ 9:30 10:30 Calprotein 12:00 ⁎⁎ ⁎ ⁎ (hours:minutes) 500 1000 1500 2000 2500 3000 3500 (ng/mL) Figure 2: Plasma levels of calprotectin during the control day (solid line) and in response to 20 min of exercise (Ex) between 08:30 and 08:50 am (dotted line). Data are means ± 95% CI. ∗Significantly different to baseline at p < 0 05. ∗∗Significantly different to baseline at p < 0 001. 10 Pain 9 8 7 6 5 4 3 2 1 0 08:30 am p = 0.035 p = 0.031 08:50 am 06:00 pm Figure 1: Pain evaluations by visual analog scale during the control day (black bar) and exercise day (white bar). Data are means ± 95% CI. 3. Results 8:30 8:50 Ex ⁎⁎ 9:30 10:30 Calprotein 12:00 ⁎⁎ ⁎ ⁎ (hours:minutes) 500 1000 1500 2000 2500 3000 3500 (ng/mL) 10 Pain 9 8 7 6 5 4 3 2 1 0 08:30 am p = 0.035 p = 0.031 08:50 am 06:00 pm Figure 1: Pain evaluations by visual analog scale during the control day (black bar) and exercise day (white bar). Data are means ± 95% CI. Figure 1: Pain evaluations by visual analog scale during the control day (black bar) and exercise day (white bar). Data are means ± 95% CI. Figure 2: Plasma levels of calprotectin during the control day (solid line) and in response to 20 min of exercise (Ex) between 08:30 and 08:50 am (dotted line). Data are means ± 95% CI. ∗Significantly different to baseline at p < 0 05. ∗∗Significantly different to baseline at p < 0 001. ±1294 to 35,943 ±1217; p = 0 06) between the nonexercise control day and the exercise day (Figure 4). 4. Discussion increase of calprotectin and no effect on IL-6. The absence of IL-6 increase could be explained by the short duration and a low intensity of the exercise bout tested. Furthermore, as exercise does not increase calprotectin levels in neutrophils [6], the likely source is skeletal muscle, since S100A8 and S100A9 mRNA rates increase in skeletal muscle during exer- cise [16]. Therefore, as our child population has a lower mus- cle mass and incomplete muscular recruitment [17], there is likely a lower production of calprotectin and IL-6 by the muscles than in adults. In addition, pubertal status, physical exercise training, and BMI seem to be factors involved in the impact of exercise-induced IL-6 release in children [18, 19]. Nevertheless, in our child population with systemic low-grade inflammation, as in studies in children with Overall, the exercise session was well tolerated in all children. There was a slight increase in pain immediately postexercise, but the effect was transient and disappeared before the eve- ning. In terms of pain, it is therefore conceivable to advocate exercise in this child population, as more and more studies are showing that physical activity improves quality of life and symptoms in JIA patients [14, 15]. In healthy adults, acute exercise is known to induce a sig- nificant secretion of calprotectin [6] and IL-6 [7] by muscle in an exercise intensity-dependent manner. In fact, exercise promotes muscle recruitment of activated neutrophils, this phenomenon is followed by a systemic counterregulation involving IL-10 and IL-1ra [7]. Here, we observed a slight 4 Mediators of Inflammation 25 (pg/mL) 20 15 10 5 0 1300 Ex sgp130 IL-6 25 (pg/mL) (ng/mL) 20 15 10 5 0 1300 1200 1100 1000 900 800 Ex Ex sgp130 IL-6 sIL-6R 400 (nmol/L) (pg/mL) 350 300 250 200 150 100 50 30 25 20 15 10 5 8 9 10 (hours) 11 12 Ex Ex Cortisol ACTH ⁎⁎ ⁎⁎ ⁎ ⁎ Cortisol 400 (nmol/L) (pg/mL) 350 300 250 200 150 100 50 30 25 20 15 10 5 8 9 10 (hours) 11 12 Ex Ex Cortisol ACTH ⁎⁎ ⁎⁎ ⁎ ⁎ Figure 4: HPA axis secretion in children with JIA. Serum cortisol level and plasma ACTH level during the control day (solid line) and in response to 20 min of exercise (Ex) between 08:30 and 08:50 am (dotted line). ∗Significantly different to baseline at p < 0 05. 4. Discussion 400 (nmol/L) (pg/mL) 350 300 250 200 150 100 50 30 25 20 15 10 5 8 9 10 (hours) 11 Ex Ex ACTH ⁎⁎ ⁎⁎ ⁎ ⁎ Figure 4: HPA axis secretion in children with JIA. Se level and plasma ACTH level during the control day and in response to 20 min of exercise (Ex) betwee 08:50 am (dotted line). ∗Significantly different to ba 0 05. ∗∗Significantly different to baseline at p < 0 0 means ± 95% CI. (nmol/L) (pg/mL) 30 25 20 15 10 5 8 9 10 (hours) 11 12 Ex ACTH (ng/mL) 1300 1200 1100 1000 900 800 Ex sgp130 sIL-6R (ng/mL) (hours:minutes) 360 320 280 240 200 8:30 8:50 9:30 10:30 12:00 Ex sIL-6R Figure 4: HPA axis secretion in children with JIA. Serum cortisol level and plasma ACTH level during the control day (solid line) and in response to 20 min of exercise (Ex) between 08:30 and 08:50 am (dotted line). ∗Significantly different to baseline at p < 0 05. ∗∗Significantly different to baseline at p < 0 001. Data are means ± 95% CI. (ng/mL) outbreak may have been feared in response to exercise in a context of proinflammatory disease, this is not the case in response to an acute exercise of moderate intensity. IL-6 is involved not only in the activation of the immune system but also in regenerative processes and in the regula- tion of metabolism, the maintenance of bone homeostasis, and many neural functions [7, 20]. IL-6 is usually concerned as a proinflammatory cytokine. However, in the setting of muscular release, it is known to induce anti-inflammatory effects with inhibition of TNF-α production and may thus prevent TNF-α-induced insulin resistance [7]. Taken together, these observations raise questions over the different role for IL-6 in children compared to adults, making it neces- sary to address whether acute physical activity is followed by systemic counterregulation in children with JIA. Figure 3: Plasma level of IL-6, sgp130, and sIL-6R during the control day (solid line) and in response to 20 min of exercise (Ex) between 08:30 and 08:50 am (dotted line). Area under the curve (AUC) for plasma sIL-6R decreased 8.4% between control day and exercise day (from 63.36 106 ± 1.06 106 to 58.03 106 ± 0.97 106; p = 0 006). 4. Discussion ∗∗Significantly different to baseline at p < 0 001. Data are means ± 95% CI. 25 (pg/mL) (ng/mL) (ng/mL) (hours:minutes) 20 15 10 5 0 1300 360 320 280 240 200 8:30 8:50 9:30 10:30 12:00 1200 1100 1000 900 800 Ex Ex Ex sgp130 IL 6 sIL-6R Figure 3: Plasma level of IL-6, sgp130, and sIL-6R during the control day (solid line) and in response to 20 min of exercise (Ex) between 08:30 and 08:50 am (dotted line). Area under the curve (AUC) for plasma sIL-6R decreased 8.4% between control day and exercise day (from 63.36 106 ± 1.06 106 to 58.03 106 ± 0.97 106; p = 0 006). AUC for plasma sgp130 increased 2.6% between control day and exercise day (from 188.96 106 ± 4.81 106 to 193.91 106 ± 5.08 106; p = 0 11). Data are means ± 95% CI. outbreak may have been feared in response to e context of proinflammatory disease, this is not response to an acute exercise of moderate intensi IL-6 is involved not only in the activation of t system but also in regenerative processes and in tion of metabolism, the maintenance of bone h and many neural functions [7, 20]. IL-6 is usually as a proinflammatory cytokine. However, in th muscular release, it is known to induce anti-in effects with inhibition of TNF-α production thus prevent TNF-α-induced insulin resistance together, these observations raise questions over t role for IL-6 in children compared to adults, maki sary to address whether acute physical activity is 25 (pg/mL) (ng/mL) (ng/mL) (hours:minutes) 20 15 10 5 0 1300 360 320 280 240 200 8:30 8:50 9:30 10:30 12:00 1200 1100 1000 900 800 Ex Ex Ex sgp130 sIL-6R Figure 3: Plasma level of IL-6, sgp130, and sIL-6R during the control day (solid line) and in response to 20 min of exercise (Ex) between 08:30 and 08:50 am (dotted line). Area under the curve (AUC) for plasma sIL-6R decreased 8.4% between control day and exercise day (from 63.36 106 ± 1.06 106 to 58.03 106 ± 0.97 106; p = 0 006). AUC for plasma sgp130 increased 2.6% between control day and exercise day (from 188.96 106 ± 4.81 106 to 193.91 106 ± 5.08 106; p = 0 11). Data are means ± 95% CI. References Concerning the soluble receptors of IL-6, our results showed no effect of exercise on plasma sgp130 and sIL-6R levels. These results can be explained by the small number of patients and the fact that within-subject variation was lower than between-subject variation for IL-6, sIL-6R, and sgp130 in response to exercise, as reported in another study [23]. This finding can also in part explain why data on the effect of exercise on the rate of sgp130 is contradictory, with reports citing either an increase or a decrease or no effect [23-28]. Furthermore, all IL-6 family cytokines use gp130 as a receptor [29], and this may partly explain the high concen- trations of this receptor and its homeostasis. Finally, our levels of sIL-6R and sgp130 are higher than reported in literature, although sIL-6R levels are high in the serum and synovial fluid of JIA patients [30]; this discrepancy could be explained by the fact that we have used plasma or by the detection kits used. [1] Y.-T. Lin, C.-T. Wang, M. E. Gershwin, and B.-L. Chiang, “The pathogenesis of oligoarticular/polyarticular vs systemic juvenile idiopathic arthritis,” Autoimmunity Reviews, vol. 10, no. 8, pp. 482–489, 2011. [2] C. Kessel, D. Holzinger, and D. Foell, “Phagocyte-derived S100 proteins in autoinflammation: putative role in patho- genesis and usefulness as biomarkers,” Clinical Immunology, vol. 147, no. 3, pp. 229–241, 2013. [3] J. Gerss, J. Roth, D. Holzinger et al., “Phagocyte-specific S100 proteins and high-sensitivity C reactive protein as biomarkers for a risk-adapted treatment to maintain remission in juvenile idiopathic arthritis: a comparative study,” Annals of the Rheu- matic Diseases, vol. 71, no. 12, pp. 1991–1997, 2012. [4] H. Moncrieffe, S. Ursu, D. Holzinger et al., “A subgroup of juvenile idiopathic arthritis patients who respond well to methotrexate are identified by the serum biomarker MRP8/14 protein,” Rheumatology, vol. 52, no. 8, pp. 1467– 1476, 2013. We did not observe any impact of physical activity on the HPA axis except for cortisol. The decrease in cortisol level observed on the second day was probably due to lower stress at this session, as the children were aware of the study envi- ronment, contrary to the first session. [5] B. Gualano, A. L. Sá Pinto, B. Perondi et al., “Evidence for pre- scribing exercise as treatment in pediatric rheumatic diseases,” Autoimmunity Reviews, vol. 9, no. 8, pp. 569–573, 2010. 5. Conclusion [9] A. Viru, L. Laaneots, K. Karelson, T. Smirnova, and M. Viru, “Exercise-induced hormone responses in girls at different stages of sexual maturation,” European Journal of Applied Physiology, vol. 77, no. 5, pp. 401–408, 1998. This study found that in children with JIA, exercise is followed by a transient mild proinflammatory systemic response and low musculoskeletal leg pain, which resolve within three hours. [10] Y. Kilian, F. Engel, P. Wahl, S. Achtzehn, B. Sperlich, and J. Mester, “Markers of biological stress in response to a single session of high-intensity interval training and high-volume training in young athletes,” European Journal of Applied Phys- iology, vol. 116, no. 11-12, pp. 2177–2186, 2016. References The relatively small number of patients could be consid- ered as a limitation. A second limitation is that it focused on the physiological response to exercise at blood level, which may not be an accurate reflection of the muscular or articular response in this population. However, muscle biopsies or articular aspirations in these children would have been complicated to perform. Nevertheless, further research is warranted to confirm our results and evaluate other markers involved in the inflammatory response to exercise in children (healthy or with an inflammatory pathology). [6] M. K. Fagerhol, H. G. Nielsen, A. Vetlesen, K. Sandvik, and T. Lyberg, “Increase in plasma calprotectin during long- distance running,” Scandinavian Journal of Clinical and Labo- ratory Investigation, vol. 65, no. 3, pp. 211–220, 2005. [7] M. Gleeson, N. C. Bishop, D. J. Stensel, M. R. Lindley, S. S. Mastana, and M. A. Nimmo, “The anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease,” Nature Reviews Immunology, vol. 11, no. 9, pp. 607–615, 2011. [8] E. Richmond and A. D. Rogol, “Endocrine responses to exercise in the developing child and adolescent,” Frontiers of Hormone Research, vol. 47, pp. 58–67, 2016. Mediators of Inflammation References Data Availability [11] P. Picco, M. Gattorno, M. P. Sormani et al., “Involvement of the hypothalamic-pituitary-adrenal axis in children with oligoarticular-onset idiopathic arthritis,” Annals of the New York Academy of Sciences, vol. 966, no. 1, pp. 369–372, 2002. The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest [12] Y. Bilginer, R. Topaloglu, A. Alikasifoglu et al., “Low cortisol levels in active juvenile idiopathic arthritis,” Clinical Rheuma- tology, vol. 29, no. 3, pp. 309–314, 2010. The authors declare that they have no conflicts of interest [13] G. Kuntze, C. Nesbitt, J. L. Whittaker et al., “Exercise therapy in juvenile idiopathic arthritis: a systematic review and meta- analysis,” Archives of Physical Medicine and Rehabilitation, vol. 99, no. 1, pp. 178–193.e1, 2018. 4. Discussion AUC for plasma sgp130 increased 2.6% between control day and exercise day (from 188.96 106 ± 4.81 106 to 193.91 106 ± 5.08 106; p = 0 11). Data are means ± 95% CI. y g IL-6 acts through a heterodimeric signaling complex consisting of the IL-6 receptor (IL-6R) and the signal- transducing subunit glycoprotein 130 (gp130). Trans- signaling of IL-6 through its soluble receptors is involved in the promotion of chronic inflammatory diseases [21, 22]. obesity or type 1 diabetes, single bout exercise is not followed by increased IL-6 levels [19]. Finally, the fact that our patients are under medication (methotrexate or TNF-α inhibitor) may also explain the discrepancy between the results found here and data in adult or healthy subjects. A calprotectin 5 Mediators of Inflammation [15] L. Brosseau, D. B. Maltais, G. P. Kenny et al., “What we can learn from existing evidence about physical activity for juvenile idiopathic arthritis?,” Rheumatology, vol. 55, no. 3, pp. 387-388, 2016. [29] L. Cron, T. Allen, and M. A. Febbraio, “The role of gp130 receptor cytokines in the regulation of metabolic homeostasis,” Journal of Experimental Biology, vol. 219, no. 2, pp. 259–265, 2016. [30] N. J. Peake, K. Khawaja, A. Myers et al., “Interleukin-6 signal- ling in juvenile idiopathic arthritis is limited by proteolytically cleaved soluble interleukin-6 receptor,” Rheumatology, vol. 45, no. 12, pp. 1485–1489, 2006. [16] O. H. Mortensen, K. Andersen, C. Fischer et al., “Calprotectin is released from human skeletal muscle tissue during exercise,” The Journal of Physiology, vol. 586, no. 14, pp. 3551–3562, 2008. [17] S. Ratel, P. Duché, and C. A. Williams, “Muscle fatigue during high-intensity exercise in children,” Sports Medicine, vol. 36, no. 12, pp. 1031–1065, 2006. [18] B. W. Timmons, “Paediatric exercise immunology: health and clinical applications,” Exercise Immunology Review, vol. 11, pp. 108–144, 2005. [19] G. Paltoglou, M. Schoina, G. Valsamakis et al., “Interrelations among the adipocytokines leptin and adiponectin, oxidative stress and aseptic inflammation markers in pre- and early- pubertal normal-weight and obese boys,” Endocrine, vol. 55, no. 3, pp. 925–933, 2017. [20] M. A. Febbraio and B. K. Pedersen, “Muscle-derived interleu- kin-6: mechanisms for activation and possible biological roles,” The FASEB Journal, vol. 16, no. 11, pp. 1335–1347, 2002. [21] H. Chen, X. Zhang, N. Liao, and F. Wen, “Increased levels of IL-6, sIL-6R, and sgp130 in the aqueous humor and serum of patients with diabetic retinopathy,” Molecular Vision, vol. 22, pp. 1005–1014, 2016. [22] J. Wolf, S. Rose-John, and C. Garbers, “Interleukin-6 and its receptors: a highly regulated and dynamic system,” Cytokine, vol. 70, no. 1, pp. 11–20, 2014. [23] I. Walshe, P. Robson-Ansley, A. St Clair Gibson, C. Lawrence, K. G. Thompson, and L. Ansley, “The reliability of the IL-6, sIL-6R and sgp130 response to a preloaded time trial,” European Journal of Applied Physiology, vol. 110, no. 3, pp. 619–625, 2010. [24] S. R. Gray, M. Robinson, and M. A. Nimmo, “Response of plasma IL-6 and its soluble receptors during submaximal exercise to fatigue in sedentary middle-aged men,” Cell Stress & Chaperones, vol. 13, no. 2, pp. 247–251, 2008. [25] M. Leggate, M. A. Nowell, S. A. Jones, and M. A. Acknowledgments This work was supported by a grant from the Clermont- Ferrand University Hospital (AOI 2015). The authors thank the pediatric nurse Alexandra Usclade; the data manager Aurélie Chausset and Khaled Messaoudi, Chloé Plantard, and Clara Martin for their help with handling the blood sampling. [14] T. Takken, M. Van Brussel, R. H. H. Engelbert, J. Van Der Net, W. Kuis, and P. J. M. Helders, “Exercise therapy in juvenile idiopathic arthritis: a Cochrane review,” European Journal of Physical and Rehabilitation Medicine, vol. 44, no. 3, pp. 287–297, 2008. 6 Mediators of Inflammation Mediators of Inflammation Nimmo, “The response of interleukin-6 and soluble interleukin-6 receptor isoforms following intermittent high intensity and continuous moderate intensity cycling,” Cell Stress & Chaperones, vol. 15, no. 6, pp. 827–833, 2010. [26] S. Patterson, S. Reid, S. Gray, and M. Nimmo, “The response of plasma interleukin-6 and its soluble receptors to exercise in the cold in humans,” Journal of Sports Sciences, vol. 26, no. 9, pp. 927–933, 2008. [27] C. Keller, A. Steensberg, A. K. Hansen, C. P. Fischer, P. Plomgaard, and B. K. Pedersen, “Effect of exercise, train- ing, and glycogen availability on IL-6 receptor expression in human skeletal muscle,” Journal of Applied Physiology, vol. 99, no. 6, pp. 2075–2079, 2005. [28] M. Robinson, S. R. Gray, M. S. Watson et al., “Plasma IL-6, its soluble receptors and F2-isoprostanes at rest and during exercise in chronic fatigue syndrome,” Scandinavian Journal of Medicine & Science in Sports, vol. 20, no. 2, pp. 282–290, 2010.
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Spanish Version of the Flourishing Scale (FS) on the Parents of Children With Cancer: A Validation Through Rasch Analysis
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Spanish Version of the Flourishing Scale (FS) on the Parents of Children With Cancer: A Validation Through Rasch Analysis Carmen Pozo Muñoz 1* and Blanca Bretones Nieto 2 1 Department of Psychology, University of Almería, Almería, Spain, 2 Psychosocial Intervention and Health (HUM-792) Research Group, University of Almería, Almería, Spain The interest in the study of flourishing is due to the fact that it has been proven that it contributes to a good adjustment to the demands of the environment, relating to indicators of health and well-being. There are many researches that have tried to find out what dimensions make it up. The goal of this study is to validate the Spanish Version of The Flourishing Scale (FS), being the first time it is applied to Spanish parents of children with cancer. A total of 138 parents of children with cancer participated in a semi-structured interview. Through IBM SPSS and Winsteps descriptive analyzes and the internal consistency of the FS were calculated. Rasch analysis was used to study the dimensionality of the scale, the adjustment of each one of the items, the reliability values for the items and for the people, the validity of the construct, the functioning of the response categories, and the differential item functioning (DIF). The external construct validity of the FS was examined with associated measures. Results found that parents indicated a flourishing attitude (M = 3.85; sd =0.63). We confirm that the FS maintains an adequate internal consistency and a unidimensional structure. We observed a good alignment between the question and the person’s abilities as soon as a high reliability for the items. Although the sample is large enough to corroborate the difficulty hierarchy of the items, the construct validity could be improved by introducing items of moderate and high difficulty. On the other hand, the answer category 3 overlaps with the 2 and 4, so we propose possible solutions. Regarding a possible DIF, this exists in relation to “gender,” “level of education” and “by the situation with respect to treatment.” Finally, the convergent validity of the FS is demonstrated, there being a significant correlation with well-being, satisfaction with social support and coping. In general, the results show adequate psychometric properties of the Spanish version of the FS, so we recommend integrating it in psychosocial interventions aimed at parents of children with cancer, in order to provide them with resources to deal with the disease. Keywords: validation, rasch analysis, flourishing, parents, childhood cancer Edited by: Eduardo A. Garza-Villarreal, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz (INPRFM), Mexico Edited by: Eduardo A. Garza-Villarreal, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz (INPRFM), Mexico Reviewed by: Kathy Ellen Green, University of Denver, United States Alessandro Giuliani, Istituto Superiore di Sanità (ISS), Italy Corrado Caudek, Università degli Studi di Firenze, Italy Reviewed by: Kathy Ellen Green, University of Denver, United States Alessandro Giuliani, Istituto Superiore di Sanità (ISS), Italy Corrado Caudek, Università degli Studi di Firenze, Italy Reviewed by: Kathy Ellen Green, University of Denver, United States Alessandro Giuliani, Istituto Superiore di Sanità (ISS), Italy Corrado Caudek, Università degli Studi di Firenze, Italy *Correspondence: Carmen Pozo Muñoz cpozo@ual.es Specialty section: This article was submitted to Clinical and Health Psychology, a section of the journal Frontiers in Psychology Received: 05 May 2018 Accepted: 08 January 2019 Published: 25 January 2019 ORIGINAL RESEARCH published: 25 January 2019 doi: 10.3389/fpsyg.2019.00035 INTRODUCTION Recently, flourishing has been analyzed as a positive coping mechanism for health and well-being in the context of childhood cancer, specifically in the parents of diagnosed children and adolescents (Bretones et al., 2016; Bretones, 2018). Agreement has not been reached regarding the dimensions that comprise the Flourishing Scale. The first results come from the application of the original Flourishing Scale (FS) by Diener et al. (2009). The FS scale was found to have a single strong factor with an eigenvalue of 4.24, which accounted for 52% of the items’ variance, with Cronbach’s alpha of 0.87 and a test-retest reliability of 0.71. Subsequent studies have obtained similar results. In Brazil, Nunes et al. (2015) support the one-dimensionality of the scale in two studies carried out, respectively with students and the general population. Similarly, Tang et al. (2016) obtained a single factor in a sample Chinese population (α > 0.90 and a good convergent and discriminant validity); as did Schotanus-Dijkstra et al. (2016) in research conducted with adults in the Netherlands (α = 0.86; adequate global reliability and a good fit of most of the elements in the Rasch analysis). Although the psychometric properties of FS have previously been studied through the Classical Test Theory (CTT) (Diener et al., 2009; Silva and Caetano, 2013; Hone et al., 2014; Sumi, 2014), on a few occasions analysis of the psychometric properties of this instrument has been used by new models of the Item Response Theory (IRT), such as the Rasch model (Pozo et al., 2016; Schotanus-Dijkstra et al., 2016). The main advantages of the Rasch Model (the most parsimonious IRT model) in comparison with CTT are the possibility of estimating the extent to which each test or item measures the skill of the participants, the joint estimates of respondents’ parameters and items, and the invariance of the parameters obtained from the different samples (Arias et al., 2013; Garzón Umerenkova et al., 2017). In contrast to the CTT, the Rasch model establishes the probability of a person’s response to a stimulus, i.e., the difference between the measure of a person’s trait and the measure of the stimulus used (Tristán, 2002). INTRODUCTION It is worth highlighting the cross-cultural research of Pozo et al. (2016), in which Colombian and Spanish university students participated. Satisfactory psychometric properties (α Colombian = 0.88; α Spanish = 0.85) and good levels of reliability were found (the Rasch person reliability was fairly acceptable -Colombia = 0.77; Spain = 0.80-, and the person separation statistic was 1.83 for Colombia and 2.02 for Spain. Rasch item reliability was 0.93 for Colombia and 0.94 for Spain, and the item separation statistic was 3.69 for Colombia and 4.13 for Spain. In both samples, the Cronbach alpha values were higher than 0.70). The central concept developed by Keyes (2007) defines the flourisher as the individual who experiences a high level of emotional, psychological and social well-being. In subsequent studies, Diener et al. (2010) consider that the flourishing consists of a sense of competence, self-acceptance, optimism, and to contribute to the well-being of others. According to Huppert and So (2009) flourishing comprises a set of central characteristics including: positive emotions, delivery, interest, meaning and purpose; and an additional set of competencies: self-esteem, optimism, resilience, vitality, self-determination, and positive relationships. Giuntoli et al. (2017) analyze the dimensionality of the construct (they found a superior fit for a two factor model) and its invariance in two studies (with students, unemployed and healthy samples) through a multigroup analysis. Concurrent validity was verified with other well-being, depression and anxiety measures. Well-being Theory is based on the Theory of Authentic Happiness, centered on the study of the latter to increase satisfaction with life. However, from the theory of well-being, positive psychology focuses its interest on the analysis of it, using flourishing for its measurement (Seligman, 2011). Flourishing has been related to psychological well-being and has been studied in conjunction with positive and negative emotions in order to elaborate scales to help us understand well-being (Diener et al., 1999). In relation to the fields of application, previous investigations have verified the association between flourishing and less limitations in daily life, as well as flourishing and better health (Howell et al., 2013; Huppert and So, 2013; Gilmour, 2015). Along the same lines, the psychosocial factor in the context of education has been studied and found to be beneficial regarding the well-being, academic performance, and abilities of university students (Fredrickson and Branigan, 2005; Howell, 2009; Wilson- Strydom and Walker, 2015), as well as for occupational health workers (Rautenbach and Rothmann, 2017). Citation: Pozo Muñoz C and Bretones Nieto B (2019) Spanish Version of the Flourishing Scale (FS) on the Parents of Children With Cancer: A Validation Through Rasch Analysis. Front. Psychol. 10:35. doi: 10.3389/fpsyg.2019.00035 January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 1 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto January 2019 | Volume 10 | Article 35 INTRODUCTION Taking into account these circumstances and since flourishing has previously been related to variables related to health and well-being in the context of illness (Bretones et al., 2016), we consider it important to verify the effects that Flourishing causes in terms of coping with the disease. For this, first of all we believe that it is necessary to continue deepening the analysis of the Flourishing Scale, as a novelty, in a sample of Spanish parents of children with cancer. that they have been considered as “invisible patients” (Hinds, 1992). On the other hand, it has been shown that the adaptation of children and adolescents is determined by factors related to the degree of adjustment of their parents, especially in the initial stages of the disease (Fedele et al., 2013). During the childhood illness the parents of children with cancer have to suffer a lot of critical moments in relation with diagnosis, treatments, side effects, surgeries, e.g., In this way, stress symptoms represent a common experience in the familiar context, also after the illness situation (Vrijmoet-Wiersma et al., 2010). Taking into account these circumstances and since flourishing has previously been related to variables related to health and well-being in the context of illness (Bretones et al., 2016), we consider it important to verify the effects that Flourishing causes in terms of coping with the disease. For this, first of all we believe that it is necessary to continue deepening the analysis of the Flourishing Scale, as a novelty, in a sample of Spanish parents of children with cancer. objective and subjective health, well-being, and stressors, the majority used coping strategies (including flourishing), as well as support from their formal and informal network. Because of the characteristics of the study, sociodemographic and clinical variables of interest were collected. Socio- demographics included: age, gender, nationality, educational level, marital status, and employment situation. Regarding the children, clinical information was required. These were: the diagnosis, date of diagnosis, type of treatment and current status post-treatment. The adaptation of the Symptom Scale (Martos et al., 2009) consists of a 9-point Likert scale that assesses perceived physical and psychological symptoms. Subjective health was assessed with the item “In general, I am in poor health.” The internal consistency in the original scale, according to Cronbach’s alpha statistic is 0.79 (Jou and Foucada, 1997), whereas in the present study it is 0.85. INTRODUCTION Likewise, the Rasch model quantifies the amount of information and error with which it is measured in each point of the dimension and allows the selection of those items that make it possible to increase the information in previously specified regions of the construct (Jiménez and Montero, 2013). Recent studies in Spain have applied the Spanish version of the FS. In a study by Ramírez et al. (2017), of university students and patients with chronic back pain, the main axis factor, and the simultaneous component analysis in both groups showed a common unidimensional structure. In addition, the omega coefficient showed high reliability. The principal conclusion was that flourishing had a mediating effect between personality variables and positive and negative affect, which explains the association between anxiety, optimism, pessimism, and positive affect. De la Fuente et al. (2017) have also supported the Spanish version of the FS as a suitable measure. Specifically, they applied it with university students from different fields of knowledge. They obtained a single factor structure of the FS, with good internal consistency. The convergent validity of the scale was demonstrated with another measure of psychological well-being and its discriminant validity with the scale of symptoms of depression, anxiety, and stress. In light of the above, it has already been supported that a flourishing attitude is related to well-being indicators. Childhood cancer is understood as a “family disease” because it affects the health of all its members and determines its ongoing functioning (Bretones, 2018). The importance of analyzing the psychosocial repercussion of this pathology in the parents is due to the fact January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 2 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto that they have been considered as “invisible patients” (Hinds, 1992). On the other hand, it has been shown that the adaptation of children and adolescents is determined by factors related to the degree of adjustment of their parents, especially in the initial stages of the disease (Fedele et al., 2013). During the childhood illness the parents of children with cancer have to suffer a lot of critical moments in relation with diagnosis, treatments, side effects, surgeries, e.g., In this way, stress symptoms represent a common experience in the familiar context, also after the illness situation (Vrijmoet-Wiersma et al., 2010). Procedure The study was presented to and approved by the Research Ethics Committee of Torrecárdenas Hospital (Almeria, Spain). Research methodology was planned considering the design of the inclusion and exclusion criteria, the sample selection, the interview design, etc. The requirements for participation in the interview were being the parent of a child diagnosed with cancer who had been treated in that hospital. Exclusion criteria was determined as the following: being the parent of a child who had died due to the disease and a parent who did not speak Spanish with sufficient command. INTRODUCTION The Spanish version of the Satisfaction with Life Scale (Goodwin and Hernández, 2000) is a 5-item Likert scale used to assess the level of well-being experienced by parents. Answers range from 1 (completely disagree) to 5 (completely agree). Cronbach’s alpha in the original scale is 0.87 (Diener et al., 1985), while it is 0.82 in this study. Objectives The purpose of this study was to validate the Spanish version of the Flourishing Scale following administration in a sample of parents of children with cancer. This was done using Rasch analysis in particular, also dimensionality, the fit of the items to the model, the functioning of the measurement scale, construct validity, reliability, and the differential item functioning (DIF). Finally, the convergent validity of the FS was determined in relation to other measures that study the following variables: well-being, coping styles and social support (these variables have been chosen because they represent indications of the state of health and quality of life of the parents of children with cancer, in this case). In order to analyze the coping styles in parents, the reduced Spanish version of the Cope Inventory (Cuestionario de Afrontamiento del Estrés, CAE) (González and Landero, 2007) was applied. This scale consists of 21 items, which are divided into seven subscales (although the total score of the scale was used): problem-solving coping, negative auto- focused coping, positive reappraisal, overt emotional expression, avoidance coping, social support seeking, and religious coping. Items are answered on a 5-point Likert scale ranging from 0 (never) to 5 (almost always). While Cronbach’s alpha for Sadín’s and Chorot’s original questionnaire reached 0.77, in CAE’s reduced version internal consistency was 0.74, and finally, in this study it reached 0.62. Sample The sample consisted of 138 adults: 60 men and 78 women, who were the parents of 94 children (aged between 1 and 14 years) diagnosed with cancer. The parents’ ages ranged from 41 to 50 years old 0.84% were of Spanish nationality and 16% of foreign origin (10% African and 6% Romanian). In this cohort 88.4% were married or living together as a couple, 60.2% had completed secondary school and 53.9% worked in the services sector (includes subsectors such as commerce, communications, call center, finance, tourism, hospitality, leisure, culture, entertainment, public administration, and so- called public services, provided by the State or private initiative (health, education, care dependency; among others). The diagnoses of the children were: leukemia (44.2%) and solid tumors (55.8%). The flourishing attitude was evaluated through the Spanish version of the Flourishing Scale (original by Diener et al., 2009). This scale is made up of eight items. The values of the answers range from 1 to 5 (1 being “strongly disagree”; 2 “disagree”; 3 “neither agree nor disagree”; 4 “agree” and 5 “totally agree”). Cronbach’s alpha in the original scale is 0.87 (Diener et al., 2009), whereas it is 0.74 in the present study. Measures This study comprises a quantitative part (on which this article focuses, with validated Likert-type scales) and a qualitative part (a series of open-ended questions designed to investigate the main problems in fundamental areas of the family’s daily life) of the data gathered during the semi-structured interviews in which they evaluated: socio-demographics and clinical characteristics, The interview was drawn up according to the research objectives and administered individually (a single session) in a January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 3 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto between 0.5 and 1.5 (the MNSQ values of Infit and Outfit) (see Table 2). private room of the hospital (approximately one hour’s duration), following detailed explanation of the study’s characteristics. Participants signed consent forms and all data was treated in accordance with current privacy laws. These were recorded and transcribed verbatim to a text file. Audio and text files were then saved and processed confidentially. The PT-Measure-CORR column allows observation of the alignment between the question and the subject’s abilities. The results indicate that there are no inverse correlations and the associations are moderate (the lowest value is set in item 7, with a correlation of 0.49, and the highest item being 4, with a value of 0.73). In addition, columns PT-Measure-EXP. show that there are no notable differences between observed and expected correlations. Data Analysis Pearson correlation coefficients were used to evaluate the convergent validity between the Spanish version of the Flourishing Scale (FS), the Spanish version of the Satisfaction with Life Scale, the reduced Spanish version of the Coping Stress Questionnaire (CAE) and the adaptation of the Perceived Social Support Scale (EASP/PSS). Data Analysis The results were analyzed using IBM SPSS software (version 22 for Windows) and Winsteps (v. 3.72.3) statistical package. The following analyses were conducted: On the other hand, the reliability for the items was 0.99 with a separation of 8.58, with high values. The values of separation are >3. In relation to the reliability for people, a value of 0.65 and a separation of 1.36 was obtained. The next step was to analyse the construct validity based on the hierarchy of the items using the Wright map (Figure 1). Unlike the results obtained in other studies (Pozo et al., 2016), in this case, there was a number of subjects in the center of the figure who do not have items in front of them. They are at a moderate level of flourishing, but the scale does not have adequate items to measure the level of the variable. – Descriptive analyses. – Internal consistency of the items calculated using Cronbach’s alpha. – The unidimensionality of the test was verified by “main components.” – The goodness of fit of each of the eight FS items (infit and outfit estimates) to the Rasch model. – Reliability values were calculated for the items and for the subjects. – The validity of the construct was verified based on the hierarchy of the items (Wright map). TABLE 1 | Items of the Spanish Version of the FS. Ítems x ± sd I am a good person and live a good life (item 6) 4.48 ± 0.81 People respect me (item 8) 4.41 ± 0.87 I am competent and capable in the activities that are important to me (item 5) 4.28 ± 1.10 I actively contribute to the happiness and wellbeing of others (item 4) 4.25 ± 1.03 I lead a purposeful and meaningful life (item 1) 4.11 ± 1.08 I am engaged and interested in my daily activities (item 3) 3.76 ± 1.25 I am optimistic about my future (item 7) 3.62 ± 1.24 My social relationships are supportive and rewarding (item 2) 1.91 ± 0.95 – The functioning of the response categories was verified through the Rating Scale Model (RSM) for polytomous items. – The possible differential item functioning (DIF) was contrasted. – Finally, FS construct validity was examined with a wide variety of measures. Descriptive Analysis TABLE 2 | Infit and outfit estimations for each item. TABLE 2 | Infit and outfit estimations for each item. INFIT OUTFIT PT-MEASURED Item Measure Model SE MNSQ MNSQ CORR. EXP. Item7 0.35 0.09 1.37 1.40 0.49 0.65 Item2 2.30 0.10 1.16 1.36 0.48 0.57 Item6 −0.88 0.12 1.11 0.97 0.48 0.52 Item5 −0.50 0.11 1.04 0.79 0.66 0.57 Item1 −0.25 0.10 1.03 1.02 0.60 0.60 Item8 −0.75 0.12 0.96 0.98 0.54 0.54 Item4 −0.46 0.11 0.89 0.75 0.65 0.58 Item3 0.19 0.09 0.83 0.81 0.73 0.64 Item mean 0.00 0.11 1.05 1.01 – – SD 0.96 0.01 0.16 0.23 – – The order of the items corresponds to the adjustment of each of the items according to the output of the table in the Rash model. Measure, Location of the latent variable; Model SE, Error estándar; INFIT-MNSQ, Infit Mean Squared; “OUTFIT-MNSQ, Outfit Mean Squared; PT Measured, Point Measure Correlation. The results exceed the midpoint of the scale (M = 3.85; sd = 0.63). This also happens in specific items, such as being considered a “good person and lead a good life,” feeling respected, subjectively perceived as “competent and capable,” contributing to the happiness and well-being of people close to them and having a “useful and meaningful” life (Table 1). On the other hand, defining oneself as optimistic and being involved in daily activities was also considered. Despite this, the lowest scores are related to parent’s perceptions about their supportive and rewarding social relations. The Rasch Model on the Flourishing Scale The Rasch Model on the Flourishing Scale Cronbach’s alpha for the Spanish version of the FS was 0.74. In this sense, it is possible to conclude that it is structured in a single dimension. The variance explained by the measure was 59.2%, with a first contrast eigenvalues of 1.6, meeting the recommendations for assuming the dimensionality (variance >0.30; first contrast eigenvalue <3.0) stated by of Bond and Fox (2007, 2012) or Smith (2012). The item fit to the Rasch Model of each of the eight items of the scale was checked. All items were January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 4 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto FIGURE 2 | Probability curves for the response categories. FIGURE 2 | Probability curves for the response categories. FIGURE 1 | Wright map of person and items. FIGURE 2 | Probability curves for the response categories. (2011), an item has a DIF associated with belonging to a group when people with the same value in the measured variable, but from different groups, have a different probability of solving that item correctly. In this way, it was established as a criterion for a possible DIF that the values of “DIF contrast” were >0.5 logits between the comparison groups and that this difference in the t values was significant (p ≤0.05; Bond and Fox, 2007, 2012). ) A possible DIF by “gender” was found for item 7 (“I am optimistic about my future”) with a contrast of 0.62 (p ≤0.05), being a more difficult item for women than for men. There was also a possible DIF by “level of education” for item 5 (“I am competent and capable in the activities that are important to me”), with a contrast of 1.41 (p ≤0.05), being a more difficult item for participants without studies than for those with higher education. It is equally important to consider a possible DIF with “the child’s situation with respect to treatment” for item 6 (“I am a good person and live a good life”), with a contrast of 0.84 (p ≤ 0.05). It is a more difficult item for parents whose children are receiving treatment, compared to participants whose children have been “out of treatment” for >5 years. The Rasch Model on the Flourishing Scale A possible DIF by “gender” was found for item 7 (“I am optimistic about my future”) with a contrast of 0.62 (p ≤0.05), being a more difficult item for women than for men. There was also a possible DIF by “level of education” for item 5 (“I am competent and capable in the activities that are important to me”), with a contrast of 1.41 (p ≤0.05), being a more difficult item for participants without studies than for those with higher education. It is equally important to consider a possible DIF with “the child’s situation with respect to treatment” for item 6 (“I am a good person and live a good life”), with a contrast of 0.84 (p ≤ 0.05). It is a more difficult item for parents whose children are receiving treatment, compared to participants whose children have been “out of treatment” for >5 years. FIGURE 1 | Wright map of person and items. Response categories’ function was studied through the Rating Scale Model (RSM) for polytomous items. The response categories were the following: (1) means “strongly disagree”; (2) “somewhat disagree”; (3) “neither agree nor disagree”; (4) “somewhat in agreement,” and (5) “totally agree.” In the probability curves of the categories it was found that category 3 overlapped with categories 2 and 4 (see Figure 2). Finally, regarding convergent and divergent validity, our results largely confirmed the correlations between FS and other measures under study with a Bonferroni-adjusted (Table 3). We found a moderate relationship between flourishing and well- being (r = 0.403, p ≤0.01). The FS also showed a low correlation with the satisfaction with the social support received (r = 0.293, p ≤0.01) and with coping strategies that parents use; specifically, “the positive re-appraisal” (r = 0.211, p ≤0.05); with “the problem-solving coping” (r = 0.186; p ≤0.05), the “negative auto-focused coping” (r = −0.248; p ≤0.01) and the “over emotional expression” (r = −0.171; p ≤0.05). The possible Differential Item Functioning (DIF) of the items was checked by “gender,” “educational level” and “the child’s situation with respect to treatment.” According to Prieto et al. January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 5 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto TABLE 3 | Correlation between Flourishing and other measures. 1 2 3 4 5 6 7 1. Flourishing 1 – – – – – – 2. DISCUSSION Originally called “item bias” (Lord, 1980), Differential Item Functioning (DIF) has been recognized as a potential source of bias in person measure. However, other authors indicate that the existence of the DIF does not necessarily imply a bias in the item (Wright and Stone, 1999). When analyzing the differential behavior of items (DIF), the results indicate a possible DIF by gender, by level of education and by situation with respect to treatment. In short, the aforementioned items should continue to be investigated in order to corroborate if there really is a bias or not. The purpose of the present study consisted of validate the Spanish version of the Flourishing Scale following administration in a sample of parents of children with cancer. As a first step, descriptive analyzes were carried out to check the tendency of the sample to show a flourishing attitude. If were considered the results of this study with those obtained in other investigations in which other groups (specifically, university students) have contributed (Pozo et al., 2016; Garzón Umerenkova et al., 2017) it is possible to affirm that the parents who have participated in this study have a flourishing attitude, because the scores obtained exceed the midpoint of the scale. Good results are maintained with regard to the internal consistency of the Spanish version of the FS. The Rasch Model on the Flourishing Scale Wellbeing 0.403** 1 – – – – – 3. Satisfaction with social support 0.293** 0.099 1 – – – – 4. Positive re–appraisal 0.211* 0.265** 0.137 1 – – – 5. Problem–solving coping 0.186* 0.158 0.010 0.177* 1 – – 6. Negative auto–focused coping −0.248** −0.188* −0.029 −0.328** −0.161 1 – 7. Over emotional expression −0.171* −0.057 −0.111 −0.043 −0.008 0.247** 1 *p ≤0.05; **p ≤0.01. TABLE 3 | Correlation between Flourishing and other measures. CONCLUSION As a strength, the Rasch analysis performed after the application of the Spanish version of the FS on the parents of children with cancer, indicates that generally the test presents a good psychometric performance. However, it would be appropriate to continue investigating the previously mentioned aspects to further improve its performance. The findings of the Rasch analysis have shown the one- dimensionality of the scale and there is a good fit to the model of each of the eight items that make up the scale. The infit and outfit mean-square (MNSQ) statistics provide evidence of construct validity when expected values are close to 1.0, with values from 0.5 to 1.5 being useful for measure, so in this case a good fit to the model is confirmed. Equally, there is a good alignment between the questions and the skills of the respondent. In relation to the convergent validity, our results are in agreement with the postulates of well-being Theory in the field of Positive Psychology (Seligman, 2011), since there is a moderate correlation between flourishing and perceived well- being. For this reason, it is important to continue researching, using flourishing as a measure. In this connection, in principle, we could interpret that flourishing is an effective coping strategy for the parents of children with cancer, because it is positively related to styles considered healthy (“satisfaction with social support,” “positive re-appraisal” and “problem-solving coping”) and in a negative way with others, that are not (“negative auto- focused coping” and “over emotional expression”). Therefore, it is possible to affirm that flourish people are more likely to develop more favorable behavior patterns in situations that may be problematic (such as the experience of having a child with cancer). It would be convenient to take these results into account in order to develop psychosocial intervention programs (planning actions that stimulate the development of Flourishing components) aimed at responding to the needs of the parents of children with cancer. In accordance with Smith (2012), the Rasch model verifies that the sample is large enough to corroborate the difficulty hierarchy of the items, namely, the construct validity of the instrument the reliability of people is considered a low index of separation for people with inferior values to 2. Therefore, the instrument is not as sensitive as one would wish, for identifying people with high and low skill. Frontiers in Psychology | www.frontiersin.org CONCLUSION The differences in this study compared to other studies may be due to the diversity of the characteristics of the samples used (Pozo et al., 2016; Garzón Umerenkova et al., 2017). The construct validity of FS can be improved by adding items of moderate and high difficulty. This would increase the sensitivity of the instrument and the reliability for people, since these two points are related. The overlap found between the response categories could be solved by eliminating category 3, reducing the Likert scale from five to four points, or the term “neither agree nor disagree” could be rewritten, which would seem to be insufficiently differentiated from the terms “somewhat in disagreement” (2) or “somewhat in agreement” (4). On the other hand, regarding the advantages of an IRT analysis with respect to the usual CCT, the use of the characteristics of the IRT method has constituted an advance for Frontiers in Psychology | www.frontiersin.org January 2019 | Volume 10 | Article 35 6 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto this study, allowing the improvement in the measurement of the scale as well as its refinement. the different strategies to face the childhood illness on the part of the mother and the father, isolating this problem from the others. On the other hand, a global approach based on dyads could be useful in this case (even for correlation with other scales and the Rasch analysis) and could be useful to complement the results obtained. It is worth highlighting some methodological limitations detected that should be taken into account in subsequent studies (low separation, no items at a mid-range of the construct, some problem with the response scale, moderate correlations with validation measures). On the other hand, due to the number of cases of childhood cancer in the study context, it has not been possible to apply a specific sampling method. In addition, a large volume of experimental mortality has been observed. Regarding this issue, it has been found that research related to “childhood cancer” is a difficult field to gather large samples (Bragado et al., 2008). The language barrier (in the immigrant population) and the families that declined the invitation to participate, were the greatest difficulties. REFERENCES Diener, E., Wirtz, D., Tov, W., Kim-Prieto, C., Choi, D., Oishi, S., et al. (2010). New measures of well-being: flourishing and positive and negative feelings. Soc. Indic. 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Doctoral thesis. University of Almería, Almería. Gilmour, H. (2015). Chronic pain, activity restriction and flourishing mental health. Health Rep. 26, 15–22. Bretones, B., Pozo, C., and Vázquez, M. A. (2016). “Influence of flourishing on the health of parents of children with cancer,” in II International Congress of the Spanish Scientific Society of Social Psychology and XIII National Congress of Social Psychology, ed J. M. Sabucedo (Elche: Spanish Scientific Society of Social Psychology). Giuntoli, L., Ceccarini, F., Sica, C., and Caudek, C. (2017). ETHICS STATEMENT This study has been developed in accordance with the Declaration of Helsinki and has been approved by the Research Ethics Committee of Torrecárdenas Hospital (Almeria, Spain). There was no conflict of interest between the authors and other institutions. CONCLUSION This is associated with the fact that, for most of the parents, submitting to the interview took great emotional effort, due to having to exercise awareness and delve into circumstances of their life that were especially painful for the child, the parents themselves, and the rest of the family. AUTHOR CONTRIBUTIONS CP was the researcher in charge of coordinating research planning, reviewing the literature, and subsequently proposing the objectives and research design. In addition, she has participated in the analysis phase of the data and in the writing of the article. Finally, the importance of these results makes it necessary to replicate this study in a population with similar characteristics in order to contrast the resulting conclusions and implement methodological improvements in relation to the scale. We even propose for a future investigation to explore through a pair of pairs of parents of the same child, so that both can individualize BB was responsible for selecting the final sample of parents who participated; she conducted the interviews, participated in the analysis phase of the data and in the writing of the article. REFERENCES Validation of the italian versions of the flourishing scale and of the scale of positive and negative experience. SAGE Open 7:2158244016682293. doi: 10.1177/21582440166 82293 González, M. T., and Landero, R. (2007). The Coping Stress Questionnaire (CAE). Validation in a Mexican sample. Revista de Psicopatología y Psicología Clínica 12, 189–198. De la Fuente, R., Parra, A., and Sánchez-Queija, I. (2017). Psychometric properties of the flourishing scale and measurement invariance between two samples of spanish university students. Eval. Health Prof. 40, 409–424. doi: 10.1177/0163278717703446 Goodwin, R., and Hernández, S. (2000). 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(2009). “New measures of well-being,” in Assessing Well-Being. Social Indicators Research Series, Vol 39, ed E. Diener (Dordrecht: Springer), 247–266. Howell, A. J. (2009). Flourishing: achievement-related correlates of students’ well-being. J. Posit. Psychol. 4, 14–13. doi: 10.1080/174397608020 43459 January 2019 | Volume 10 | Article 35 7 Frontiers in Psychology | www.frontiersin.org Pozo Muñoz and Bretones Nieto Flourishing Scale in Childhood Cancer (FWS-SF): Results and implications of a South African study. J. Psychol Afr. 27, 303–309. doi: 10.1080/14330237.2017.1347748 Howell, A. J., Keyes, C. L. M., and Passmore, H. A. (2013). “Flourishing among children and adolescents: structure and correlates of positive mental health, and interventions for its enhancement,” in Research, Applications, and Interventions for Children and Adolescents: A Positive Psychology Perspective, eds C. Proctor and P.A. Linley (New York, NY: Springer). p. 59–80. Schotanus-Dijkstra, M., Pieterse, M. E., Drossaert, C. H. C., Westerhof, G. J., de Graaf, R., ten Have, M., et al. (2016). What factors are associated with flourishing? Results from a large representative national sample. J. Happiness Stud. 17, 1351–1370. doi: 10.1007/s10902-015-9647-3 Stud. 17, 1351–1370. doi: 10.1007/s10902-015-9647-3 Huppert, F. REFERENCES A., and So, T. T. (2013). Flourishing across europe: application of a new conceptual framework for defining well-being. Soc. Indic. Res. 110, 837–861. doi: 10.1007/s11205-011-9966-7 Seligman, M. E. P. (2011). “Flourish,” in What is wellbeing? ed M. E. P Seligman (Barcelona: Ediciones B), 18–43. Huppert, F. A., and So, T. T. C. (2009). “What percentage of people in Europe are flourishing and what characterizes them?,” in Presented at the Meeting of the OECD/ISQOLS Meeting (Florence). Silva, A., and Caetano, A. (2013). Validation of the flourishing scale and scale of positive and negative experience in Portugal. Soc. Indic. 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Moral Educ. 44, 310–324. doi: 10.1080/03057240.2015.1043878 Nunes, P., da Silva, B., Gomes, L. H., Correa, K., and Veloso, V. (2015). Flourishing scale: evidence of its suitability to the brazilian context. Soc. Inq. Well Being 1, 33–40. January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org REFERENCES doi: 10.13165/SIIW-15-1-2-07 Wright, B., and Stone, M. (1999). Measurement Essentials. Wilmington: Wide Range. g Pozo, C., Garzón, A., Bretones, B., and Ligia, C. (2016). Psychometric properties and dimensionality of the “Flourishing Scale” in Spanish speaking population. Electr. J. Res. Ed. Psychol. 14, 175–192. doi: 10.14204/ejrep.38.15044 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Prieto, G., Delgado, A. R., Perea, M. V., and Ladera, V. (2011). Differential functioning of mini-mental test items according to disease. Neurología 26, 474–480. doi: 10.1016/j.nrl.2011.01.013 Copyright © 2019 Pozo Muñoz and Bretones Nieto. 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. Ramírez, C., Correa, M., Ricas, T., López, A. E., Serrano, E. R., and Esteve, R. (2017). Psychometric characteristics of the Flourishing Scale-Spanish Version (FS-SV). The factorial structure in two samples: Students and patients with chronic pain. Pers. Indiv. Differ. 117, 30–36. doi: 10.1016/j.paid.2017.05.035 Rautenbach, C., and Rothmann, S. (2017). Special section: measures validation psychometric validation of the Flourishing-at-Work Scale – Short Form January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 8
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Archigregarines of the English Channel revisited: New molecular data on Selenidium species including early described and new species and the uncertainties of phylogenetic relationships
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RESEARCH ARTICLE Editor: Ross Frederick Waller, University of Cambridge, UNITED KINGDOM Editor: Ross Frederick Waller, University of Cambridge, UNITED KINGDOM Received: May 30, 2017 Accepted: October 19, 2017 Published: November 3, 2017 Copyright: © 2017 Rueckert, Hora´k. 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. Archigregarines of the English Channel revisited: New molecular data on Selenidium species including early described and new species and the uncertainties of phylogenetic relationships Sonja Rueckert1*, Alesˇ Hora´k2,3 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 School of Applied Sciences, Edinburgh Napier University, Edinburgh, United Kingdom, 2 Biology Centre of CAS, Institute of Parasitology, Česke´ Budějovice, Czech Republic, 3 University of South Bohemia, Faculty of Science, Česke´ Budějovice, Czech Republic * s.rueckert@napier.ac.uk OPEN ACCESS Citation: Rueckert S, Hora´k A (2017) Archigregarines of the English Channel revisited: New molecular data on Selenidium species including early described and new species and the uncertainties of phylogenetic relationships. PLoS ONE 12(11): e0187430. https://doi.org/10.1371/ journal.pone.0187430 Background Gregarines represent an important transition step from free-living predatory (colpodellids s.l.) and/or photosynthetic (Chromera and Vitrella) apicomplexan lineages to the most important pathogens, obligate intracellular parasites of humans and domestic animals such as coccidians and haemosporidians (Plasmodium, Toxoplasma, Eimeria, Babesia, etc.). While dozens of genomes of other apicomplexan groups are available, gregarines are barely entering the molecular age. Among the gregarines, archigregarines possess a unique mixture of ancestral (myzocytosis) and derived (lack of apicoplast, presence of subpellicular microtubules) features. Editor: Ross Frederick Waller, University of Cambridge, UNITED KINGDOM Received: May 30, 2017 Accepted: October 19, 2017 Published: November 3, 2017 Copyright: © 2017 Rueckert, Hora´k. 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 Gregarine apicomplexans are obligate, unicellular parasites of freshwater, marine and terres- trial invertebrates infecting the intestine, coelom and reproductive vesicles. As gregarines do not generally cause any harmful diseases compared to their well-studied closely related sis- tergroups known as notorious parasites of humans and domestic animals (e.g. Plasmodium, Cryptosporidium and Toxoplasma), they are still an undersampled and little described group of protists. There is a strong need to overhaul the complete gregarine taxonomy and Cavalier- Smith [1] made an attempt in doing so. Unfortunately, his way to tackle the problem by erect- ing 18 new taxa mainly based on a single SSU rDNA analysis is rather preliminary. The poor support of most of the clades and branching orders in question does not allow for these taxo- nomic conclusions to be made. At this point, we will therefore stick with the separation of the gregarines into the three major groups (archigregarines, eugregarines and neogregarines) based on trophozoite morphology, host affinity and life history (e.g. [2–9]) The first time a gregarine was officially described was in 1828 by Dufour, who erected the genus Gregarina [2]. While in the early years of gregarine discovery, morphological descrip- tions were based exclusively on light microscopic and histologic studies, the importance of ultrastructural studies using electron microscopy became quickly apparent and shed important light on differences e.g. between archigregarines and eugregarines (e.g. [10–15]). These data showed that archigregarines have wide, longitudinal epicytic folds with an underlying layer of subpellicular microtubules, while eugregarines have narrow epicytic folds with apical filaments and rippled dense structures (see [16]). Nonetheless, available data are still quite limited. In the last decades the molecular approach for phylogenetic analyses started to boom and several genomes of the crown apicomplexans including Plasmodium, Toxoplasma, Crytosporidium, Babesia, etc. are already available (e.g. [17,18]). The genomes of photosynthetic apicomplexan lineages including Chromera velia and Vitrella brassicaformis have been published recently [19], but gregarine apicomplexans have hardly entered the genomic age (apart from a very par- tial genome draft of Ascogregarina taiwanensis [20]). The importance of molecular phyloge- netic data and their value in inferring relationships between gregarine species has lead to an increase in available sequence data in public databases such as GenBank, but these are still very limited in numbers and mostly restricted to SSU rDNA sequences. Archigregarines of the English Channel Conclusions/Significance No. 15-17643S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Our phylogenies using the extended gregarine sampling show that the archigregarines are indeed not monophyletic with one strongly supported clade of Selenidium sequences around the type species S. pendula. We suggest the revision of the whole archigregarine taxonomy with only the species within this clade remaining in the genus Selenidium, while the other species should be moved into newly erected genera. However, the SSU rDNA phylogenies show very clearly that the tree topology and therefore the inferred relationships within and in between clades are unstable and such revision would be problematic without additional sequence data. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Methodology/Principal findings In this study we revisited five of the early-described species of the genus Selenidium includ- ing the type species Selenidium pendula, with special focus on surface ultrastructure and molecular data. We were also able to describe three new species within this genus. All spe- cies were characterized at morphological (light and scanning electron microscopy data) and molecular (SSU rDNA sequence data) levels. Gregarine specimens were isolated from poly- chaete hosts collected from the English Channel near the Station Biologique de Roscoff, France: Selenidium pendula from Scolelepis squamata, S. hollandei and S. sabellariae from Sabellaria alveolata, S. sabellae from Sabella pavonina, Selenidium fallax from Cirriformia tentaculata, S. spiralis sp. n. and S. antevariabilis sp. n. from Amphitritides gracilis, and S. opheliae sp. n. from Ophelia roscoffensis. Molecular phylogenetic analyses of these data showed archigregarines clustering into five separate clades and support previous doubts about their monophyly. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This project was supported by ASSEMBLE EU FP7 research infrastructure initiative (EU-RI-227799) call 7 (2013) and call 9 (2014), the research infrastructure funded from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n˚ 316304 as well as the Czech Grant Agency project 1 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Collection and isolation of organisms Different polychaete species were collected by hand from rocks and sandy beaches from the English Channel near the Station Biologique Roscoff (SBR), France, in May 2013 and in June 2014. No specific permits were required for the sampling locations as they are not privately owned or protected. This study did not involve any endangered or protected species. The collected polychaetes were identified based on morphological characteristics. Selenidium pendula was iso- lated from the intestines of Scolelepis squamata (Mueller, 1806) (Spionida) collected by hand from a beach close to the SBR (48˚43´31´´N, 3˚59´26´´W). Selenidium hollandei and S. sabellariae were isolated from the intestines of Sabellaria alveolata (Linnaeus, 1767) (Sabellida) collected by hand from Saint Efflam (48˚68´48´´N, 3˚61´13´´W). Selenidium sabellae was isolated from the intes- tines of Sabella pavonina Savigny, 1822 (also Sabellida) hand-collected from Pempoul (48˚68´23 ´´N, 3˚95´14´´W). Selenidium fallax was isolated from Cirriformia tentaculata (Montagu, 1808), S. spiralis sp. n. and S. antevariabilis sp. n. were isolated from Amphitritides gracilis (Grube, 1860) (both Terebellida) both polychaete species were collected by hand from rock assemblages close to the SBR (48˚43’44”N, 3˚59’23”W). Selenidium opheliae sp. n. was isolated from Ophelia roscoffensis Augener, 1910 (Opheliidae) hand-collected from Le Guillec (48˚68´56´´N, 4˚06´78´´W). The trophozoites of each species were released in seawater by teasing apart the intestines of the respective host with fine-tipped forceps under a dissecting microscope (Zeiss Stemi 2000). The gut material was examined under an inverted microscope (Zeiss Axiovert A1) and para- sites were isolated with a hand drawn glass pipette and washed three times in filtered seawater, before being examined and photographed under the inverted microscope or prepared for DNA extraction. Introduction One general problem with molecular phylogenies is that the outcome often heavily depends on the original species dataset utilized for the analyses [21]. Archigregarines, which are found only in marine invertebrates, especially polychaetes, are of particular interest, as they are supposed to form the paraphyletic stem group from which all other gregarines and maybe even all apicomplexans have evolved [2,22–26]. Archigregarines PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 2 / 24 Archigregarines of the English Channel possess a unique mixture of ancestral (myzocytosis; e.g. [27]) and derived (lack of apicoplast, presence of subpellicular microtubules; e.g. [3, 28,29]) features. There are around ~80 archigre- garine species described to date [11,25,26,29]. Around 65 described species belong to the genus Selenidium. Levine [3] proposed to erect a new genus Selenidioides to accommodate those 11 species that show merogony, belonging truly to the archigregarines, while the rest of the Seleni- dium species was shuffled into the order Eugregarinorida. Because it is difficult to prove or dis- prove the presence or absence of merogony/schizogony (asexual reproduction of trophozoite stage) within a lifecycle, we and many other authors do not believe it to be a good indicator for the relationships among archigregarines [16,25,26,29–32]. Therefore, we will not follow this split of Selenidium species and thus treat Selenidium as the main genus of archigregarines. Just recently Schre´vel et al. [33] published for the first time molecular data for the type species Seleni- dium pendula. The presented phylogenies show one clade of true Selenidiidae around S. pendula and two other clades of archigregarines, all reflecting their respective host species. In this study we set out to obtain molecular data of morphologically and ultrastructurally well-described species including the type species Selenidium pendula, S. hollandei, S. sabellar- iae, S. sabellae and S. fallax. In addition, we were able to discover three new species. We will present morphological characterizations of all species by light and scanning electron micros- copy and discuss the phylogenetic relationships of archigregarines within the context of a broad apicomplexan phylogeny based on SSU rDNA data. Archigregarines of the English Channel Between 8 and 60 specimens each of the isolated gregarine species were prepared for scan- ning electron microscopy (SEM). Some individuals were deposited directly into the threaded hole of separate Swinnex filter holders, containing a 10 μm polycarbonate membrane filter (Millipore Corp., Billerica, MA), that was submerged in 10 ml of seawater within a small canis- ter (2 cm diam. and 3.5 cm tall). A piece of Whatman No. 1 filter paper was mounted on the inside base of a beaker (4 cm diam. and 5 cm tall) that was slightly larger than the canister. The Whatman filter paper was saturated with 4% (w/v) OsO4 and the beaker was turned over the canister. The parasites were fixed by OsO4 vapors for 30 min. Ten drops of 4% (w/v) OsO4 were added directly to the seawater and the parasites were fixed for an additional 30 min. A 10-ml syringe filled with distilled water was screwed to the Swinnex filter holder and the entire apparatus was removed from the canister containing seawater and fixative. Other individuals were deposited in hand-made baskets (top end of a 1000μl pipette tip fixed with silicon to the same 10 μm filters) and placed in 24-well culture plates. These filters were prefixed in 2.5% (v/ v) glutaraldehyde in 0.05 M cacodylate buffer (pH = 7.4) for one hour, washed and post-fixed with 1% (w/v) OsO4 for 45 minutes. All filters were washed with water and dehydrated with a graded series of ethyl alcohol. Filters prepared in the Swinnex filter holders were critical point dried with CO2, filters from the hand-made baskets were air-dried. Filters were mounted on stubs, sputter coated with 5 nm of gold, and viewed under a scanning electron microscope (JEOL JBM7401F or Phenom 806). Some SEM data were presented on a black background using Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, CA). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Light and scanning electron microscopy Differential interference contrast (DIC) and phase contrast (PC) light micrographs of all spe- cies were taken with a 5 megapixel CMOS camera AxioCam ERc 5s, attached to an inverted microscope (Zeiss Axiovert 1). 3 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Molecular phylogenetic analysis The eight new SSU rDNA sequences were aligned with 163 other SSU rDNA sequences, repre- senting the major lineages of apicomplexans (with an emphasis on gregarines) and relevant outgroups (ciliates, dinozoans and chromerids and colpodellids), using local alignment with generalized affine gap costs (E-INS-I) as implemented in MAFFT [35]. Sites comprised mostly of gaps and ambiguously aligned regions were manually excluded from the 171-taxon align- ment in SEAVIEW 4 [36] resulting in 1,488 unambiguously aligned sites (a NEXUS file of this alignment is available upon request). Alternatively, we have used trimAl 1.2 [37] for automatic ambiguous sites detection/exclusion under relaxed (parameters set as ’-gt 0.3’ and ’-st 0.001’; dataset R, 1805 sites) as well as strict (-gappyout option engaged; dataset G, 945 sites) settings. Based on the results of a preliminary analyses and in agreement with previously published studies, we have also created a smaller dataset (S) based on the exhaustive sampling of Seleni- diidae s.s. (as defined by [33]), as well as other archigregarine taxa (81 taxa, 1601 sites) rooted with marine eugregarines. Homogeneity of base-composition was tested using Tree-Puzzle 5.3 [38]. Root-to-tip distances for main clades of the dataset were measured using the TreeStat 1.2 (http://tree.bio.ed.ac.uk/software/treestat/). Because the 18S rDNA of several gregarine taxa did not pass the homogeneity test, the Bayesian inference was carried out with exchange rates defined by the general time-reversible model and the number of categories limited to 40 (GTR + C40), implemented in Phylobayes 4.1 [39]. This model was chosen as a compromise between the robustness of CAT admixture model with an infinite number of rate categories, which is however mostly suitable for large phylogenomic datasets due to its complexity, and simplicity of time-tested GTR model. Two chains were run until they converged (i.e. the maximum observed discrepancy below 0.2 and effective number of model parameters reached 100). Posterior probabilities of the branching were reconstructed after burn-in of the first fifth of the generations. Alternatively, we have also performed a maximum likelihood analysis under the gamma-corrected GTR model using the RAxML 8.2a [40]. The highest-scoring topology was estimated using the rapid-bootstrapping algorithm from 1000 replicates, the branching support was assessed using the non-parametric bootstrapping and the ‘thorough’ algorithm from 1000 replicates using the same model and software. The putative monophyly of archigregarines was tested using the approximate likeli- hood ratio test as implemented in Consel [41]. DNA isolation, PCR, cloning, and sequencing Individual trophozoites of each species were isolated from the dissected hosts, washed three times in filtered seawater, and deposited into a 1.5-ml microcentrifuge tube: 23 trophozoites of S. pen- dula from S. squamata, 27 trophozoites of S. hollandei and 75 trophozoites of S. sabellariae from Sabellaria alveolata, 8 trophozoites of S. sabellae from Sabella pavonina, 45 cells of Selenidium fal- lax from Cirriformia tentaculata, 11 and 12 trophozoites of S. spiralis sp. n. and S. antevariabilis sp. n. from Amphitritides gracilis, and 31 trophozoites of S. opheliae sp. n. from Ophelia sp. DNA was extracted using the MasterPureTM Complete DNA and RNA Purification Kit (Epicentre Biotechnologies, Madison, WI). Small subunit rDNA (SSU rDNA) sequences were PCR-amplified either using a total volume of 50 μl containing 1 μl of primer, 5 μl of DNA tem- plate, 25 μl of OneTaq Mastermix (New England Biolabs, Inc., Ipswich, USA), or using a total volume of 25μl containing 2 μl of primer, 2.5 μl of DNA template, 20.5 μl of dH2O and one PuReTaq Ready-to-go PCR Bead (GE Healthcare, Quebec, Canada). The SSU rDNA sequences from these species were amplified in one fragment (~1800 bp) using universal eukaryotic PCR primers F1 5´-GCGCTACCTGGTTGATCCTGCC-3´ and R1 5´-GATCCTTCTGCAGGTTCACCTAC-3´ [34] and internal primers designed to match exist- ing eukaryotic SSU sequences F2 5´-AAGTCTGGTGCCAGCAGCC-3´ and R2 5´-TTTAAG TTTCAGCCTTGCG-3´.PCR was performed using MJ MiniTM Gradient Thermal Cycler (Bio- Rad) and the following protocol: After 4 cycles of initial denaturation at 94 oC for 4.5 min, 45 oC for 1 min and 72 oC for 1.75 min, 34 cycles of 94 oC for 30 sec (denaturation), 50 oC for 1 min (annealing), 72 oC for 1.75 min (extension), followed by a final extension period at 72 oC for 10 min. PCR products corresponding to the expected size were gel isolated using the Ultra- CleanTM 15 DNA Purification kit (MO Bio, Carlsbad, California) and cloned into the pCR 2.1 vector using the TOPO TA cloning kit (Invitrogen, Frederick, MD). Eight cloned plasmids were digested with EcoRI and screened for size. One or two clones for each species were se- quenced with ABI big dye reaction mix using vector primers and internal primers oriented in both directions. DNA isolation, PCR, cloning, and sequencing PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 4 / 24 Archigregarines of the English Channel The new SSU rDNA sequences were initially identified by BLAST analysis and subsequently verified with molecular phylogenetic analyses (GenBank Accession numbers: Selenidium pen- dula MF882901, Selenidium hollandei MF882899, Selenidium sabellariae MF882900, Seleni- dium sabellae MF882906, Selenidium fallax MF882905, Selenidium spiralis sp. n. MF882902, Selenidium antevariabilis sp. n. MF882903, Selenidium opheliae sp. n. MF882904). Molecular phylogenetic analysis Prior to the analysis, we have optimized the topology and branch lengths of the starting tree with forced monophyly of archigregarine sequences in RAxML. Morphological observations All given measurements are based on light micrographs from fresh material, as some of the gregarines seemed to have shrunk during preparation for scanning electron microscopy. All trophozoites were brownish in colour under the LM, reflecting an accumulation of amylopec- tin granules within the cytoplasm. Selenidium pendula (Fig 1, Table 1). Trophozoites were isolated from the polychaete Scolelepis squamata (Mueller, 1806). The morphology matched the original description of Fig 1. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium pendula isolated from the polychaete Scolelepis squamata. A-C. DIC micrographs showing a spindle-shaped trophozoite in different positions of the pendular movement. The mucron at the anterior end is rounded (arrowhead) and visually separated from the rest of the cell with a slight restriction. The axial canal (ac) is conspicuous at the anterior end, but ran along the entire cell. The oval nucleus (n) is situated in the middle of the cell. D-E. Two gamonts (G1, G2) in caudal syzygy. The mucrons (arrowhead) are visible. The double arrow marks the junction between the two gamonts. Gamonts are changing from elongated to stumpy. F. Young gametocyst, the junction (double arrow) is still visible. G. Gametocysts in different focal planes, packed with round oocysts. H-K. SEM micrographs of trophozoites showing the epicytic longitudinal folds (arrow), the mucron (arrowhead) free of folds and some transverse striations in the inner curvature of the cell. Scale bars: Fig 1A-E, 30 μm; Fig 1F, 50 μm; Fig 1G, 70 μm; Fig 1H-I, 5μm; Fig J-K, 2μm. https://doi org/10 1371/journal pone 0187430 g001 Fig 1. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium pendula isolated from the polychaete Scolelepis squamata. A-C. DIC micrographs showing a spindle-shaped trophozoite in different positions of the pendular movement. The mucron at the anterior end is rounded (arrowhead) and visually separated from the rest of the cell with a slight restriction. The axial canal (ac) is conspicuous at the anterior end, but ran along the entire cell. The oval nucleus (n) is situated in the middle of the cell. D-E. Two gamonts (G1, G2) in caudal syzygy. The mucrons (arrowhead) are visible. The double arrow marks the junction between the two gamonts. Gamonts are changing from elongated to stumpy. F. Nomenclatural acts The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomen- clatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated infor- mation viewed through any standard web browser by appending the LSID to the prefix "http:// zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:91B0E976-C459- 5 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Archigregarines of the English Channel 46E5-9B93-FA96177BC9CA. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, Edinburgh Napier Repository (http://www.napier.ac.uk/research-and- innovation/repository). 46E5-9B93-FA96177BC9CA. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, Edinburgh Napier Repository (http://www.napier.ac.uk/research-and- innovation/repository). Archigregarines of the English Channel Table 1. Morphological comparison of Selenidium species presented in this study and other species that belong to the same clades in the phylogenetic tree (data from this study highlighted in bold). species Host Locality Host tissue Trophozoite shape Trophozoite size (L x W, μm) Nucleus shape Nucleus size (L x W, μm) Position of nucleus Motility # of long. epicytic folds Trans. surface folds Shape of mucron SSU rDNA Ref. S. pendula Nerine cirratulus E. Atlantic Intestine spindle-shaped 180 x 30–40 round to oval 18–33 x 13–32 middle bending, twisting, pendulum-like 20–30 ? pointed - [3,43] S. pendula Scolelepis squamata E. Atlantic Intestine spindle-shaped 147–260 x 12– 48 oval 16–28 x 7–22 middle bending, twisting, pendulum-like 30–34 + rounded + this study S. terebellae Terebella lapidaris E. Pacific, E. Atlantic Intestine spindle-shaped 30–330 x 12–40 oval (ellipsoidal) 35 x 15 posterior half Bending, twisting 46.00 + Pointed to slightly round + [3, 26] S. hollandei Sabellaria alveolata E. Atlantic Intestine spindle-shaped, elongate, flattened up to 500 x 20– 30 spherical or ovoid 16 x 6–8 ? bending, twisting about 16 ? heart-shaped - [3] S. hollandei Sabellaria alveolata E. Atlantic Intestine elongated, flattened 97–411 x 13–43 spherical or ovoid 4–19 x 6–17 middle to posterior half bending, twisting, rapid rolling 18 + heart-shaped, sometimes rounded + this study S. sabellariae Sabellaria alveolata E. Atlantic Intestine spindle-shaped 220–500 x 16– 25 ovoid 16 x 13 ? bending, twisting ? + pointed, spine present - [3] S. sabellariae Sabellaria alveolata E. Atlantic Intestine spindle shaped, slightly flattened 68–227 x 8–23 ovoid 7–15 x 6–13 middle quick coiling & uncoiling 14-Dec + pointed + this study S. neosabellaria Neosabellaria cementarium W. Pacific Intestine vermiform 125–350 x 9–12 spherical 10 middle bending, twisting, undulated 12-Oct + nipple-shaped + [49] S. sabellae Sabella pavonina E. Atlantic Intestine 24–120 x 8–60 spherical ? ? ? 24–40 ? conical - [3] S. sabellae Sabella pavonina E. Atlantic Intestine rectangualar’, flattened 40–85 x 10–16 oval 6–12 x 5–11 middle bending, contracting 20–40 - cone-shaped + this study S. fallax Cirriformia tentaculata E. Atlantic Intestine spindle-shaped 300–500 x 10–30 ? ? ? bending, twisting 90 ? knob-like - [3,26] S. fallax Cirriformia tentaculata E. Atlantic Intestine spindle-shaped 116–264 x 10–26 ellipsoid 3–9 x 6–17 anterior third twisting. Quick coiling 40–45 - flat-topped tip + this study S. idanthyrsae Idanthyrsus alveolata W. Morphological observations Young gametocyst, the junction (double arrow) is still visible. G. Gametocysts in different focal planes, packed with round oocysts. H-K. SEM micrographs of trophozoites showing the epicytic longitudinal folds (arrow), the mucron (arrowhead) free of folds and some transverse striations in the inner curvature of the cell. Scale bars: Fig 1A-E, 30 μm; Fig 1F, 50 μm; Fig 1G, 70 μm; Fig 1H-I, 5μm; Fig J-K, 2μm. 6 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 The two individuals in syzygy underwent continuous change from elongated cells, which showed active movement to more stumpy cells (Fig 1E), which did show restricted movement. Different developmental stages of the gamontocyst and gametocyst were observed (Fig 1F and 1G). The gametocyst was oval [129 (103–146) μm x 88 (61–106) μm, n = 5]. The junction between the two cells was still visible in the young gametocyst. (Fig 1F). At the end of sporogony the gametocyst was packed with spherical oocysts (Fig 1G). The oocysts contain- ing 4 sporozoites were 13.1 μm in diameter (11.8–14.5μm, n = 22). The SEM micrographs demonstrated that there were around 30–34 longitudinal epicytic folds inscribing the surface of the trophozoites. The anterior end was free of folds (Fig 1H, 1I and 1K), while some of the folds terminated towards the posterior end. In bended and shortened cells, transverse folds appeared on the longitudinal epicytic folds (Fig 1K). In the middle of the cell, the density of longitudinal folds was 1 fold/micron (Fig 1J). Single trophozoites and two individuals in early syzygy were capable of pendular movement, which gave this type species of Selendium its name S. pendula [42]. Selenidium hollandei (Fig 2, Table 1). The trophozoites of this archigregarine were isolated from the intestines of the polychaete Sabellaria alveolata (Linnaeus, 1767). The morphology matched the original description of S. hollandei by Vivier and Schre´vel [13] and several accounts in studies on different aspects that followed [3,10,11]. Trophozoites were mostly elongated and extremely flattened (Fig 2A, 2B, 2H and 2I). They were 250 μm (97–411 μm, n = 25) long and 22 μm (13–43 μm, n = 25) wide (Fig 2A–2D). The anterior and posterior end were both heart-shaped, but the anterior end was often a lot narrower than the posterior end (Fig 2B and 2I). The anterior end showed a slight middle ridge in some specimens (Fig 2). In some cases the anterior end was rounded and swollen and the posterior end was blunt (Fig 2D). The nucleus was spherical or ovoid and measured 9 x 10 μm (4–19 x 6–17 μm, n = 6) (Fig 2C–2E), situated in the middle of the trophozoite or slightly shifted towards the posterior end. The SEM showed broad epicytic folds covering the cell (Fig 2F–2I). The density of the folds was up to 1 fold/micron (Fig 2H and 2J). Pacific Intestine spindle-shaped, partially flattened 450–543 x 9–11 spherical 13–16 x 9–10 middle to posterior half bending, twisting 20–22 + pointed + [26] S. melongena Thelepus japonicus W. Pacific Coelom oval, ’eggplant- shaped’ 30–155 x 10–41 spherical 19-Jul middle none 30 - neck-like, pointed + [50] S. cf. mesnili Myxicola infundibulum W. Pacific Intestine spindle-shaped 85–157 x 18–24 ellipsoidal 7–9 x 10–11 anterior to posterior bending, coiling 22–24 + pointed to round + [26] S. boccardiellae Boccardiella ligerica W. Pacific Intestine spindle-shaped, partially flattened 87–250 x 10–12 ellipsoidal 10–12 x 4–6 anterior half bending, coiling, thrashing 12-Oct - pointed + [26] S. sensimae Spirobranchus giganteus E. Pacific Intestine spindle shaped 130–170 x 10–13 ellipsoidal 10 x 4–6 middle slow bending, twisting 16–18 - rounded to blunt + [49] S. cf.echinatum Dodecaceria concharum W. Pacific Intestine spindle-shaped 95–205 x 8–11 spherical 10 anterior half bending, twisting 12-Oct - nipple-shaped + [49] S. spiralis sp. n. Amphitritides gracilis E. Atlantic Intestine spindle-shaped 105–194 x 21–37 ovoid 10–21 x 15–25 posterior half little bending 6 (12) + rounded + this study S. antevariabilis sp. n. Amphitritides gracilis E. Atlantic Intestine spindle-shaped 131–190 x 22–31 ovoid 13–21 x 15–20 middle little bending, plastic mucron 6 ? plastic + this study S. opheliae sp. n. Ophelia roscoffensis E. Atlantic Intestine sugar snap pea- like’ 123–177 x 10–16 ovoid 10–16 x 7–11 middle to posterior half little bending none + elongated, rounded + this study PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 7 / 24 Archigregarines of the English Channel S. pendula by Giard [42] and several accounts in studies on different aspects that followed [10,11,33,43]). The cells were elongated and spindle-shaped (Fig 1). Trophozoites were 200 μm (147–260 μm, n = 15) long and 25 μm (12–48 μm, n = 15) wide. There was a slight restriction between the anterior end and the rest of the body. The anterior end was globular and rounded, while the posterior end was tapering into a pointed tip (Fig 1A–1C, 1H and 1I). The axial canal was visible along the entire cell starting from the restriction at the anterior end to the tip of the posterior end (Fig 1B). The oval nucleus [20 (16–28) μm x 13 (7–22), n = 10] was situated in the middle of the cell (Fig 1A and 1B). Mature trophozoites (or gamonts) pair up in caudal syz- ygy (Fig 1D). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 None of the folds terminated on either end of the cell. When the trophozoites were maximally contracted there were densely packed transverse striations/folds visible (Fig 2F). Micropores were situated in the shallow grooves between the epicytic folds (Fig 2J). Syzygy between two cells was observed showing a dorso-ventrally overlap of the two posterior ends (Fig 2E and 2G). Single trophozoites showed rapid rolling movements. Selenidium sabellariae (Fig 3, Table 1). Trophozoites of this archigregarine species were also isolated from the polychaete Sabellaria alveolata. The morphology matched the original description of S. sabellariae by Schre´vel [43] and accounts in later studies (e.g. [3]). The tro- phozoites were spindle-shaped and slightly flattened dorso-ventrally (Fig 3A–3C and 3F). Tro- phozoites were 137 μm (68–227 μm, n = 19) long and 16 μm (8–23 μm, n = 19) wide (Fig 3A and 3B). The anterior and posterior end both tapered into pointed tips. The ovoid nucleus 11 x 9 μm (7–15 x 6–13 μm, n = 15) situated in the middle of the cell contained a circular nucleolus [5 (4–6) μm in diameter, n = 10) (Fig 3B and 3C). The SEM revealed around 14 epi- cytic longitudinal folds (Fig 3F–3H). These folds split ‘superficially’ into two folds shortly after PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 8 / 24 Archigregarines of the English Channel Fig 2. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium hollandei isolated from the polychaete Sabellaria alveolata. A-B. DIC micrographs of elongated and flattened trophozoites. The anterior end (mucron area, arrowhead) is narrower than the posterior end of the cell. C-D. Contracted trophozoites during peristaltic movement. The ovoid nucleus is visible (n). Broad epicytic folds (arrow) inscribe the surface of the entire cell. E-F. DIC and SEM micrographs of two gamonts (G1, G2) in lateral syzygy with overlapping posterior ends (double arrow). G-I. Trophozoites in different stages of movement. When contracted or bended, transverse striations (double arrow) form on the surface. The mucron (arrowhead) at the anterior end is free of folds. The broad longitudinal epicytic folds (arrows) cover most of the body. The lateral view shows dorso-ventrally extremely flattened trophozoites. J. Longitudinal epicytic folds (arrow) with visible pores (arrowheads) in the grooves between the folds. Scale bars: Fig 2A, C-D, G-H, 20 μm; Fig 2B, E-F, 30 μm; Fig 2I, 25 μm; Fig 2J, 5 μm. Fig 2. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium hollandei isolated from the polychaete Sabellaria alveolata. A-B. DIC micrographs of elongated and flattened trophozoites. The anterior end (mucron area, arrowhead) is narrower than the posterior end of the cell. C-D. Contracted trophozoites during peristaltic movement. The ovoid nucleus is visible (n). Broad epicytic folds (arrow) inscribe the surface of the entire cell. E-F. DIC and SEM micrographs of two gamonts (G1, G2) in lateral syzygy with overlapping posterior ends (double arrow). G-I. Trophozoites in different stages of movement. When contracted or bended, transverse striations (double arrow) form on the surface. The mucron (arrowhead) at the anterior end is free of folds. The broad longitudinal epicytic folds (arrows) cover most of the body. The lateral view shows dorso-ventrally extremely flattened trophozoites. J. Longitudinal epicytic folds (arrow) with visible pores (arrowheads) in the grooves between the folds. Scale bars: Fig 2A, C-D, G-H, 20 μm; Fig 2B, E-F, 30 μm; Fig 2I, 25 μm; Fig 2J, 5 μm. Fig 2. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium hollandei isolated from the polychaete Sabellaria alveolata. A-B. DIC micrographs of elongated and flattened trophozoites. The anterior end (mucron area, arrowhead) is narrower than the posterior end of the cell. C-D. Contracted trophozoites during peristaltic movement. The ovoid nucleus is visible (n). Broad epicytic folds (arrow) inscribe the surface of the entire cell. E-F. DIC and SEM micrographs of two gamonts (G1, G2) in lateral syzygy with overlapping posterior ends (double arrow). G-I. Trophozoites in different stages of movement. When contracted or bended, transverse striations (double arrow) form on the surface. The mucron (arrowhead) at the anterior end is free of folds. The broad longitudinal epicytic folds (arrows) cover most of the body. The lateral view shows dorso-ventrally extremely flattened trophozoites. J. Longitudinal epicytic folds (arrow) with visible pores (arrowheads) in the grooves between the folds. Scale bars: Fig 2A, C-D, G-H, 20 μm; Fig 2B, E-F, 30 μm; Fig 2I, 25 μm; Fig 2J, 5 μm. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 https://doi.org/10.1371/journal.pone.0187430.g002 the mucron with rounded tip, which was free of folds, and merged again shortly before the posterior end (Fig 3G and 3H). There were numerous micropores visible in the grooves of the main folds, and none in the “superficially’ or secondary ones (Fig 3E). The density of the folds was 1 fold/micron at the anterior end (Fig 3G and 3H). Several transverse folds formed the base of the mucron (Fig 3G). Two cells in syzygy were laterally attached to each other with the gamonts oriented in opposite directions (Fig 3D). Single trophozoites showed quick coiling and uncoiling movements. Selenidium fallax (Fig 4, Table 1). Trophozoites of this archigregarine species infect the intestines of the polychaete Cirriformia tentaculata (Montagu, 1808). The morphology matched the original description of S. fallax by MacGregor & Thomasson [44] and accounts on different aspects that followed (e.g. [3,28]). The trophozoites were spindle-shaped (Fig 4A– 4D). Trophozoites were 174 μm (116–264 μm, n = 23) long and 16 μm (10–26 μm, n = 23) wide (Fig 4A–4D). The anterior end tapered into a flat-topped tip with a nipple-like structure in the middle (Fig 4A, 4B and 4E). The posterior end tapered into a pointy tip (Fig 4A–4D and 4F). The ellipsoid nucleus 5 x 11 μm (3–9 x 6–17 μm, n = 21) situated in the anterior third of the cell (Fig 1A–1C) contained a circular nucleolus [5 (4–5) μm in diameter, n = 7]. The SEM revealed around 40 to 45 epicytic longitudinal folds (Fig 4D and 4G) and the mucron being free of folds (Fig 4E). The surface right beneath the bulging rim of the mucron was inscribed PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 9 / 24 Archigregarines of the English Channel Fig 3. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium sabellariae isolated from the polychaete Sabellaria alveolata. A- C. DIC micrographs of trophozoites in different focal planes and different stages of movement. The mucron (arrowhead) is pointed to rounded and the ovoid nucleus (n) is situated in the middle of the cell. D. Two gamonts (G1, G2) in lateral syzygy with their orientation in opposite directions. The anterior ends are marked with arrowheads. Both gamonts are widest in the area of the junction (double arrow). E. Higher magnification SEM of the surface ultrastructure. There are numerous micropores (arrowheads) along the grooves of the main folds (arrow), whereas the grooves of the secondary folds (double arrow) do not display any micropores. F. Trophozoite, showing a rounded mucron (arrowhead) and longitudinal epicytic folds (arrow) with secondary folds expanding over parts of the cell’s length. G. Anterior end showing the rounded mucron (arrowhead) that is free of folds, but has a basal cluster of transverse striations (double arrowhead). Shortly after the mucron the main folds (arrow) start splitting (double arrowhead) into secondary folds. H. Posterior end of the trophozoite, showing the merging secondary folds (arrow), before they reach the posterior end. Scale bars: Fig 3A-D, 30 μm; Fig 3F, 20 μm; Fig 3E, G-H, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g003 Fig 3. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium sabellariae isolated from the polychaete Sabellaria alveolata. A- C. DIC micrographs of trophozoites in different focal planes and different stages of movement. The mucron (arrowhead) is pointed to rounded and the ovoid nucleus (n) is situated in the middle of the cell. D. Two gamonts (G1, G2) in lateral syzygy with their orientation in opposite directions. The anterior ends are marked with arrowheads. Both gamonts are widest in the area of the junction (double arrow). E. Higher magnification SEM of the surface ultrastructure. There are numerous micropores (arrowheads) along the grooves of the main folds (arrow), whereas the grooves of the secondary folds (double arrow) do not display any micropores. F. Trophozoite, showing a rounded mucron (arrowhead) and longitudinal epicytic folds (arrow) with secondary folds expanding over parts of the cell’s length. G. Anterior end showing the rounded mucron (arrowhead) that is free of folds, but has a basal cluster of transverse striations (double arrowhead). Shortly after the mucron the main folds (arrow) start splitting (double arrowhead) into secondary folds. H. Posterior end of the trophozoite, showing the merging secondary folds (arrow), before they reach the posterior end. Scale bars: Fig 3A-D, 30 μm; Fig 3F, 20 μm; Fig 3E, G-H, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g003 https://doi.org/10.1371/journal.pone.0187430.g003 by short, narrow, superficial, longitudinal folds (4E). The main folds emerged just after this area, split subsequently into two folds and merged again shortly before the posterior end (4F). The main grooves also called ‘primary’ grooves were deeper and wider than the superficial ‘secondary’ grooves (Fig 4E and 4G). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Micropores were only visible in the ‘primary grooves’. The density of the folds was up to 2 doublefolds/micron (Fig 4G). Free trophozoites showed quick coiling and uncoiling movements (Fig 4C). The motion also involved some helical twist- ing. The anterior end of the cell was always the center of the formed spiral (Fig 4C). After the trophozoites had been isolated for some time, they often remained in the coiled position. Selenidium sabellae (Fig 5, Table 1). Trophozoites of this archigregarine species were iso- lated from the intestines of the polychaete Sabella pavonina Savigny, 1822. The morphology matched the original description of S. sabellae by Lankester [45], its re-description by Ray [46], and several accounts in studies that followed (e.g. [3]). The cells were short and slightly dorso- ventrally flattened (Fig 5A, 5E and 5F). Trophozoites were 61 μm (40–85 μm, n = 22) long and 14 μm (10–16 μm, n = 23) wide. The anterior end tapered into a narrow, but long, cone-shaped tip. The posterior end was broadly rounded or blunt (Fig 5A and 5D–5F). The ovoid nucleus 9 x 7 μm (6–12 x 5–11 μm, n = 21) situated in the middle of the cell (Fig 5A and 5B), contained a circular nucleolus [4 (3–6) μm in diameter, n = 18]. In some cases the trophozoites were attached to a globular body (Fig 5C). The SEM revealed around 20–40 epicytic longitudinal folds (Fig 5E). The folds were quite narrow starting at the base of the mucron (free of folds), PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 10 / 24 Archigregarines of the English Channel Fig 4. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium fallax isolated from the polychaete Cirriformia tentaculata. A-C. DIC micrographs of trophozoites in different stages of movement. Trophozoits can be elongated to curled. The mucron (arrowhead) is rounded and sometimes quite flattened at the tip. The ellipsoid nucleus (n) is situated in the anterior part of the cell. D. SEM micrograph of a trophozoite, showing a flattened mucron (arrowhead) and longitudinal epicytic folds (arrows) with secondary folds expanding over parts of the cell’s length. The posterior end is very pointed. E. Anterior end showing the flat-topped mucron (arrowhead) that is free of folds, but has a nipple-like structure (arrow) in the middle. The upper rim appears to be a bit bulgy (double arrowhead). The region directly after the bulge is inscribed by short, narrow, superficial folds. The broader main folds start splitting (asterisks) into secondary folds shortly after. F. Posterior end of the trophozoite showing the tip free of folds, but with some indentations. G. Higher magnification SEM of the surface ultrastructure. The grooves between the secondary folds (asterisks) are often narrower than the grooves between the main folds (arrows). Scale bars: Fig 4A-B, 25 μm; Fig 4C, 15 μm; Fig 4D, 10 μm; Fig 4E-G, 1 μm. Fig 4. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium fallax isolated from the polychaete Cirriformia tentaculata. A-C. DIC micrographs of trophozoites in different stages of movement. Trophozoits can be elongated to curled. The mucron (arrowhead) is rounded and sometimes quite flattened at the tip. The ellipsoid nucleus (n) is situated in the anterior part of the cell. D. SEM micrograph of a trophozoite, showing a flattened mucron (arrowhead) and longitudinal epicytic folds (arrows) with secondary folds expanding over parts of the cell’s length. The posterior end is very pointed. E. Anterior end showing the flat-topped mucron (arrowhead) that is free of folds, but has a nipple-like structure (arrow) in the middle. The upper rim appears to be a bit bulgy (double arrowhead). The region directly after the bulge is inscribed by short, narrow, superficial folds. The broader main folds start splitting (asterisks) into secondary folds shortly after. F. Posterior end of the trophozoite showing the tip free of folds, but with some indentations. G. Higher magnification SEM of the surface ultrastructure. The grooves between the secondary folds (asterisks) are often narrower than the grooves between the main folds (arrows). Scale bars: Fig 4A-B, 25 μm; Fig 4C, 15 μm; Fig 4D, 10 μm; Fig 4E-G, 1 μm. https://doi.org/10.1371/journal.pone.0187430.g004 and enlarged towards the posterior end, where they terminated gradually (Fig 5D–5F). There were numerous micropores scattered along the grooves of the epicytic folds (Fig 5F and 5G). The density of the folds was 1 fold/micron at the anterior end (Fig 5E). Syzygy between two cells was observed showing a dorso-ventrally overlap of the two posterior ends. Trophozoites were able to bend, as well as expand and contract along their length (Fig 5B). Selenidium spiralis sp. n. (Fig 6, Table 1). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Trophozoites were isolated from the polychaete Amphitritides gracilis (Grube, 1860). The cells were spindle-shaped (Fig 6A–6C). Trophozoites were 139 μm (105–194 μm, n = 18) long and 31 μm (21–37 μm, n = 18) wide. The anterior end narrowed into a rounded mucron (Fig 6B and 6D) with a slight indentation setting it apart from the rest of the cell. The posterior end tapered into a more pointed tip (Fig 6E). The cells were wider in the posterior part of the trophozoite compared to the anterior part (Fig 6A–6C). The ovoid nucleus 12 x 20 μm (10–15 x 15–25 μm, n = 18) situated in the posterior half of the cell (Fig 6A and 6B), contained a circular nucleolus [7 (5–9) μm in diameter, n = 9). The tro- phozoites appeared to have a fishnet pattern on the surface in some of the micrographs (Fig 6A and 6B). The SEM revealed six epicytic longitudinal folds (Fig 6B and 6C). These folds split into two folds shortly after the mucron, which was free of folds, and merged again shortly before the posterior end (Fig 6A–6F). The main grooves also called ‘primary’ grooves were deeper than the superficial ‘secondary’ grooves (Fig 6C). The grooves between the secondary folds become gradually shallower towards the posterior end at which point they disappear PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 11 / 24 Archigregarines of the English Channel Fig 5. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium sabellae isolated from the polychaete Sabella pavonina. A. DIC micrographs of trophozoites in different focal planes. The mucron (arrowhead) is cone-shaped. The ellipsoid nucleus (n) is situated in the middle of the cell. Longitudinal epicytic folds (arrow) are visible along the cell. The posterior part ends in a dorso-ventrally flattened, blunt end. B. PC micrographs showing the plasticity of the cell. The nucleus (n) keeps its position in the middle of the trophozoite. C. DIC micrograph of an attached trophozoite with visible epicytic folds (arrow). D. High magnification SEM of the anterior end showing the cone-shaped mucron (arrowhead) that is free of folds. The longitudinal epicytic folds (arrows) start to broaden after their emergence. E. Trophozoite, showing the cone-shaped mucron (arrowhead) and longitudinal epicytic folds (arrows) that fan out right after the mucron. The posterior end is broadly rounded or blunt. F. Posterior end of trophozoite in lateral view, showing that the very tip is free of folds. The cell is dorso-ventrally flattened. There are micropores (arrowheads) visible in between the folds. G. Surface ultrastructure towards the posterior end. The epicytic folds (arrow) enlarge towards the posterior end, and gradually terminate. There are numerous micropores (arrowheads) along the grooves between the folds. Scale bars: Fig 5A, C, 20 μm; Fig 5B, 30 μm; Fig 5E, 10 μm; Fig 5F, D, 3 μm; Fig 5G, 1 μm. Fig 5. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium sabellae isolated from the polychaete Sabella pavonina. A. DIC micrographs of trophozoites in different focal planes. The mucron (arrowhead) is cone-shaped. The ellipsoid nucleus (n) is situated in the middle of the cell. Longitudinal epicytic folds (arrow) are visible along the cell. The posterior part ends in a dorso-ventrally flattened, blunt end. B. PC micrographs showing the plasticity of the cell. The nucleus (n) keeps its position in the middle of the trophozoite. C. DIC micrograph of an attached trophozoite with visible epicytic folds (arrow). D. High magnification SEM of the anterior end showing the cone-shaped mucron (arrowhead) that is free of folds. The longitudinal epicytic folds (arrows) start to broaden after their emergence. E. Trophozoite, showing the cone-shaped mucron (arrowhead) and longitudinal epicytic folds (arrows) that fan out right after the mucron. The posterior end is broadly rounded or blunt. F. Posterior end of trophozoite in lateral view, showing that the very tip is free of folds. The cell is dorso-ventrally flattened. There are micropores (arrowheads) visible in between the folds. G. Surface ultrastructure towards the posterior end. The epicytic folds (arrow) enlarge towards the posterior end, and gradually terminate. There are numerous micropores (arrowheads) along the grooves between the folds. Scale bars: Fig 5A, C, 20 μm; Fig 5B, 30 μm; Fig 5E, 10 μm; Fig 5F, D, 3 μm; Fig 5G, 1 μm. https://doi.org/10.1371/journal.pone.0187430.g005 completely (Fig 6C). The folds were more or less helically arranged along the cells body (Fig 6B and 6C). Micropores were observed in the ‘primary grooves’. The density of the folds was less than 1 fold/micron (Fig 6F). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 The main epicytic folds (double arrowheads) start right after the mucron and split very early into secondary folds. The grooves (asterisks) between the secondary folds appear quite deep from the anterior end and become gradually shallower towards the posterior end at which point they disappear completely. The cell is adorned with transverse striations (arrows) along its whole length apart from the anterior and posterior tip. D. The mucron (arrowhead) is free of folds. The very prominent transverse striations (arrow) start immediately posterior to the mucron, similar to the main epicytic folds (double arrowhead) and the split (asterisks) into secondary folds soon after. E. The transverse striations (arrows) continue to almost the tip of the posterior end, but are less prominent compared to the anterior end. One slight indentation (asterisk) of a secondary fold is visible on one of the main epicytic folds (double arrowhead). F. High magnification SEM of the transverse striations (arrow) and the grooves (asterisk) between the folds. Scale bars: Fig 6A-B, 25 μm; Fig 6C, 10 μm; Fig 6D-F, 2 μm. Fig 6. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium spiralis sp. n. isolated from the polychaete Amphitritides gracilis. A. PC micrograph showing the general spindle-like shape of the trophozoites with spirally arranged epicytic folds (double arrowhead) that appear to overlap each other in a crisscross pattern. The ovoid nucleus (n) with its round nucleolus (nu) is situated in the posterior half of the cell. The mucron (arrowhead) is set apart from the rest of the cell through a slight indentation. B. DIC micrographs of the same cell as in A in different focal planes. The main epicytic folds (double arrowhead) and the secondary epicytic folds (asterisks) are spirally arranged along the longitudinal axis of the cell. C. SEM micrograph showing the surface ultrastructure of the trophozoite. The mucron (arrowhead) is free of folds. The main epicytic folds (double arrowheads) start right after the mucron and split very early into secondary folds. The grooves (asterisks) between the secondary folds appear quite deep from the anterior end and become gradually shallower towards the posterior end at which point they disappear completely. The cell is adorned with transverse striations (arrows) along its whole length apart from the anterior and posterior tip. D. The mucron (arrowhead) is free of folds. The epicytic folds were covered in thick, evenly spread trans- verse striations (2–3 transverse striations/micron), which started right after the mucron (Fig 6C–6F). The posterior end was free of the transverse striations. The striations became less prominent over the length of the cell (4D-F). Trophozoites were not very active and showed some bending movements. Selenidium antevariabilis sp. n. (Fig 7, Table 1). Trophozoites were isolated from the poly- chaete Amphitritides gracilis (Grube, 1860). The cells were spindle-shaped, sometimes with a slight indentation at the anterior end superficially separating the mucron area from the rest of the cell (Fig 7A). Trophozoites were 165 μm (131–190 μm, n = 7) long and 29 μm (22–31 μm, n = 7) wide (at the widest part of the cell). The anterior end was very plastic in shape, changing from almost rounded to flat with finger-like protrusions, while the posterior end was rounded (Fig 7A–7C). The ovoid nucleus [17 x 19 μm (13–21 x 15–20 μm) in diameter, n = 7] was situ- ated in the middle of the cell (Fig 7A–7C). The trophozoites were widest at the position of the nucleus. The nucleus contained a circular nucleolus [9 (7–11) μm in diameter, n = 4]. The tro- phozoites appeared to have a fishnet pattern on the surface in some of the micrographs (Fig PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 12 / 24 Archigregarines of the English Channel Fig 6. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium spiralis sp. n. isolated from the polychaete Amphitritides gracilis. A. PC micrograph showing the general spindle-like shape of the trophozoites with spirally arranged epicytic folds (double arrowhead) that appear to overlap each other in a crisscross pattern. The ovoid nucleus (n) with its round nucleolus (nu) is situated in the posterior half of the cell. The mucron (arrowhead) is set apart from the rest of the cell through a slight indentation. B. DIC micrographs of the same cell as in A in different focal planes. The main epicytic folds (double arrowhead) and the secondary epicytic folds (asterisks) are spirally arranged along the longitudinal axis of the cell. C. SEM micrograph showing the surface ultrastructure of the trophozoite. The mucron (arrowhead) is free of folds. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 The very prominent transverse striations (arrow) start immediately posterior to the mucron, similar to the main epicytic folds (double arrowhead) and the split (asterisks) into secondary folds soon after. E. The transverse striations (arrows) continue to almost the tip of the posterior end, but are less prominent compared to the anterior end. One slight indentation (asterisk) of a secondary fold is visible on one of the main epicytic folds (double arrowhead). F. High magnification SEM of the transverse striations (arrow) and the grooves (asterisk) between the folds. Scale bars: Fig 6A-B, 25 μm; Fig 6C, 10 μm; Fig 6D-F, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g006 7A and 7B). Six epicytic longitudinal folds were more or less helically arranged along the cells body (Fig 7B and 7C). This helical arrangement of the folds leads to the visible fishnet pattern, due to a crisscross overlap of the longitudinal folds on opposite cell surfaces. The density of folds was less than 1 fold/micron (Fig 7B). Trophozoites showed some bending movements and very active change of the morphology of the anterior end (Fig 7A and 7C). Selenidium opheliae sp. n. (Fig 8, Table 1). Trophozoites were isolated from the polychaete Ophelia roscoffensis Augener, 1910. The cell shape was generally elongated to slightly reminis- cent of a pea pod (Fig 8A–8C and 8E). Trophozoites were 151 μm (123–177 μm, n = 19) long and 13 μm (10–16 μm, n = 19) wide. The anterior end tapered into an elongated, but at the tip mostly rounded mucron (Fig 8A–8C and 8G). The posterior end was wider with a heart- shaped or blunt end (Fig 8C, 8E and 8H). The ovoid nucleus 13 x 9 μm (10–16 x 7–11 μm, n = 19) situated in the middle of the cell or slightly shifted to the posterior end contained a cir- cular nucleolus [6 (5–8) μm in diameter, n = 9] (Fig 8A–8D). The SEM revealed no epicytic folds (Fig 8F–8H). The surface of some trophozoites appeared a bit crinkled (Fig 8E and 8H). On some of the trophozoites transverse striations were visible (Fig 8F). Syzygy of two cells was PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 13 / 24 Archigregarines of the English Channel Fig 7. Differential interference contrast (DIC) light micrographs showing the general morphology of the gregarine Selenidium antevariabilis sp. n. isolated from the polychaete Amphitritides gracilis. A-C. DIC micrographs of spindle-shaped trophozoites with plastic anterior (arrowhead) and rounded posterior ends. The arrowhead marks the mucron area, which can be rounded or flattened, but often showing finger-like protrusions (double arrowhead). The ovoid nucleus (n) is situated in the middle of the cell or slightly shifted to the anterior end. Broad longitudinal epicytic folds (asterisks) run along the cells anterior-posterior axis with a helical turn, which appears like a criss-cross pattern on the surface (arrow). Scale bars: Fig 7A-C, 30 μm. Fig 7. Differential interference contrast (DIC) light micrographs showing the general morphology of the gregarine Selenidium antevariabilis sp. n. isolated from the polychaete Amphitritides gracilis. A-C. DIC micrographs of spindle-shaped trophozoites with plastic anterior (arrowhead) and rounded posterior ends. The arrowhead marks the mucron area, which can be rounded or flattened, but often showing finger-like protrusions (double arrowhead). The ovoid nucleus (n) is situated in the middle of the cell or slightly shifted to the anterior end. Broad longitudinal epicytic folds (asterisks) run along the cells anterior-posterior axis with a helical turn, which appears like a criss-cross pattern on the surface (arrow). Scale bars: Fig 7A-C, 30 μm. https://doi.org/10.1371/journal.pone.0187430.g007 observed, in which the outer posterior ends were attached through overlap (Fig 8D). Tropho- zoites were not very active and showed some bending movements. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Molecular phylogenetic analyses The preliminary phylogenetic analysis of the pan-apicomplexan dataset revealed an increased rate of evolution in gregarines compared to the already highly divergent crown apicomplexan lineages. The averaged root-to-tip distance of gregarines as a whole, or archigregarines, was significantly higher than in other lineages (S1 Fig). Together with the non-homogenous base composition observed in several gregarine species, this makes the correct phylogenetic reconstruction of api- complexans very challenging. Therefore, together with the widely-used Maximum likelihood inference under the gamma-corrected GTR model, we have employed also the CAT admixture model (or rather the ‘C40’ approximation of CAT; see relevant Methods part for details and rea- soning), which is supposed to be more robust to phylogenetic artifacts stemming from varying rates of evolution along the phylogenetic tree, namely long-branch attraction (LBA). The resulting phylogeny (Fig 9) shows the expected topology with monophyletic myzozo- ans (dinozoans, chrompodellids [47] and apicomplexans) and ciliates as an outgroup. Both dinozoans and apicomplexans are monophyletic, while chrompodellids split into two clades. One, represented by Alphamonas and Vitrella, which branch out as the basal-most myzozoan lineage, whereas the rest (Chromera, Voromonas and Colpodella) are a sister group to the api- complexans s.s. Apicomplexans are further split into two clades. The first contains ‘true’ coccidians, hae- mosporidians with Nephromyces spp. and the adeleorinid coccidians (Adelina and Hepato- zoon). The second clade is comprised of gregarines including the cryptosporidians and two highly divergent apicomplexans Platyproteum vivax and Filipodium phascolosomae, which show typical archigregarine features, but were classified as an independent myzozoan lineages by a previous study of Cavalier-Smith [1]. Within the gregarine clade, we were able to identify the three main currently recognized lineages (Archigregarinorida, Eugregarinorida, Neogregarinorida), as well as Cryptosporidium as sister clade to the gregarines from terrestrial hosts. Bayesian inference (Fig 9A) split grega- rines into two main clades. The first contains neogregarines, cryptosporidia, cephaloidophor- ids and terrestrial gregarines. The second comprised of archigregarines and marine PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 14 / 24 Archigregarines of the English Channel Fig 8. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium opheliae sp. n. isolated from the polychaete Ophelia roscoffensis. A-C. DIC micrographs of different trophozoite cells showing the general elongated cell shape that is slightly reminiscent of a pea pod. Molecular phylogenetic analyses The mucron (arrowhead) anterior end is slightly elongated and ends in a rounded tip, while the posterior end (arrow) is wider and either blunt or heart-shaped. The ovoid nucleus (n) is situated in the middle of the cell or shifted slightly to the posterior end. D. Two gamonts (G1, G2) in syzygy. This species forms a caudo-lateral syzygy with the two posterior ends overlapping (arrow). Both gamonts have a visible nucleus (n) in the middle. E. SEM micrograph showing the general morphology and ultrastructure of the trophozoite, with an elongated mucron (arrowhead) and a heart- shaped (arrow) posterior end. The surface seemed to be crinkled in places (double arrow). F. Some surface areas showed few transverse striations (asterisks) but in no obvious pattern. G. Anterior end with elongated mucron (arrowhead). H. Posterior end with visible crinkles (double arrow) on the surface. Scale bars: Fig 3A–3D, 30 μm; Fig 3E, 20 μm; Fig 3F–3H, 2 μm. htt //d i /10 1371/j l 0187430 008 Fig 8. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium opheliae sp. n. isolated from the polychaete Ophelia roscoffensis. A-C. DIC micrographs of different trophozoite cells showing the general elongated cell shape that is slightly reminiscent of a pea pod. The mucron (arrowhead) anterior end is slightly elongated and ends in a rounded tip, while the posterior end (arrow) is wider and either blunt or heart-shaped. The ovoid nucleus (n) is situated in the middle of the cell or shifted slightly to the posterior end. D. Two gamonts (G1, G2) in syzygy. This species forms a caudo-lateral syzygy with the two posterior ends overlapping (arrow). Both gamonts have a visible nucleus (n) in the middle. E. SEM micrograph showing the general morphology and ultrastructure of the trophozoite, with an elongated mucron (arrowhead) and a heart- shaped (arrow) posterior end. The surface seemed to be crinkled in places (double arrow). F. Some surface areas showed few transverse striations (asterisks) but in no obvious pattern. G. Anterior end with elongated mucron (arrowhead). H. Posterior end with visible crinkles (double arrow) on the surface. Scale bars: Fig 3A–3D, 30 μm; Fig 3E, 20 μm; Fig 3F–3H, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g008 eugregarines. Finally, S. Archigregarines of the English Channel Platyproteus vivax AY196708 Filipodium phascolosomae FJ832163 Paralecudina polymorpha AY196706 Veloxidium leptosynaptae JN857966 Pterospora schizosoma DQ093793 Pterospora floridiensis DQ093794 Lithocystis sp. DQ093795 Difficilina tubulani FJ832160 Difficilina paranemertis FJ832159 Lecudina tuzetae AF457128 Lecudina longissima FJ832157 Lankesteria abbotti DQ093796 Lankesteria cystodytae EU670241 Lankesteria chelyosomae EU670240 Lecudina phyllochaetopteri FJ832156 Selenidium pisinnus FJ832162 Selenidium orientale FJ832161 Selenidium pendula LN901443 Selenidium cf. echinatum KC110874 Selenidium boccardiellae JN857969 Selenidium sp. 1 KC110866 Selenidium serpulae DQ683562 Selenidium sp. 2 KC110864 Selenidium sensimae KC110869 Selenidium cf. mesnili JN857968 Selenidium sabellariae Selenidium idanthyrsae JN857967 Selenidium idanthyrsae JN857967 Selenidium neosabellariae KC110873 Selenidium hollandei Polyplicarium translucidae JX535351 Polyplicarium lacrymae JX535347 Polyplicarium curvarae JX535341 Selenidium terebellae AY196709 Selenidium antevariabilis sp. n. Selenidium melongena KC890799 Cryptosporidium fragile EU162753 Cryptosporidium baileyi DQ898161 Cryptosporidium parvum Cryptosporidium parvum S40330 Cryptosporidium andersoni AB513869 Xiphocephalus triplogemmatus FJ459763 Xiphocephalus ellisi FJ459762 Syncystis mirabilis DQ176427 Prismatospora evansi FJ459756 Monocystis agilis AF457127 Hoplorhynchus acanthatholius FJ459750 Mattesia gemina AY334568 Mattesia sp. SV-2003 AY334569 Ophriocysti elektroscirrha AF129883 Paraschneideria metamorphosa FJ459755 Ascogregarina taiwanensis DQ462455 Ascogregarina culicis DQ462456 Apicomplexan Acarus siro pathogen AY490099 Gregarina polymorpha FJ459748 Gregarina niphandrodes AF129882 Gregarina coronata FJ459743 Apicomplexa sp. FJ481523 Gregarina kingi FJ459746 Protomagalhaensia granulosae FJ459757 Leidyana migrator AF457130 Leidyana haasi FJ459753 Gregarina blattarum FJ459741 Apicomplexa sp. KC890798 Amoebogregarina nigra FJ459737 Ganymedes sp. FJ976721 Heliospora cf. longissima HQ891115 Heliospora caprellae HQ876007 Thiriotia pugettiae HQ876006 Cephaloidophora cf. communis HQ876008 0.2 ciliates (6) dinoflagellates incl. Perkinsus (22) chrompodellids (7) coccidians (28) haemosporidians and piroplasmids incl. Nephromyces spp. (23) Selenidium spiralis sp. n. Selenidium sabellae Selenidium fallax Selenidium opheliae sp. n. Selenidium pendula Selenidium hollandei LN901442 0.2 1/3 of actual size cephaloidophoroids eugregarines neogregarines and eugregarines cryptosporidia A1 A3 A2 marine eugregarines A4 cephaloidophoroids marine eugregarines A1 A2 A3 A4 cryptosporidia eugregarines neogregarines and eugregarines EF100227 EF100292 Difficilina paranemetrtes FJ832159 Lankesteria chelyosomae EU670240 Lankesteria abbotti DQ093796 Lecudina cf. tuzetae JF264877 EU087268 Selenidium melongena KC890799 Selenidium melongena KC890800 Selenidium terebellae KC890803 Selenidium terebellae KC890805 Selenidium pisinnus FJ832162 Selenidium orientale FJ832161 Platyproteus vivax AY196708 Filipodium phascolosomae FJ832163 Selenidium fallax JF972216 Gulf of Mexico Selenidium pendula Selenidium pendula LN901443 Selenidium pendula LN901444 JF972220 Gulf of Mexico JQ243319 Papua New Guinea Selenidium opheliae sp. n. AB275007 Honshu coast, Japan AY046619 Baja California AY046806 Baja California EF100333 Greenland EF100387 Greenland JF972228 Gulf of Mexico Selenidium cf. echinatum KC110874 Selenidium cf. Molecular phylogenetic analyses fallax, whose phylogenetic position is for the first time presented here, as well as Platyproteum vivax and Filipodium phascolosomae were the most basal gregarines and sister to the two above mentioned clades. Maximum likelihood analysis split gregarines according to the ecology: terrestrial clade containing cryptosporidia, neogregarines and terrestrial eugregarines and the marine clade comprised of cephaloidophorids branching with marine eugregarines and several archigregar- ine lineages (Fig 9B). Both phylogenetic methods unequivocally and consistently placed the newly obtained sequences of Selenidium in three separate clades (Fig 9, S2 Fig, S3 Fig). Seleni- dium pendula branched within the well supported ‘true’ Selenidium clade with S. sabellariae, S. sabellae, S. hollandei, S. opheliae and nine other representatives, while S. pisinnus formed a weakly supported clade with S. orientale basal to it. Another putatively archigregarine clade PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 15 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 mesnili JN857968 Selenidium sabellariae Selenidium idanthyrsae JN857967 Selenidium idanthyrsae JN857967 Selenidium neosabellariae KC110873 Selenidium hollandei Polyplicarium translucidae JX535351 Polyplicarium lacrymae JX535347 Polyplicarium curvarae JX535341 Selenidium terebellae AY196709 Selenidium antevariabilis sp. n. Selenidium melongena KC890799 Cryptosporidium fragile EU162753 Cryptosporidium baileyi DQ898161 Cryptosporidium parvum Cryptosporidium parvum S40330 Cryptosporidium andersoni AB513869 Xiphocephalus triplogemmatus FJ459763 Xiphocephalus ellisi FJ459762 Syncystis mirabilis DQ176427 Prismatospora evansi FJ459756 Monocystis agilis AF457127 Hoplorhynchus acanthatholius FJ459750 Mattesia gemina AY334568 Mattesia sp. SV-2003 AY334569 Ophriocysti elektroscirrha AF129883 Paraschneideria metamorphosa FJ459755 Ascogregarina taiwanensis DQ462455 Ascogregarina culicis DQ462456 Apicomplexan Acarus siro pathogen AY490099 Gregarina polymorpha FJ459748 Gregarina niphandrodes AF129882 Gregarina coronata FJ459743 Apicomplexa sp. FJ481523 Gregarina kingi FJ459746 Protomagalhaensia granulosae FJ459757 Leidyana migrator AF457130 Leidyana haasi FJ459753 Gregarina blattarum FJ459741 Apicomplexa sp. KC890798 Amoebogregarina nigra FJ459737 Ganymedes sp. FJ976721 Heliospora cf. longissima HQ891115 Heliospora caprellae HQ876007 Thiriotia pugettiae HQ876006 Cephaloidophora cf. communis HQ876008 Selenidium spiralis sp. n. Selenidium sabellae Selenidium fallax Selenidium opheliae sp. n. Selenidium pendula Selenidium hollandei LN901442 0.2 1/3 of actual size cephaloidophoroids eugregarines neogregarines and eugregarines cryptosporidia A1 A3 A2 marine eugregarines A4 A5 ABSOLUTE SU A B A cephaloidophoroids marine eugregarines outgroup (95) A3 Selenidium fallax A5 ABSOLUTE SUPPORT (BI 1.0, ML 100) STRONG SUPPORT (BI >0.95, ML >85) haemosporidians and piroplasmids incl. Nephromyces spp. (23) coccidians (28) dinoflagellates incl. Perkinsus (22) Fig 9. Phylogenetic relationships of apicomplexans. A. Bayesian tree of apicomplexans inferred using the GTR + C40 model of substitution in Phylobayes on an alignment of 171 small subunit (SSU) rDNA sequences and 1488 unambiguously aligned sites. Thickened lines at branches denote Bayesian posterior probabilities and bootstrap support (see legend in the center of the figure for details). The eight sequences derived from this study are highlighted in bold. A1 to A5 show the positions of the five ‘archigregarine’clades. B. Schematized Maximum likelihood phylogeny analysis of the same dataset inferred under the GTR+G model as implemented in RAxML 8.2. The outgroup composition and topology is the same as in Fig 9A and is simplified due to space limitations. C. Bayesian topology of the dataset S with exhaustive sampling of Selenidiidae, also enriched for environmental sequences inferred in Phylobayes under the GTR + C40 model of substitution. The branching support follows parameters of tree A. Fig 9. Phylogenetic relationships of apicomplexans. A. echinatum KC110875 EU087194 East Sea JQ243944 Papua New Guinea Selenidium sabellaria Selenidium hollandei Selenidium hollandei LN901445 Selenidium neosabellariae KC110871 Selenidium neosabellariae KC110872 Selenidium neosabellariae KC110873 JQ244240 Papua New Guinea Selenidium cf. mesnili JN857968 Selenidium sabellae FJ221514 NY coast, USA Selenidium sp. 1 KC110866 Selenidium sp. 1 KC110867 Selenidium sp. 1 KC110863 Selenidium serpulae DQ683562 Selenidium sensimae KC110869 Selenidium sensimae KC110870 Selenidium sp. 2 KC110865 Selenidium sp. 2 KC110864 Selenidium sp. 2 KC110868 0.1 Lecudina phyllochaetopteri FJ832156 Pterospora floridiensis DQ093794 Pterospora schizosoma DQ093793 Lithocystis sp. DQ093795 Lecudina longissima FJ832157 Veloxidium leptosynaptae JN857966 Selenidium spiralis sp. n. Selenidium antevariabilis sp. n. Selenidium boccardiellae JN857969 Selenidium idanthyrsae JN857967 B C A5 1/3 of actual size ABSOLUTE SUPPORT (BI 1.0, ML 100) STRONG SUPPORT (BI >0.95, ML >85) A5 Paralecudina polymorpha AY196706 A outgroup (95) Polyplicarium translucidae JX535351 Polyplicarium lacrymae JX535347 Polyplicarium curvarae JX535341 Alphamonas edax AY234843 Vitrella brassicaformis DQ174732 Fig 9. Phylogenetic relationships of apicomplexans. A. Bayesian tree of apicomplexans inferred using the GTR + C40 model of substitution in Phylobayes on an alignment of 171 small subunit (SSU) rDNA sequences and 1488 unambiguously aligned sites. Thickened lines at branches denote Bayesian posterior probabilities and bootstrap support (see legend in the center of the figure for details). The eight sequences derived from this study are highlighted in bold. A1 to A5 show the positions of the five ‘archigregarine’clades. B. Schematized Maximum likelihood phylogeny analysis of the same dataset inferred under the GTR+G model as implemented in RAxML 8.2. The outgroup composition and topology is the same as in Fig 9A and is simplified due to space limitations. C. Bayesian topology of the dataset S with exhaustive sampling of Selenidiidae, also enriched for environmental sequences inferred in Phylobayes under the GTR + C40 model of substitution. The branching support follows parameters of tree A. Platyproteus vivax AY196708 Filipodium phascolosomae FJ832163 Paralecudina polymorpha AY196706 Veloxidium leptosynaptae JN857966 Pterospora schizosoma DQ093793 Pterospora floridiensis DQ093794 Lithocystis sp. DQ093795 Difficilina tubulani FJ832160 Difficilina paranemertis FJ832159 Lecudina tuzetae AF457128 Lecudina longissima FJ832157 Lankesteria abbotti DQ093796 Lankesteria cystodytae EU670241 Lankesteria chelyosomae EU670240 Lecudina phyllochaetopteri FJ832156 Selenidium pisinnus FJ832162 Selenidium orientale FJ832161 Selenidium pendula LN901443 Selenidium cf. echinatum KC110874 Selenidium boccardiellae JN857969 Selenidium sp. 1 KC110866 Selenidium serpulae DQ683562 Selenidium sp. 2 KC110864 Selenidium sensimae KC110869 Selenidium cf. Formal taxonomic description Phylum Apicomplexa Levine, 1970 Subphylum Sporozoa Leuckart, 1879 Class Gregarinea J.A.O. Bu¨tschli, 1882, stat. nov. Grasse´, 1953 Order: Archgregarinorida Grasse´, 1953 Family Selenidiidae Brasil, 1907 Genus Selenidium Giard, 1884 Selenidium spiralis Rueckert and Hora´k sp. n. urn:lsid:zoobank.org:act:0687AD8C-E669-47B4-8EC2-9FC22A039B urn:lsid:zoobank.org:act:0687AD8C-E669-47B4-8EC2-9FC22A039B42 Diagnosis. Trophozoites are spindle-shaped and 105–194 μm long (mean length 147 μm) and 21–37 μm wide (mean width 30 μm). Cell tapers into a rounded mucron at the anterior and a pointed tip at the posterior end. The ovoid nucleus (13 x 20 μm) is situated in the posterior half of the trophozoite. Trophozoites are brownish in colour under the LM due to the accumulation of amylopectin granules within the cytoplasm. Six longitudinally and helically oriented broad epicy- tic folds are present on the cell surface, except the mucron and the posterior tip. They are sepa- rated by deep, primary grooves and also show shallow secondary grooves in between. Epicytic folds are covered by thick transverse striations. The trophozoites are capable of some bending. DNA sequence. The SSU rDNA sequence; GenBank Accession No. MF882900. Type locality. Rock assemblages close to Station Biologique Roscoff (48˚43’44”N, 3˚ 59’23”W), Roscoff, France. Type habitat. Marine. Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). Location in host. Intestinal lumen. Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British Columbia, Vancouver, Canada (collection number MI-PR135). Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British Columbia, Vancouver, Canada (collection number MI-PR135). Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British Columbia, Vancouver, Canada (collection number MI-PR135). Iconotype. Fig 6B. Iconotype. Fig 6B. Etymology. The species name spiralis refers to the helically arranged longitudinal epicytic folds. It is interesting that while most archigregarine species are described from temperate regions, many of these environmental sequences originate from tropical and subtropical waters clearly show- ing their cosmopolitan distribution and research bias. Bayesian tree of apicomplexans inferred using the GTR + C40 model of substitution in Phylobayes on an alignment of 171 small subunit (SSU) rDNA sequences and 1488 unambiguously aligned sites. Thickened lines at branches denote Bayesian posterior probabilities and bootstrap support (see legend in the center of the figure for details). The eight sequences derived from this study are highlighted in bold. A1 to A5 show the positions of the five ‘archigregarine’clades. B. Schematized Maximum likelihood phylogeny analysis of the same dataset inferred under the GTR+G model as implemented in RAxML 8.2. The outgroup composition and topology is the same as in Fig 9A and is simplified due to space limitations. C. Bayesian topology of the dataset S with exhaustive sampling of Selenidiidae, also enriched for environmental sequences inferred in Phylobayes under the GTR + C40 model of substitution. The branching support follows parameters of tree A. https://doi.org/10.1371/journal.pone.0187430.g009 https://doi.org/10.1371/journal.pone.0187430.g009 with sequences related to S. terebellae (including two of our new isolates from Amphitritides gracilis) is found elsewhere among the gregarines. Selenidium fallax probably represents an independent (archi-) gregarine lineage of uncertain position. However, analysis of auxiliary datasets R and G (S2 Fig, S3 Fig) shows the gregarine topology is unstable and highly depen- dent on the phylogenetic method and site selection. It is worth mentioning that the archigre- garine monophyly was also rejected using the au-test. While all the respective clades are robustly supported in all analyses, their mutual relationships are unclear to say the least and the support for deeper branching is missing completely. A pairwise distance calculation based on the Kimura two-parameter model [48] of 1656 nt (with pairwise exclusion of the indels) resulted in sequence divergences between 2% and 35.8% between all Selenidium species. The sequence divergence ranges were 2–15.6% (A1); 5.8–10.5% (A2) and 15.3 (A3) within clades PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 16 / 24 Archigregarines of the English Channel A1-A3 and 18.5–35.8% between species from the different clades (S1 Table). Since the sequence of the archigregarine type species S. pendula has been recently published [33], we have also attempted to explore the diversity and phylogenetic structure of the genus Selenidium s.s. Our exhaustive sampling revealed that almost half of the diversity is comprised of environ- mental sequences without any morphology and host-specificity record. Selenidium pendula is found near the base of the Selenidium clade, which lacks a strong phylogenetic structure. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Selenidium antevariabilis Rueckert and Hora´k sp. n. Selenidium antevariabilis Rueckert and Hora´k sp. n. urn:lsid:zoobank.org:act:F830245F-4CF9-4925-9F20-CFFB8699DBCB posterior tip. The trophozoites are capable of some bending and very active change of the mor- phology of the anterior end. Type habitat. Marine. Type habitat. Marine. Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). L ti i h t I t ti l l Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). Holotype. The name-bearing type of this species is the specimen shown in Fig 7B (see Ico- notype). This is in accordance with Declaration 45 recommendations to article 73 of the ICZN. An explanation is provided in the remarks. Iconotype. Fig 7. Etymology. The species epithet antevariabilis refers to the plastic morphology of the ante- rior end and stems from the Latin words ‘ante’ meaning front/before and ‘variabilis’ meaning changeable. Remarks. In accordance with Declaration 45 of the ICZN we use the specimen presented in the light micrographs (7B) as name-bearing type material (Holotype). Here we provide reason- ing, why no preserved specimen was used as name-bearing type for the new species. This new gregarine species occurred in very low numbers. The few specimens we were able to isolate were used for DNA extraction, prepared for SEM and for LM analyses. Trophozoites fixed for SEM often do represent a gregarine’s morphology better than a fixed and flattened cell on an object slide. Light micrographs (Iconotype) were obtained from freshly isolated cells and rep- resent the morphology of the trophozoite and especially of the plasticity of the anterior end better than a fixed specimen on an object slide could. We were not able to find any gregarines on the filter prepared for SEM, therefore the only documentation of this species are the light micrographs and the cell measurements. We do provide the SSU rDNA sequence, which sepa- rates S. antevariabilis n. sp. clearly from S. spiralis n. sp. isolated from the same host. Therefore, we feel that we provide enough evidence for the description of a new species. Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe- lia roscoffensis Augener, 1910. urn:lsid:zoobank.org:act:F830245F-4CF9-4925-9F20-CFFB8699DBCB Diagnosis. Trophozoites are spindle-shaped and 131–190 μm long (mean length 165 μm) and 22–31 μm wide (mean width 29 μm). Cell tapers into a rounded posterior end. The ante- rior end is plastic in shape, being rounded, or flat, or showing finger-like protrusions. The ovoid nucleus (17 x 19 μm) is situated in the middle of the trophozoite and contains a circular nucleolus (9 μm in diameter). Trophozoites are brownish in colour under the LM due to the accumulation of amylopectin granules within the cytoplasm. Six longitudinally and helically oriented broad epicytic folds are present on the cell surface, except the mucron and the PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 17 / 24 Archigregarines of the English Channel posterior tip. The trophozoites are capable of some bending and very active change of the mor- phology of the anterior end. posterior tip. The trophozoites are capable of some bending and very active change of the mor- phology of the anterior end. Selenidium opheliae Rueckert and Hora´k sp. n. Selenidium opheliae Rueckert and Hora´k sp. n. Justification for newly erected species Trophozoites of Selenidium spiralis sp. n. and S. antevariabilis sp. n. were isolated from Amphi- tritides gracilis. No gregarines have so far been reported for this terebellid polychaete. The anal- yses of the SSU rDNA sequences available for gregarines of the genus Selenidium placed S. spiralis sp. n. and S. antevariabilis sp. n. into a clade with other Selenidium species from terebel- lid worms (Thelepus japonicus), namely Selenidium terebellae and Selenidium melongena. While S. terebellae is a typical archigregarine of the genus Selenidium, with few longitudinal epicytic folds, bending/twisting and coiling movements and transverse striations, S. melongena is quite different (see Table 1 and Wakeman et al. 2014). One of the most distinct features is that the 30–40 epicytic folds are helically arranged along the longitudinal axis of the cell and that no movement could be observed. The new species described here present different degrees of helically arranged epicytic folds, but a lot less in numbers (six broad folds, in one case with secondary grooves). Along the whole length of the epicytic folds of S. spiralis sp. n. there are thick transverse folds at regular intervals. While S. spiralis sp. n. and S. antevariabilis sp. n. were found within the intestine, S. melongena was predominantly found attached to the outer wall of the intestine [50]. The calculated sequence divergences ranged between 8.7–10.5% for S. spiralis sp n., S. terebellae and S. antevirabilis sp. n. compared to S. melongena (compare S1 Table). The lowest sequence divergence (2%) was actually found between two established spe- cies S. idanthyrsae and S. neosabellariae. Apart from their sequence divergence (6.8%), the big- gest difference between the two new species is the morphology of the anterior end. While S. spiralis sp. n. has a rounded mucron, the anterior end of S. antevariabilis sp. n. is very plastic and changing in form constantly. Combining all evidence we are convinced that both isolated trophozoites represent new Selenidium species. Ophelia roscoffensis has never been described as hosting any gregarine species. There are two Rhytidocystis species (Agamococcidiorida) described from opheliid worms, namely R. opheliae from O. bicornis [51] and R. henneguyi from O. neglecta [52], but their phylogenetic position is not quite certain yet [9]. Rotari et al. [53] described metchnikovellids (hyperpara- sites of gregarines) from a Selenidium species infecting Ophelia limacina, but the gregarine spe- cies was not formally described and only a line drawing is available. Discussion In the last decade more and more information has been published about the archigregarines, which occur exclusively in marine habitats and are most likely the stem group from which all gregarine and maybe even all apicomplexans have derived [24–26,29,32,49,50]). Most of these publications synergize data on morphology, ultrastructure and SSU rDNA sequences to newly describe or re-describe species in the most comprehensive way possible to date. Almost all of the work has been done on the trophozoite stages, the most prominent and abundant life cycle stages. Recently work on other life-cycle stages of S. pendula has been published [33]. Molecu- lar data of archigregarines are still very limited compared to other taxa. urn:lsid:zoobank.org:act:8316105E-9419-4F06-9463-E12553F9E2C5 urn:lsid:zoobank.org:act:8316105E-9419-4F06-9463-E12553F9E2C5 Diagnosis. Trophozoites elongated ellipsoid to pea pod shape and 123–177 μm long (mean length 151 μm) and 10–16 μm wide (mean width 13 μm). Anterior end is elongated with rounded mucron, posterior end is wide with heart-shaped or blunt end. The ovoid nucleus measures13 x 9 μm (10–16 x 7–11 μm, n = 19) and is situated in the middle of the cell or slightly shifted to the posterior end. Trophozoites are brownish in colour reflecting an accu- mulation of amylopectin granules within the cytoplasm. Epicytic folds are lacking. Surface of trophozoites can appear crinkled and some show transverse striations. Trophozoites are capa- ble of bending movements. DNA sequence. The SSU rDNA sequence; GenBank Accession No. MF882904. Type locality. Le Guillec (48˚68´56´´N, 4˚06´78´´W), France. Type habitat. Marine. Type habitat. Marine. Type host. Ophelia roscoffensis Augener, 1910 (Annelida, Polychaeta, Opheliidae). Location in host. Intestinal lumen. Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British Columbia, Vancouver, Canada (collection number MI-PR136). Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe- lia roscoffensis Augener, 1910. Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe- lia roscoffensis Augener, 1910. Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe- lia roscoffensis Augener, 1910. 18 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Archigregarines of the English Channel PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 True Selenidiidae vs. Selenidioides Levine [3] split the genus Selenidium and its species into two: 1) Selenidioides within the family Selenidioididae in the order Archigregarinorida encompassing species with merogony and 2) Selenidium within the family Selenidiidae in the order Eugregarinorida without merogony. There have been many discussions before this split and ever since the split [16,25,26,29,30,32] about the taxonomy of gregarine species described as Selenidium, as it is difficult to prove or disprove the existence of merogony within a gregarine life-cycle. No sequence data were avail- able back then and still the available DNA sequences are quite limited. Our phylogenetic trees based on SSU rDNA sequence data (Fig 9) show that S. hollandei, which after Levine [3] should belong to the genus Selenidioides, clusters within the big clade around the type species of the genus Selenidium, S. pendula, which was moved to the Eugregarinorida due to the ‘absence’ of merogony. Therefore, S. pendula as well as S. hollandei belong to the true Selendiidae, which has also been shown by Schre´vel et al. [33], inferring that the split of the genus Selenidium was premature, because it is not backed up by molecular sequence data and should therefore be ignored from hereon. While S. sabellariae and S. sabellae also cluster within the clade A1 together with S. pendula, the species S. fallax that should belong into this genus as well clusters out with any of the other ‘Selenidium’ sequences, which supports our suggestion to revise this genus at some point, when better/more sequence data and morphological descriptions of other species within this genus become available. Justification for newly erected species Compared to the other known Selenidium species our newly described S. opheliae sp. n. from O. roscoffensis has very different morphological features, as this is the only species that does not show any longitudinal epicytic folds (compare Table 1). The crinkled surface and the few transverse striations could be the result of the SEM fixation process. The sequence divergence showed values between 6.9% and 15.4% when comparing S. opheliae sp. n. with all other Selenidium species in clade A1. The distances between all Selenidium species in clade A1 ranged from 2% to 15.6% (S1 Table). The phylogenetic tree based on gregarine SSU rDNA sequences alone (Fig 9) shows clearly that this species has a distinct position from the other Selenidium species within the S. pendula clade of true Selenidium species, therefore validating the erection of the new species. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 19 / 24 Archigregarines of the English Channel Archigregarine phylogeny The so called archigregarines (i.e. apicomplexans mostly with vermiform or spindle-shaped actively moving trophozoits), represented here by all species with available SSU rDNA sequences, apparently comprise of five independent lineages: 1) a clade around the Selenidium type-species S. pendula, 2) a clade containing S. pisinnus and S. orientale, 3) a clade with para- sites isolated from polychaetes Amphitritides gracilis and Thelepus japonicus and 4) a clade made up of two very divergent species Platyproteum vivax and Filipodium phascolosomae, and 5) the single sequence of S. fallax at the base of all other gregarines. Although all these lineages share similar morphology (see Table 1) and possibly (according to the limited insight available) also general biology, phylogenetic analysis of SSU rDNA clearly reveals well defined and sepa- rated lineages. However, the deeper branching reflecting their mutual relationship is again very unstable and poorly supported. All recent studies [1,25,26,33,49,50], as well as the phylog- eny of our main dataset presented here (Fig 9) suggest their paraphyly. Archigregarine mono- phyly was also rejected by the approximately-unbiased test. However, short internal branches suggest rapid archigregarine diversification, which is always hard to capture using phyloge- netic reconstruction. In our opinion, while improbable, the archigregarine monophyly cannot be reliably rejected based on current taxon sampling and SSU rDNA. One obvious outcome is that only the archigregarines of the clade around S. pendula should retain the genus name Sele- nidium and thorough revision of the whole archigregarine concept is required. Suitability of SSU rDNA for gregarine phylogeny Molecular diversity is almost exclusively represented by SSU rDNA sequences for gregarine apicomplexans. Yet, even the most elaborate phylogenetic models designed for phylogenies of highly divergent datasets [54,55], failed to cope with the extreme diversity of gregarine SSU rDNA. The topology is unstable and is highly dependent on the particular model used (GTR + C40 vs. GTR) as well as taxon and site sampling. Therefore, we intentionally refrain from any taxonomic revisions of gregarines s.l. as recently proposed by Cavalier-Smith [1], as we do not find it justified by the available data. It is our opinion that we have to leave the question of PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 20 / 24 Archigregarines of the English Channel relationships among the key gregarine lineages open until new sets of markers of sufficient sampling will be available. Supporting information S1 Fig. Boxplots showing averaged root-to-tip distances of main clades used in phyloge- netic analyses. Three groups of gregarines (representatives of Selenidium s.s., archigregarines and gregarines as a whole) show significantly longer branch lengths and therefore also increased rates of evolution as revealed by pairwise t-test comparison to other clades presented in the dataset. (EPS) S2 Fig. Phylogenetic topology of auxiliary dataset G (945 sites) as revealed by Bayesian inference under the GTR + C40 model as implemented in Phylobayes. Thickened lines show branching supported by Bayesian posterior probabilities (dark, p.p. 1.0, grey p.p. above 0.94). See relevant parts of methods for details. (EPS) S3 Fig. Phylogenetic topology of auxiliary dataset R (1805 sites) as revealed by Bayesian inference under the GTR + C40 model as implemented in Phylobayes. Thickened lines show branching supported by Bayesian posterior probabilities (dark, p.p. 1.0, grey p.p. above 0.94). See relevant parts of methods for details. (EPS) S3 Fig. Phylogenetic topology of auxiliary dataset R (1805 sites) as revealed by Bayesian inference under the GTR + C40 model as implemented in Phylobayes. Thickened lines show branching supported by Bayesian posterior probabilities (dark, p.p. 1.0, grey p.p. above 0.94). See relevant parts of methods for details. (EPS) S1 Table. Estimates of evolutionary divergence between sequences. The numbers of base substitutions per site between sequences are shown. The analysis involved 50 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1654 positions in the final dataset. The sequences for taxa in bold were derived from this study, taxa highlighted in light grey belong to Selenidium s.s. and taxa highlighted in dark grey are novel species for the first time described here. (XLSX) Acknowledgments SR and AH would like to thank the marine team from Station Biologique de Roscoff for col- lecting some of the polychaete species. Both authors would like to thank the SEM facility at the Institute of Parasitology, Biology Centre of CAS for their technical support and expertise. The authors would also like to thank Olga Flegontova, for her help in the field and in the laboratory. References 1. Cavalier-Smith T. Gregarine site-heterogeneous18S rDNA trees, revision of gregarine higher classifica- tion, and the evolutionary diversification of Sporozoa. EJOP. 2014; 50: 472–495. 2. Grasse´ P-P. Classe des gre´garinomorphes (Gregarinomorpha, N. nov., Gregarinae Haeckel, 1866; gregarinidea Lankester, 1885; gre´garines des auteurs). In: Grasse´ P-P editor. 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Data curation: Sonja Rueckert, Alesˇ Hora´k. Formal analysis: Sonja Rueckert, Alesˇ Hora´k. Funding acquisition: Sonja Rueckert, Alesˇ Hora´k. Investigation: Sonja Rueckert, Alesˇ Hora´k. Methodology: Sonja Rueckert, Alesˇ Hora´k. Writing – original draft: Sonja Rueckert, Alesˇ Hora´k. Data curation: Sonja Rueckert, Alesˇ Hora´k. Formal analysis: Sonja Rueckert, Alesˇ Hora´k. 21 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430 November 3, 2017 Archigregarines of the English Channel Writing – review & editing: Sonja Rueckert, Alesˇ Hora´k. References Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002; 419: 498–511. https://doi.org/10.1038/ nature01097 PMID: 12368864 18. Kissinger JC, Brunk BP, Crabtree J, Fraunholz MJ, Gajria B, Milgram AJ et al. The Plasmodium genome database: Designing and mining a eukaryotic genomics resource. 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Determination of the Performance of Some Apple Cultivars Matured in Different Periods in the Mediterranean Transitional Zone
Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi/Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi
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Determination of the Performance of Some Apple Cultivars Matured in Different Periods in the Mediterranean Transitional Zone Determination of the Performance of Some Apple Cultivars Matured in Different Periods in the Mediterranean Transitional Zone Abstract: Apple is a temperate climate fruit grown in different ecology and a wide range of area. However, to determine the suitable variety for every ecology, it is of great importance to investigate the performance of these varieties. This study was carried out in 2012-2013 in order to determine the performance of Mondial Gala, Fuji and Scarlet Spur apple varieties grown in Mediterranean transitional zone and matured in different periods. In this study, some phenological properties of cultivars and yield and fruit quality characteristics were investigated. According to the results, full bloom occurred in 21 to 25 April and maturation on 11 of August and 5 October. In cultivars, fruit weights in ranged between 137.50 g and 217.30 g, fruit diameter 66.57 mm and 80.67 mm. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi Cilt 29, Sayı 2, 28.06. 2019 Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi Cilt 29, Sayı 2, 28.06. 2019 Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi (YYU Journal of Agricultural Science) Araştırma Makalesi (Research Article) Öz: Elma, farklı ekolojilerde ve oldukça geniş bir alanda yetiştiriciliği yapılan bir ılıman iklim meyvesidir. Ancak, yetiştiricilik yapılacak ekolojiye uygun çeşit önerisi için, o ekolojideki performansların incelenmesi büyük önem taşımaktadır. Çalışma, Akdeniz geçit kuşağında yetiştirilen ve değişik dönemlerde olgunlaşan Mondial Gala, Fuji ve Scarlet Spur elma çeşitlerinin performanslarının belirlenmesi amacıyla 2012-2013 yıllarında yürütülmüştür. Araştırmada, çeşitlerin bazı fenolojik özellikleri ile verim ve meyve kalite özellikleri incelenmiştir. Bulgulara göre çeşitlerde tam çiçeklenme 11 Nisan (Mondial Gala)-25 Nisan (Fuji) ve meyve olgunlaşması ise 11 Ağustos (Mondial Gala) - 5 Ekim (Fuji) tarihleri arasında gerçekleşmiştir. Çeşitlerin meyve ağırlıkları 137.50 g (Scarlet Spur) - 217.30 g (Fuji), meyve boyu 61.65 mm (Mondial Gala) - 71.03 mm (Scarlet Spur) ve meyve çapı 66.57 mm (Scarlet Spur) - 80.67 mm (Fuji) arasında değişim göstermiştir. Elma çeşitlerinde suda çözünebilir kuru madde miktarı % 13.50 (Scarlet Spur) - % 15.17 (Mondial Gala), titre edilebilir asit miktarı % 0.23 (Scarlet Spur) - % 0.40 (Fuji), indirgen şeker kapsamı % 8.93 (Scarlet Spur) - % 10.17 (Fuji), toplam şeker düzeyi % 12.08 (Scarlet Spur) – % 13.87 (Fuji) ve toplam fenol içerikleri ise 113.40 mg/100 g (Scarlet Spur) - 148.40 mg/100 g (Fuji) arasında yer almıştır. Öte yandan, elma çeşitlerinde ağaç başına verim 4.92 (Scarlet Spur) – 9.32 kg/ağaç (Fuji) ve birim gövde kesit alanına düşen verim 0.28 (Scarlet Spur) – 0.66 kg/cm2 (Mondial Gala) arasında değişim göstermiştir. Yaz dönemi açısından Mondial Gala, erken güz dönemi açısından Scarlet Spur ve güz dönemi için ise Fuji çeşidinin bölgede ürün çeşitliğinin sağlanması açısından üreticilere katkı sağlayacağı düşünülmektedir. Geliş: 17.10.2018 Kabul: 11.04.2019 Online Yayınlanma 28.06. 2019 DOI: 10.29133/yyutbd.471552 Anahtar kelimeler Fenoloji, Malus domestica, Meyve kalitesi, Pomoloji, Verim etkinliği 1. Giriş Dünya üzerinde Antarktika hariç, bütün kıtalarda, ılıman iklime sahip bölgelerde ve tropik bölgelerin yüksek rakımlı yerlerinde doğal olarak yetişen ve 30 kadar türü bulunan elma (Malus domestica Borkh.), ılıman iklim meyveleri içerisinde en fazla üretim ve tüketim miktarlarına sahip olan türdür (Özongun ve ark., 2014). Dünyada elma yetiştiriciliği kuzey ve güney yarı kürenin 30°-50° enlemler arasındaki ılıman iklime sahip bölgelerine yayılmıştır (Soylu, 2003). Türkiye’de ise Ege Bölgesi’nde 500 metreden, Akdeniz ve Güneydoğu Anadolu Bölgesi’nde ise 800 metreden daha yüksek yerlerde yetişmektedir (Kaygısız, 2004; Özçağıran ve ark., 2005). Dünya elma üretimi yaklaşık 83 milyon ton olup, Türkiye 3 032 164 ton ile dünya elma üretiminde Çin ve ABD’nin ardından 3. sırada gelmektedir (FAOSTAT 2017). Elma bitkisinin birçok kültür çeşidi ve yabani formları soğuk ve sert iklim koşullarına önemli düzeyde dayanım göstermektedir. Elmanın odun dokuları kış döneminde - 35 ile - 40 oC’ye dayanabilmektedir. Ancak, bahar döneminde açmış çiçekler - 2.2 ile - 2.3 oC, küçük meyveler ise - 1.1 ile - 2.2 oC düzeylerindeki sıcaklıklardan zarar görebilmektedir (Özcağıran ve ark., 2005; Gerçekcioğlu ve ark., 2012). Elmada sağlıklı bir çiçeklenme meydana gelmesi ve buna dayalı olarak düzenli tozlanma ve döllenme koşullarının oluşabilmesi için tür ve çeşitlere göre değişmekle birlikte + 7.2 oC’nin altında 2 000 ile 3 000 saat arasındaki kış soğuklanma ihtiyacının karşılanması gerekmektedir (Özbek 1978; Kaygısız, 2004; Gerçekcioğlu ve ark., 2012). Ancak, son dönemlerde ıslah çalışmaları sonucunda düşük soğuklanma isteğine sahip (400-600 saat gibi) oldukça fazla sayıda çeşit elde edilmiş ve bu sayede elmadaki yetiştiricilik alanı subtropik bölgelere doğru önemli yayılma göstermiştir (Gerçekçioğlu ve ark., 2012). Elma bitkisi yaz dönemindeki yüksek sıcaklıklara belirli düzeye kadar bir dayanım göstermekle birlikte, genellikle 40 oC ve üzerindeki uzun süreli sıcaklıklardan ve düşük hava neminden olumsuz olarak etkilenmektedir (Özbek, 1978; Özcağıran ve ark., 2005; Gerçekcioğlu ve ark., 2012). Son 25-30 yıllık dönemde ülkemizde elma tarımında önemli gelişmeler meydana gelmiştir. Elma yetiştiriciliğinde uygulanan modern yetiştirme teknikleri sadece bu türün yetiştiriciliğindeki gelişmelerle kalmayıp, diğer meyve türlerinde de modern teknoloji kullanımına öncülük etmiştir. Elma yetiştiriciliğinde en önemli gelişme klasik çöğür anaçları yerine değişik kuvvette büyüme özelliğine sahip klonal anaçların kullanımı alanında yaşanmıştır. Bu sayede ülkemizin hemen her bölgesinde bodur veya yarı bodur büyüme özelliğine sahip klonal anaçlarla bahçeler kurulmuştur (Öztürk ve ark. 2005; Ikinci ve Bolat 2016; Atay ve Atay, 2018). Ülkemizde toplam elma üretiminin % 36.2’lik bölümü Akdeniz Bölgesi’nden sağlanmaktadır. Article Info Received: 17.10.2018 Accepted: 11.04.2019 Online Published 28.06.2019 DOI: 10.29133/yyutbd.471552 Keywords Phenology, Malus domestica, Keywords Phenology, Malus domestica, 258 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Fruit quality, Pomology, Yield efficiency In varieties, total soluble solids showed change as 13.50 % and 15.17 %, the amount of titratable acidity 0.23 % and 0.40 %, reducing sugar content 8.93 % and 10.17 %, total sugar level 12.08 % and 13.87 % and the total phenol content 113.40 mg/100 g and 148.40 mg/100 g. On the other hand, trunk cross-sectional area of apple cultivars was found to vary between 13.20 cm2 and 22.67 cm2, yield per tree: 4.92 kg/tree and 9.32 kg/tree and the yield efficiency: 0.28 kg/cm2 and 0.66 kg/cm2. Mondial Gala for the summer period, Scarlet spur for the early fall period and Fuji apple cultivars for the fall period will contribute to producers in the region in terms of product diversity is thought to contribute. **Mustafa YILMAZ’ın “Akdeniz Geçit Kuşağında Değişik Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi” isimli tezinden üretilmiştir. 2.1. Yöntem Araştırmada ele alınan elma çeşitleri üzerinde yapılan gözlemler ve ölçümler ile uygulanan metotlar aşağıda belirtilmiştir. Araştırmada ele alınan elma çeşitleri üzerinde yapılan gözlemler ve ölçümler ile uygulanan metotlar aşağıda belirtilmiştir. 1. Giriş Bu bölgeyi % 18.0’lik üretim payları ile Batı ve Orta Anadolu Bölgeleri ve % 9.8 ile Ege Bölgesi takip etmektedir (TÜİK, 2016). Akdeniz ve Güneydoğu Anadolu bölgeleri arasında bir geçiş noktasında bulunan Osmaniye ilinde 2 079 da alanda 3 259 ton elma üretimi yapılmakta olup, ağaç başına ortalama verim 37.4 kg’dır (TÜİK, 2016). Osmaniye ili Akdeniz Bölgesi’nde yer almakta ve etrafının büyük bir bölümü Toros Dağları ile çevrili bulunmaktadır. Osmaniye ili Merkez ilçesi 123 m rakıma sahip olmakla birlikte, etrafını çeviren dağlarda yükselti 1 575 m (Zorkun) - 2 246 m (Düldül Dağları) aralığında değişim göstermektedir. 259 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Osmaniye Merkez ilçesi ve yakın çevresi Akdeniz iklimi özelliğine sahiptir. İlin yüksek rakımlı kısımlarında ise kışın daha soğuk ve yazın daha serin özelliğe sahip Akdeniz-Karasal bölgeler arasındaki geçit iklimi hüküm sürmektedir (Anonim, 2018a). Osmaniye Merkez ilçesi ve yakın çevresi Akdeniz iklimi özelliğine sahiptir. İlin yüksek rakımlı kısımlarında ise kışın daha soğuk ve yazın daha serin özelliğe sahip Akdeniz-Karasal bölgeler arasındaki geçit iklimi hüküm sürmektedir (Anonim, 2018a). Osmaniye ili Merkez ilçesinde +7.2 oC’nin altındaki toplam soğuklama süresinin 679-970 saat arasında değişim gösterdiği bildirilmektedir (Yelmen, 2007). İlde meyvecilik tarımının yoğun olarak yapıldığı Toros dağları etekleri, Düziçi, Bahçe ve Hasanbeyli ilçeleri gibi daha yüksek rakımlı bölgelerin soğuklama sürelerine ait veri bulunmamakla birlikte, bu kesimlerin Merkez ilçeden daha uzun bir soğuklamaya sahip olduğu düşünülmektedir. Bu çalışmada Osmaniye ili Bahçe ilçesi sınırları içerisinde bulunan, geçit iklimi özelliğine sahip alanda M9 anacı üzerine aşılı ve sık dikim uygulaması yapılan üç ayrı elma çeşidinin performanslarının incelenmesi amaçlanmıştır. Toprak özellikleri Araştırmanın yürütüldüğü bahçe toprağının kimyasal özellikleri Çizelge 1.’de verilmiştir. Araştırmanın yürütüldüğü bahçe toprağının kimyasal özellikleri Çizelge 1.’de verilmiştir. Çizelge 1. Araştırmanın yürütüldüğü bahçenin toprak analizi sonuçları (Bozkurt ve ark. 2001) Çizelge 1. Araştırmanın yürütüldüğü bahçenin toprak analizi sonuçları (Bozkurt ve ark. 2001) Toplam Azot (N) (%) Alınabilir fosfor (P2O5) (kg/da) Alınabilir potasyum (K2O) (kg/da) Alınabilir kalsiyum (CaO) (kg/da) Alınabilir magnezyum (MgO) (kg/da) Alınabilir demir (Fe) (kg/da) Alınabilir çinko (Zn) (kg/da) Toprak (pH) 0.56 7.0 25.1 2720 213 12.5 0.71 8.00 Orta Orta Yeterli Çok yüksek Çok yüksek Orta Orta Bazik 2. Materyal ve Yöntem Araştırma, 2012-2013 yılları arasında Osmaniye ili Bahçe ilçesi Burgaçlı köyü Payamlı mevkiinde bulunan bir üretici bahçesinde (37o10’ Kuzey ve 36o25’ Doğu; rakımı 393 m) yürütülmüştür. Araştırma bahçesi, M9 klonal anacı üzerine aşılı Mondial Gala, Scarlet Spur ve Fuji çeşitleri ile 2008 yılı kış döneminde 3x1 m sıra arası ve sıra üzeri mesafede kurulmuştur. Tozlayıcı çeşit olarak Golden Delicious kullanılmıştır. Ağaçlara merkezi lider terbiye sistemi uygulanmıştır. Bahçedeki ağaçların üzeri güneş yanıklığından korunması için gölgeleme materyali ile kaplanmıştır. Araştırmanın yürütüldüğü bahçe Nisan-Ekim ayları arasında damla sulama sistemi ile sulanmış, gübreleme ve ilaçlama gibi teknik ve kültürel işlemler düzenli bir şekilde yerine getirilmiştir. İklim özellikleri Araştırmanın yürütüldüğü Osmaniye ilinde 1986-2014 yıllarında arasında belirlenen ortalama sıcaklık 18.3 oC, maksimum sıcaklık 34.2 oC (Ağustos), minimum sıcaklık 3.4 oC (Ocak), toplam yağış 802 mm ve ortalama nispi nem değeri ise % 62.3 olarak belirtilmiştir (Anonim, 2018b). Verim Özellikleri Elma çeşitlerine ait ağaçlarda ağaç başına düşen verim (kg ağaç-1), birim gövde kesit alanına düşen verim (kg cm-2) ve dekara verim (kg da-1) miktarı değerleri belirlenmiştir (Yaşasın ve ark., 2006, Ikinci ve Bolat, 2016). Meyve Özelliklerin Belirlenmesi İncelenen elma çeşitlerinde, çeşidin özelliklerini temsil edecek şekilde her çeşide ait her bir ağaçtan hasat döneminde alınan 10’ar meyvede; meyve ağırlığı (g) 0.01 g’a duyarlı hassas terazi (Precisa BJ 6100D) ile; meyve eni (mm), meyve boyu (mm) 0.01 mm’ye duyarlı dijital kumpas (Mitutoyo CD- 20CPX) ile; meyve eti sertliği (kg cm-2) el penetrometresi ile; meyve hacmi, ağzına kadar saf su dolu ölçü silindirinin içine teker teker elmalar bırakılarak, taşan suyun ölçü silindirinde ölçülmesi ile (cm3) ve meyve tohum sayısı, meyvelerden çıkartılan tohumların sayılarak, ortalamalarının alınması ile (adet) belirlenmiştir (Burak ve ark., 1998; Yaşasın ve ark. 2006, Ikinci ve Bolat, 2016). Fenolojik gözlemler Fenolojik özellikler olarak; tomurcuk kabarması, tomurcuk patlaması, çiçeklenme başlangıcı, tam çiçeklenme, çiçeklenme sonu, meyvenin hasat olum tarihi ve yaprak döküm tarihleri tüm çeşitler için ayrı ayrı belirlenmiştir. Çiçeklenme başlangıcı; çiçeklerin % 5’nin açtığı, tam çiçeklenme; çiçeklerin % 60-70’inin açtığı ve çiçeklenme sonu; çiçeklerin taç yapraklarının % 90’dan fazlasının döküldüğü tarihler olarak belirlenmiştir (Chapman and Catlin, 1976; Erdoğan ve Bolat, 2002; Yaşasın ve ark., 2006; Ikinci ve Bolat, 2016). 260 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Kimyasal Özelliklerin Belirlenmesi Çeşitlere ait meyvelerde toplam suda eriyebilir kuru madde (SÇKM) miktarı el refraktometresi ile % olarak ölçülerek (Burak ve ark., 1998; Karaçalı, 2004; Yaşasın ve ark., 2006; Özrenk ve ark., 2011; Abacı ve Sevindik, 2014; Ikinci ve Bolat, 2016), titre edilebilir asitlik (TA) meyve suyu örneğinin 0.1N NaOH ile titre edilmesi ve harcanan baz miktarına göre malik asit cinsinden hesaplanma ile % olarak (Karaçalı, 2004; Ikinci ve Bolat, 2016), kabuklu meyve örneğinde toplam fenolik madde (mg/100 g taze ağırlık) tayini Folin-Ciocalteu yöntemi kullanılarak (Hayoğlu ve Türkoğlu, 2007; Abacı ve Sevindik, 2014), indirgen ve toplam şeker tayinleri ise Lane-Eynon metoduna göre belirlenmiştir (Hayoğlu ve Türkoğlu, 2007; Cemeroğlu, 2010). İstatistik Analiz Araştırma tesadüf blokları deneme desenine göre 3 tekerrürlü ve her tekerrürde 3 ağaç olacak şekilde kurulmuştur. Denemeden elde edilen sonuçların değerlendirilmesinde JMP 8.0 paket programı kullanılmıştır. Ortalamalar arasındaki farklılıkların karşılaştırılmasında ‘LSD Testi’ (p<0.05 ve p<0.01) kullanılmıştır. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi 3. Bulgular ve Tartışma Denemede yer alan çeşitlerde 2012 ve 2013 yıllarında yapılan fenolojik gözlemlerde; tomurcuk kabarması 14 Mart (Mondial Gala)-23 Mart (Fuji) ve tomurcuk patlaması ise 20 Mart (Mondial Gala) - 31 Mart (Fuji) tarihleri arasında gerçekleşmiştir (Çizelge 2.). Çeşitler arasında en erken çiçeklenmeye Mondial Gala (3 Nisan), en geç ise Fuji (17 Nisan) başlamıştır. İncelenen üç elma çeşidinde tam çiçeklenme 11 Nisan (Mondial Gala) - 25 Nisan (Fuji) tarihleri arasında gerçekleşirken, çiçeklenme sonu ise 17 Nisan (Mondial Gala) – 05 Mayıs (Fuji) tarihleri arasında meydana gelmiştir. y ( j ) y g Bahçe (Osmaniye) İlçesi koşullarında yapılan gözlemlerde, elma çeşitlerinde ilk hasat olgunluğuna 11 Ağustos’ta Mondial Gala çeşidi gelirken, en geç ise 5 Ekim’de Fuji çeşidi gelmiştir (Çizelge 2.). Elma çeşitlerinde tam çiçeklenmeden hasada kadar geçen süre 122 gün (Mondial Gala) ile 167 gün (Fuji) arasında değişmiştir. Elma çeşitlerinin yaprak dökümü 8 Aralık - 27 Aralık tarihleri arasında meydana gelmiştir. 261 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Çizelge 2. Mondial Gala, Fuji ve Scarlet Spur elma çeşitlerinde 2012 ve 2013 yılına ait bazı fenolojik gözlem sonuçları Çeşitler Yıl T.K. T.P. Ç.B. T.Ç. Ç.S. H.T. T.Ç.H.G. Mondial Gala 2012 20.03 27.03 12.04 21.04 30.04 22.08 123 2013 14.03 20.03 03.04 11.04 17.04 11.08 122 Scarlet Spur 2012 21.03 29.03 14.04 21.04 29.04 10.09 142 2013 15.03 21.03 05.04 12.04 20.04 30.08 141 Fuji 2012 23.03 31.03 17.04 25.04 05.05 05.10 163 2013 17.03 24.03 08.04 15.04 24.04 29.09 167 T.K: Tomurcuk kabarması. T.P: Tomurcuk patlaması, Ç.B: Çiçeklenme başlangıcı, T.Ç: Tam çiçeklenme, Ç.S: Çiçekleneme sonu, H.T: Hasat tarihi, T.Ç.G.S: Tam çiçeklenmeden hasada kadar geçen gün sayısı Mondial Gala, Fuji ve Scarlet Spur elma çeşitlerinde 2012 ve 2013 yılına ait bazı fenolojik gözlem sonuçları 2013 17.03 24.03 08.04 15.04 24.04 29.09 167 T.K: Tomurcuk kabarması. T.P: Tomurcuk patlaması, Ç.B: Çiçeklenme başlangıcı, T.Ç: Tam çiçeklenme, Ç.S: Çiçekleneme sonu, H.T: Hasat tarihi, T.Ç.G.S: Tam çiçeklenmeden hasada kadar geçen gün sayısı Tomurcuk kabarması. T.P: Tomurcuk patlaması, Ç.B: Çiçeklenme başlangıcı, T.Ç: Tam çiçeklenme, Ç.S: Çiçeklenem H.T: Hasat tarihi, T.Ç.G.S: Tam çiçeklenmeden hasada kadar geçen gün sayısı Burak ve ark. (1994), Yalova koşullarında çöğür anacına aşılı Jerseymac çeşidinde tomurcuk kabarmasının 15 Mart, tomurcuk patlamasının 29 Mart, ilk çiçeklenmenin 16 Nisan, tam çiçeklenmenin 19 Nisan ve çiçeklenme sonunun ise 30 Nisan tarihlerinde gerçekleştiğini bildirmişlerdir. 3. Bulgular ve Tartışma Benzer ekolojide yapılan bir başka araştırmada ise elma çeşitlerinin çiçeklenme başlangıcının 16 Mart - 21 Nisan tarihleri arasında meydana geldiği tespit edilmiştir (Yaşasın ve ark., 2006). Tokat ekolojik koşullarında yetiştirilen dört farklı elma çeşidinde ilk çiçeklenme 29 Mart -26 Nisan tarihleri arasında (Polat, 1997) olurken, Eğirdir (Isparta) ekolojik koşullarında ise ilk çiçeklenme 16 Nisan - 18 Nisan tarihleri arasında meydana gelmiştir (Atay ve ark., 2010). Atay ve ark. (2010), Eğirdir koşullarında MM 106 anacı üzerine aşılı Jerseymac, Galaxy Gala ve Braeburn çeşitlerinin 2006 yılında sırasıyla; 20, 22 ve 20 Nisan tarihlerinde tam çiçeklenme aşamasında olduğunu, 2007 yılında ise sırasıyla; 28 Nisan, 1 Mayıs ve 28 Nisan tarihlerinde tam çiçeklenme aşamasına ulaştıklarını bildirmişlerdir. Van ekolojik koşulları altında 2005-2007 yılları arasında yöresel elma genotiplerinde yapılan gözlemlerde tam çiçeklenmenin 30 Nisan - 11 Mayıs (Kaya ve Balta, 2009), Çoruh Vadisi’nde yetişen elma çeşitlerinde 8 - 22 Mayıs (Erdoğan ve Bolat, 2002) ve Tokat ekolojik koşullarında yetiştirilen elma çeşitlerinde ise 5-29 Nisan (Polat, 1997) tarihleri arasında meydana geldiği belirlenmiştir. Özongun ve ark. (2014), Eğirdir/Isparta ekolojisinde MM 106 anacına aşılı 10 elma çeşidinde 2003-2009 yılları arasındaki fenolojik gözlem sonuçlarına göre çiçeklenme sonunun 01-05 Mayıs tarihlerinde olduğunu bildirmişlerdir. Eğirdir koşullarında yıllara göre yapılan fenolojik gözlem sonuçlarına göre M9, M26, MM 106 ve MM 111 anacı üzerine aşılı Mondial Gala elma çeşidinde tomurcuk kabarma tarihi 20-25 Mart, tomurcuk patlaması 29 Mart-5 Nisan, ilk çiçeklenme 17-21 Nisan, tam çiçeklenme 23-29 Nisan ve çiçeklenme sonu 1-7 Mayıs olarak belirlenmiştir (Özongun ve ark., 2016). Benzer şekilde ülkemizin farklı bölgelerinde değişik elma çeşitleri üzerinde yapılan çalışmalarda da çiçeklenme sonunun bölgeye ve çeşit özelliğine bağlı olarak değişim gösterdiği saptanmıştır (Polat. 1997; Atay ve ark.. 2010). Tokat koşullarında farklı elma çeşitlerinden meyve olgunlaşmasının 5-19 Eylül (Polat 1997), Van koşullarında 22 Ağustos - 10 Ekim (Kaya ve Balta, 2009), Ordu koşullarında 25 Eylül - 17 Ekim (Yarılgaç ve ark., 2009) ve Eğirdir koşullarında 25 Ağustos- 1 Eylül (Özongun ve ark., 2016) tarihleri arasında gerçekleştiği saptanmıştır. Araştırmamızda incelemiş olduğumuz elma çeşitlerinde 2012 yılında tam çiçeklenmeden - hasada kadar geçen süre 123 gün (Mondial Gala) ile 163 gün (Fuji), 2013 yılında ise 122 gün (Mondial Gala) ile 167 gün (Fuji) olarak tespit edilmiştir (Çizelge 2.). 3. Bulgular ve Tartışma Ülkemizin farklı bölgelerinde değişik elma çeşitleri üzerinde yapılan çalışmalarda tam çiçeklenmeden hasada kadar geçen süre Niğde ekolojik koşullarında ise 122 - 164 gün (Ceylan, 2008), Van koşullarında 102 - 150 gün (Kaya ve Balta, 2009), Tokat koşullarında 98 - 180 gün (Atay ve ark. 2010) ve Eğirdir koşullarında 142-169 gün (Seymen ve Polat, 2015) arasında değişim gösterdiği saptanmıştır. Ülkemizin değişik yörelerinde yapılan çalışmalarda, fenolojik gözlemler bakımından birbirinden farklı sonuçlar elde edilmiştir. Meyve yetiştiriciliğinde çiçeklenme zamanı ve süresi türlere, çeşitlere, anaçlara, ekolojiye, yetiştiriciliğin yapıldığı yerin enlem-boylamına, rakımına, sıcak ve soğuk rüzgarlara, yıllara ve kültürel uygulamalara göre değişkenlik göstermektedir (Karaçalı, 2004). Osmaniye koşullarında değişik dönemlerde olgunlaşan 3 elma çeşidi ait bazı meyve özellikleri Çizelge 3.'te verilmiştir. İncelenen meyve özellikleri bakımından çeşitler arasında istatistiksel olarak önemli (p<0.01 ve p<0.05) düzeyde farklılıkların olduğu tespit edilmiştir. Çeşitlerin ortalama meyve ağırlığı 157.5 g (Scarlet Spur) – 212.8 g (Fuji), ortalama meyve eni 68.2 mm (Mondial Gala) – 79.3 mm 262 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi (Fuji), ortalama meyve boyu 62.7 g (Mondial Gala) -67.5 (Scarlet Spur), ortalama meyve hacmi 166.7 ml (Mondial Gala) – 260 ml (Fuji) ve ortalama çekirdek sayısı 6.6 adet (Scarlet Spur) – 8.7 adet (Fuji) arasında saptanmıştır. Meyve eti sertliği bakımından Fuji çeşidi (8.2 kg/cm2) en yüksek değeri, Scarlet Spur çeşidi ise en düşük değeri (5.1 kg/cm2) vermiştir (Çizelge 3.). Suda çözünebilir kuru madde yönünden en yüksek değer Mondial Gala çeşidinden (% 14.7) elde edilirken, bu çeşidi Fuji (% 14.1) ve Scarlet Spur (% 13.9) çeşitleri takip etmiştir. En yüksek titre edilebilir asit miktarı Fuji çeşidinde (% 0.39), en düşük ise Scarlet Spur (% 0.28) çeşidinde belirlenmiştir. Eğirdir (Isparta) koşullarında MM 106 anacı üzerine aşılı 10 elma çeşidinin yer aldığı bir çeşit adaptasyon denemesinde, çeşitlerin meyve ağırlıkları 162 (Rubinstein) - 287 g (Crown Gold), meyve eni 69 (Early Red One) - 84 mm (Novaja), meyve boyu 57 (Gala Selecta) – 77 mm (Topred), meyve eti sertliği 6.94 (Crown Gold) - 9.55 kg/cm2 (Cripps Pink (Pink LadyTM)), SÇKM içeriği % 12.20 (Scarlet spur) – 16.40 (Cripps Pink (Pink LadyTM)) ve asitlik değeri % 0.31 (Topred) – % 1.12 (Cripps Pink (Pink LadyTM)) arasında belirlenmiştir (Özongun ve ark., 2014). 3. Bulgular ve Tartışma Konya ekolojisine uygun yeni elma çeşitlerinin belirlenmesi amacıyla yürütülen bir çalışmada, M9 anacına aşılı Fuji elma çeşidinde ortalama meyve ağırlığı 142.33 g, meyve eni 68.43 mm, meyve boyu 58.59 mm, çekirdek sayısı 8.83 adet, meyve eti sertliği 4.96 kg/cm², SÇKM miktarı % 12.40 ve titre edilebilir asit miktarı ise % 0.48 olarak belirlenmiştir (Arıkan ve ark., 2015). Samsun ekolojik şartlarında yetiştirilen bazı elma çeşitlerinde ortalama meyve ağırlığı 112.3 (Jersey Mac) - 173.9 g (Starkrimson Delicious), meyve eni 64.83 (Golden Delicious) - 74.27 mm (Granny Smith), meyve boyu 54.55 (Jersey Mac) - 63.74 mm (Red Chief), meyve eti sertliği 4.94 (Jersey Mac)-7.98 kg/cm² (Granny Smith), SÇKM % 10.46 (Starkrimson Delicious) - % 13.45 (Cooper 7 SB2 ve Golden Delicious) ve TA % 0.39 (Süper Chief) - % 0.90 (Granny Smith) arasında değişim göstermiştir (Öztürk ve Öztürk, 2016). Eğirdir koşullarında M9 anacı üzerine aşılı Mondial Gala çeşidinin meyve ağırlığı, meyve eni, meyve boyu, meyve eti sertliği, SÇKM ve TA sırasıyla; 147 g, 69.04 mm, 61.73 mm, 9.25 kg/cm2, % 13.03, 3.70 ve % 0.30 arasında tespit edilmiştir (Özongun ve ark., 2016). p ş ( g , ) En önemli sekonder metabolitlerinden olan toplam fenolik madde içeriği incelenen çeşitlerde 119.10 mg/100 g (Scarlet Spur) – 145.25 mg/100 g (Fuji) arasında değişim göstermiştir (Çizelge 3.). Benzer şekilde Ardahan ekolojik koşullarında yetiştirilen elma çeşitlerinin meyve kabuğunda fenolik madde miktarları 209.7-578.9 mg/100 g, meyve etinde - ise 50.2 ile 112.2 mg/100 g arasında değişim gösterdiği saptanmıştır (Abacı ve Sevindik, 2014). Diğer taraftan Özden ve Özden (2014), olgunluk dönemindeki elma meyvelerindeki toplam fenolik madde içeriğini 698.67 mg/GAE/kg (Granny Smith) – 810.48 mg/GAE/kg (Gala) arasında yer aldığını belirlemişlerdir. Üç farklı elma çeşidinde indirgen şeker kapsamının % 9.13 (Scarlet Spur) - % 10.09 (Fuji) arasında değiştiği belirlenmiştir (Çizelge 3.). İndirgen şeker kapsamı üzerine yapılan istatistiki analizlerde, Mondial Gala ve Scarlet Spur çeşitleri aynı grupta, Fuji çeşidi ise farklı grupta yer almıştır. Ülkemizin farklı bölgelerinde değişik elma çeşitlerinde yapılan çalışmalarda da meyvedeki indirgen şeker kapsamı bakımından çeşitlere göre farklılıklar olduğu tespit edilmiştir. Nitekim, indirgen şeker kapsamı Çoruh Vadisi’nde yetişen elma çeşitlerinde % 5.30 (Fındık) - % 8.69 (Golden Delicious) (Erdoğan ve Bolat, 2002) arasında; Erzurum, Kars, Erzincan ve Gümüşhane koşullarında yetiştirilen elmalarda % 8.11 (Amasya) - % 10.06 (Göbek) (Keleş, 1979) arasında ve Erzincan koşullarında yetiştirilen elmalarda ise % 6.96 (Granny Smith) - % 8.97 (Golden Delicious) (Güleryüz ve ark., 2001) arasında değiştiği belirlenmiştir. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Bahçe (Osmaniye) İlçesi koşullarında M9 anacı üzerine aşılı 4 yaşlı Mondial Gala, Scarlet Spur ve Fuji elma çeşitleri arasında, ağaçlardan elde edilen veya hesaplama ile belirlenen tüm verim değerleri arasında istatistiksel anlamda önemli fark bulunmuştur (Çizelge 4.). Elma çeşitlerinden 2012-2013 yıllarında elde edilen ağaç başına ortalama verim değerlerinin 6.40 kg/ağaç (Scarlet Spur) – 7.89 kg/ağaç (Fuji) arasında değişim gösterdiği saptanmıştır. Ağaç başına verim değerini Niğde koşullarında değişik elma çeşitleri üzerinde yapılan benzer bir çalışmada -10.48 kg/ağaç (Galaxy Gala/M9) - 17.34 kg/ağaç (Granny Smith/M9) (Ceylan, 2008), Tokat koşullarında 3.24 kg/ağaç - 6.82 kg/ağaç (Gala/M9) (Küçüker ve ark., 2011) ve Çorum koşullarında 10.10 kg/ağaç (Red Chief/M9) - 16.60 kg/ağaç (Granny Smith/M9) (Çulha, 2010) arasında yer aldığı tespit edilmiştir. Denemede yer alan elma çeşitlerine ait ağaçlarda 14.51 cm2 (Mondial Gala) – 20.74 cm2 (Fuji) arasında gövde enine kesit alanı değerleri ölçülmüştür. Farklı elma çeşitlerinde Tokat koşullarında 2.09 cm2 (Delcorf) - 3.69 cm2 (Red Star) (Özkan ve ark., 2009), Yalova koşullarında 28.10 cm2 (Red Chief) - 494.63 cm2 (Mutsu) (Akçay ve ark., 2009) ve Tokat koşullarında 4.36 cm2 (Gala) - 20.51 cm2 (Gala) (Küçüker ve ark., 2011) arasında gövde enine kesit alanı tespit edilmiştir. Birim gövde kesit alanına düşen verim miktarları (verim etkisi =etkili verim) göz önüne alındığında, en yüksek değer Mondial Gala (0.50 kg/cm2) çeşidinden elde edilirken, en düşük ise Fuji çeşidinde (0.39 kg/cm2) tespit edilmiştir. Görükle (Bursa) ekolojik koşulları altında farklı elma çeşitlerinin etkili verim değerinin 0.15 kg/cm2 (Elstar, Topred) - 0.39 kg/cm2 (Granny Smith) (Soylu ve ark., 2003), Tokat koşullarında 0.16 kg/cm2 (Jonagold) - 0.40 kg/cm2 (Gala) (Baytekin ve Akça 2011), Yalova koşullarında 0.49 kg/cm2 (Stark Earliest) - 2.79 kg/cm2 (Starkrimson Delicious) (Akçay ve ark., 2009) arasında yer aldığı bildirilmiştir. Elma çeşitlerinde iki yılın ortalamasına göre belirlenen dekara verim değerlerine göre; en yüksek dekara verim 2628 kg ile Fuji çeşidinde belirlenirken, bu çeşidi Mondial Gala (2367 kg) ve Scarlet Spur (2135 kg) çeşitleri izlemiştir. Tokat koşullarında elma ağaçlardan elde edilen dekara verim değerleri 1 238 - 1 925 kg/da (Küçüker ve ark., 2011) arasında, Niğde koşullarında 2 324 kg/da (Scarlet Spur) – 3 326 kg/da (Super Chief) (Özdemir ve ark. 2009) ve Samsun koşullarında ise 791 kg/da (Skyline Supreme/MM 111) – 6 655 kg/da (Granny Smith/MM 106) (Kaplan ve Macit, 2009) arasında değişim gösterdiği belirtilmiştir. 3. Bulgular ve Tartışma Çeşitlerin toplam şeker içerikleri % 12.38 (Scarlet Spur) –% 13.74 (Fuji) aralığında değişim göstermiştir (Çizelge 3.). Ülkemizin farklı bölgelerinde değişik elma çeşitleri üzerinde yapılan çalışmalarda; Çoruh Vadisi koşullarında yetiştirilen elma çeşitlerinde % 8.38 (Fındık) - % 12.72 (Golden Delicious) (Erdoğan ve Bolat, 2002) ve Erzincan koşullarında yetiştirilen elma çeşitlerinde ise % 9.04 (Granny Smith) - % 11.84 (Sakı) (Güleryüz ve ark., 2001) arasında toplam şeker içeriği tespit edilmiştir. Diğer taraftan Teletar (1985), olgunluk dönemindeki elma meyvelerindeki toplam şeker içeriğinin % 9.56 (Amasya) - % 13.09 (Hüryemez) arasında değiştiğini saptamıştır. Ü Ülkemizin farklı ekolojilere sahip olan yörelerinde yürütülen araştırmalarda, aynı çeşit ve anaç bileşenleri kullanılmış olsa dahi, elma çeşitlerinin meyve kalite özellikleri ve kimyasal içeriklerinde kısmen birbirinden farklı sonuçlar elde edilmiştir. Değişik çalışmalarla, yürütülmüş olan bu çalışma arasındaki farklılığın çeşit, anaç, meyve tutum oranlarının farklı olmasından, kültürel uygulamalardan, iklim ve toprak özelliklerinden, yetiştiriciliğin yapıldığı yerin enlem-boylam ve rakımından kaynaklandığı düşünülmektedir. 263 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Çalışmamızda; verim değerleri ve gövde enine kesit alanı ile ilgili elde edilen bulgular, ülkemizin farklı yörelerinde yürütülen çalışmalarda elde edilen bulgularla bazı durumlarda benzerlik, bazı durumlarda ise farklılık gösterdiği belirlenmiştir. Bulgular arasındaki farklılığın; çalışmalardaki anaç, çeşit, kültürel uygulamalar, iklim, toprak ve ağacın yaşı gibi özelliklerin farklılık göstermesinden kaynaklandığı düşünülmektedir. 264 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 K ğ d F kl Dö l d Ol l B El Ç i l i i P YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin P Çizelge 3. Mondial Gala, Scarlet Spur ve Fuji elma çeşitlerinin bazı meyve özellikleri (2012-2013 yılı ortalaması) Çeşitler Meyve ağırlığı (g) Meyve eni (mm) Meyve boyu (mm) Meyve hacmi (cm3) Çekirdek sayısı (adet) Meyve eti sertliği (kg/cm2) SÇKM (Briks) Titre edilebilir asitlik (%) Toplam fenol (mg/100g taze ağırlık) İndirgen şeker kapsamı (%) Toplam şeker (%) Mondial Gala 169.8 bz 68.2 b 62.7 b 166.7 c 7.3 b 7.4 b 14.7 a 0.36 ab 122.80 b 9.40 b 12.97 ab Scarlet Spur 157.5 b 68.7 b 67.5 a 225.5 b 6.6 b 5.1 c 13.9 b 0.28 b 119.10 b 9.13 b 12.38 b Fuji 212.8 a 79.3 a 65.4 ab 260.0 a 8.7 a 8.2 a 14.1 b 0.39 a 145.25 a 10.09 a 13.74 a LSD 0.05 15.17* 6.60* 3.63** 34.36* 1.06** 0.75** 0.34** 0.07** 17.30** 0.43** 0.86** z Aynı sütunda aynı harfle işaretlenmiş ortalamalar arasında istatistiksel bakımdan fark yoktur (P<0.05). *:% 5 ve **: % 1 düzeylerinde önemli. Ö.D.:Önemli Değil ve Fuji elma çeşitlerinin bazı meyve özellikleri (2012-2013 yılı ortalaması) z Aynı sütunda aynı harfle işaretlenmiş ortalamalar arasında istatistiksel bakımdan fark yoktur (P<0.05). *:% 5 ve **: % 1 düzeylerinde önemli. Ö.D.:Önemli Değil Çizelge 4. Mondial Gala, Scarlet Spur ve Fuji elma çeşitlerine ait ağaçların gövde enine kesit alanı, ağaç başına verimi, dekara verimi ve etkili verim değerleri (2012-2013 yılı ort.) Çizelge 4. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Mondial Gala, Scarlet Spur ve Fuji elma çeşitlerine ait ağaçların gövde enine kesit alanı, ağaç başına verimi, dekara verimi ve etkili verim değerleri (2012-2013 yılı ort.) Çeşitler Verim (kg/ağaç) Gövde enine kesit alanı (cm2) Birim gövde kesit alanına düşen verim (kg cm-2) Birim alana düşen verim (kg da-1) Mondial Gala 7.10 b z 14.51 c 0.50 a 2 367.0 b Scarlet Spur 6.40 c 15.99 b 0.41 b 2 135.5 c Fuji 7.89 a 20.74 a 0.39 b 2 628.0 a LSD 0.05 0.36** 0.46** 0.065** 120.19** z Aynı sütunda aynı harfle işaretlenmiş ortalamalar arasında istatistiksel bakımdan fark yoktur (P<0.05). **: % 1 düzeylerinde önemli. 265 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi 4. Sonuç Doğu Akdeniz Bölgesi’nde yer alan Osmaniye ilinde genellikle kışlar ılıman geçmektedir. Osmaniye’de öteden beri ağırlıklı olarak tarla tarımı yapılmakta, meyvecilik sektörünün tarımsal üretimdeki payı ise oldukça düşük düzeydedir. İlde eskiden elma yetiştiriciliği hemen hemen hiç yapılmamaktaydı. Ancak, son yıllarda ülkemize giren yeni çeşitler, Osmaniye ili üreticilerinin de dikkatini çekmiş ve uygun alanlarda özellikle bodur anaçlarının kullanıldığı, soğuklama isteği fazla olmayan elma çeşitlerinin kullanıldığı ve sık dikim sistemlerinin uygulandığı elma bahçeleri tesis edilmeye başlanmıştır. Mondial Gala, Scarlet Spur ve Fuji elma çeşitleri ülkemizde son dönemlerde önemli derecede yaygınlık gösteren ve pazar değerleri yüksek olan çeşitlerdir. Bu çeşitler ülkemizde hemen her bölgede yetiştirilmekle birlikte, Akdeniz Bölgesi ve İç Anadolu Bölgesi arasındaki geçit konumu özelliği gösteren Doğu Akdeniz havzasının yüksek kesimleri için (özellikle soğuklama ve renklenme ile ilgili sorunların ortadan kalkması nedeniyle) çok daha büyük bir potansiyel taşımaktadır. Araştırmanın yürütüldüğü geçit iklimi özelliği gösteren lokasyonda, üzerinde çalışılan üç elma çeşidinde de bitki gelişimi, verim ve meyve kalitesi açısından önemli bir sorunla karşılaşılmamıştır. Gerek kendi verilerimizden ve gerekse diğer bölgelerde yapılan çalışmalardan elde edilen bulguların ışığında, yaz dönemi açısından Mondial Gala, erken güz dönemi açısından Scarlet Spur ve güz dönemi açısından ise Fuji elma çeşidinin, bölgedeki elma tarımı yapılabilecek benzer alanlarda ürün çeşitliğinin sağlanması açısından üreticilere katkı sağlayacağı düşünülmektedir. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Kaynakça Abacı, Z. T., & Sevindik, E. (2014). Ardahan bölgesinde yetiştirilen elma çeşitlerinin biyoaktif bileşiklerinin ve toplam antioksidan kapasitesinin belirlenmesi. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi, 24(2), 175-184. Akçay, M. E., Doğan, A., Burak, M., Yaşasın, A. S., & Öz, F. (2009). Bazı elma çeşitlerinin Marmara Bölgesinde yapılan adaptasyon çalışmaları. Tarım Bilimleri Araştırma Dergisi, 2(2), 65-71. Anonim. (2018a). http, //www.osmaniye.gov.tr Erişim tarihi: 06 Ekim 2018. İ İ Anonim. (2018b). Osmaniye Meteoroloji İl Müdürlüğü İklim verileri. İ Arıkan, Ş., İpek, M., & Pırlak, L. (2015). Konya ekolojik şartlarında bazı elma çeşitlerinin fenolojik ve pomolojik özelliklerinin belirlenmesi. Türk Tarım – Gıda Bilim ve Teknoloji Dergisi, 3(10), 811- 815. Atay, A. N., & Atay, E. (2018). Elma ıslahında ve çeşit yönetiminde yenilikçi eğilimler. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi, 28(2), 234-240. Atay, E., Pırlak, L., & Atay, A. N. (2010). Determination of fruit growth in some apple varieties. J. of Agric. Sci., 16, 1-8. Baytekin, S., & Akça, Y. (2011). M9 elma anacı üzerine aşılı farklı elma çeşitlerinin performanslarının belirlenmesi. Gaziosmanpaşa Üniversitesi Ziraat Fakültesi Dergisi, 28(1), 45-51. Burak, M., Büyükyılmaz, M., & Öz, F, (1998). Marmara Bölgesi için ümitvar elma çeşitlerinin seçimi. Bahçe, 27, 107-119. Bozkurt, M. A., Yarılgaç, T., & Çimrin, K. M. (2001). Çeşitli meyve ağaçlarında beslenme durumlarının belirlenmesi. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi, 11(1), 39-45. Ceylan, F. B., (2008). Bodur ve yarı bodur anaçlar üzerine aşılı bazı elma çeşitlerinin Niğde ekolojik şartlarında fenolojik ve pomolojik özelliklerinin tespiti (Yayımlanmamış yüksek lisans tezi). Selçuk Üniversitesi, Fen Bilimleri Enstitüsü, Konya, 56s. Çulha, A. E. (2010). Çorum ekolojik şartlarında M9 anacına aşılı bazı elma çeşitlerinin fenolojik ve pomolojik özelliklerinin tespiti (Yayımlanmamış yüksek lisans tezi). Selçuk Üniversitesi, Fen Bilimleri Enstitüsü, Konya, 54s. y STAT. (2017). FAO Statistical database. http, // faostat.fao.org/site/567/DesktopDefault.asp Erişim tarihi: 02 Mart 2019. Gerçekçioğlu, R., Bilgener, Ş., & Soylu, A. (2012). Genel meyvecilik (Meyve yetiştiriciliğin esasları). Nobel Akademik Yayıncılık Eğitim ve Danışmanlık Tic. Ltd. Şti., Ankara, 486s. 266 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267 Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Güleryüz, M., Ercişli, S., & Erkan, E. (2001). Erzincan ovasında yetiştirilen bazı elma çeşitlerinin meyve gelişimi dönemlerinde meydana gelen fiziksel ve kimyasal değişimler ile bunlar arasındaki ilişkiler. Atatürk Üniversitesi Ziraat Fakültesi Dergisi, 32(1), 51-59. Hayoğlu, İ., & Türkoğlu, H. (2007). Meyve-sebze işleme teknolojisi dersi uygulama ders notları. Harran Üniversitesi Ziraat Fakültesi, Gıda Mühendisliği Bölümü, Şanlıurfa, 42s. Kaynakça Ikinci, A., & Bolat, I. (2016, October). Determination of phenological, pomological and yield characteristics of low chilling apple cultivars budded on M9 and MM 106 rootstocks. VIII International Scientific Agricultural Symposium "Agrosym 2016" içinde (627-636. ss.). Jahorina, Bosnia and Herzegovina. Kaplan, N., & Macit, İ. (2009). Samsun koşullarında bazı elma çeşitlerinin bitkisel geli verimliliği üzerine elma klon anaçlarının etkisi. Tar. Bil. Araş. Derg., 2(2), 159-166. Kaplan, N., & Macit, İ. (2009). Samsun koşullarında bazı elma çeşitlerinin bitkisel gelişimi ve verimliliği üzerine elma klon anaçlarının etkisi. Tar. Bil. Araş. Derg., 2(2), 159-166. Karaçalı, İ. (2004). Bahçe ürünlerinin muhafaza ve pazarlanması (4. Baskı). Ege Üniv. Ziraat Fakültesi l İ i çalı, İ. (2004). Bahçe ürünlerinin muhafaza ve pazarlanması (4. Baskı). Ege Üniv. Ziraat Fakülte Yayınları No: 494, İzmir, 413s. Kaya, T., & Balta, F. (2009). Van yöresi elma seleksiyonları 1: Periyodisite göstermeyen genotipler. Tarım Bilimleri Araştırma Dergisi, 2(2), 25-30. İ ş g ( ) Kaygısız, H. (2004). Elma yetiştiriciliği. Hasad Yayıncılık Ltd. Şti., İstanbul, 148 s. Ö Küçüker, E., Özkan. Y., & Yıldız, K. (2011). Farklı terbiye sistemi uygulanmış M9 anacına aşılı Gala (Malus domestica Borkh.) elma çeşidinde erken dönem performansının belirlenmesi. Batı Akdeniz Tarımsal Araştırma Enstitüsü Derim Dergisi, 28(1), 25-36. Akdeniz Tarımsal Araştırma Enstitüsü Derim Dergisi, 28(1), 25 36. Özbek, S. (1978). Elma yetiştiriciliği. Özel meyvecilik (Kışın yaprağını döken meyve türleri) (s. 15-94). Çukurova Üniversitesi Ziraat Fakültesi Yayınları Yayın No: 128 Ders Kitabı: 11 Adana g Özbek, S. (1978). Elma yetiştiriciliği. Özel meyvecilik (Kışın yaprağını döken meyve türleri) (s. 15-94). Çukurova Üniversitesi Ziraat Fakültesi Yayınları, Yayın No: 128, Ders Kitabı: 11, Adana. Ö Ü Ö İ Özçağıran, R., Ünal, A., Özeker, E., & İsfendiyaroğlu, M. (2005). Ilıman iklim meyve türleri, Yumuşak çekirdekli meyveler. Cilt, II. Ege Üniversitesi Ziraat Fakültesi Yayınları No: 556. Bornova, İzmir, 220s. Özden, M., & Özden, A. E. (2014). Farklı renkteki meyvelerin toplam antosiyanin, toplam fenolik kapsamlarıyla toplam antioksidan kapasitelerinin karşılaştırılması. Gıda Teknolojileri Elektronik Dergisi, 9(2), 1-12. Özongun, Ş., Dolunay, E. M., Öztürk, G., & Pektaş, M. (2014). Eğirdir (Isparta) şartlarında bazı elma çeşitlerinin performansları. Meyve Bilimi, 1(2), 21-29. Özongun, Ş., Dolunay, E. M., Pektaş, M., Öztürk, G., & Çalhan, Ö. (2016). Farklı klon anaçları üzerinde bazı elma çeşitlerinin verim ve kalite değişimleri. Ege Üniv. Ziraat Fak. Derg., 53(1), 35-42. Özrenk, K., Gündoğdu, M., Kaya, T., & Kan, T. (2011). Çatak ve Tatvan yörelerinde yetiştirilen yerel elma çeşitlerinin pomolojik özellikleri. Tarım Bilimleri Dergisi, 21(1), 57-63. Kaynakça Özongun, Ş., Dolunay, E. M., Pektaş, M., Öztürk, G., & Çalhan, Ö. (2016). Farklı klon anaçları üzerinde bazı elma çeşitlerinin verim ve kalite değişimleri. Ege Üniv. Ziraat Fak. Derg., 53(1), 35-42. Özrenk K Gündoğdu M Kaya T & Kan T (2011) Çatak ve Tatvan yörelerinde yetiştirilen yerel Özrenk, K., Gündoğdu, M., Kaya, T., & Kan, T. (2011). Çatak ve Tatvan yörelerinde yetiştiril elma çeşitlerinin pomolojik özellikleri. Tarım Bilimleri Dergisi, 21(1), 57-63. Ö Ö Öztürk, A., & Öztürk, B. (2016). Samsun ekolojisinde yetiştirilen standart bazı elma çeşitlerinin fenolojik ve pomolojik özelliklerinin belirlenmesi. Anadolu Tarım Bilimleri Dergisi, 31, 1-8. Ö Ö İ Öztürk, G., Özongun, S., Akgül, H., Kaymak, K., & Eren, İ, (2005). Bodur elma yetiştiriciliği. Eğirdir Bahçe Kültürleri Araştırma Enstitüsü, Çiftçi Eğitim Serisi 13, Isparta, 44s. Polat, M. (1997). Tokat koşullarında farklı gelişme kuvvetlerine sahip anaçlar üzerine aşılanmış elma çeşitlerinin fenolojik ve pomolojik özellikleri üzerine bir araştırma (Yayımlanmamış yüksek lisans tezi). Gaziosmanpaşa Üniversitesi, Fen Bilimleri Enstitüsü, Tokat, 101s. Seymen, T., & Polat, M. (2015). Bazı Amasya elma tiplerinin fenolojik, pomolojik özelli belirlenmesi ve morfolojik karakterizasyonu. Harran Tarım ve Gıda Bil. Derg., 19(3), 1 Seymen, T., & Polat, M. (2015). Bazı Amasya elma tiplerinin fenolojik, pomolojik özelliklerinin belirlenmesi ve morfolojik karakterizasyonu. Harran Tarım ve Gıda Bil. Derg., 19(3), 122-129. Soylu, A. (2003). Elma. Ilıman iklim meyveleri-II, Yumuşak çekirdekli meyveler (s.1-75). Uludağ Üniversitesi Ziraat Fakültesi Ders Notları, No: 72, Bursa. Soylu, A. (2003). Elma. Ilıman iklim meyveleri-II, Yumuşak çekirdekli meyveler (s.1-75). Üniversitesi Ziraat Fakültesi Ders Notları, No: 72, Bursa. Soylu, A., Ertürk, Ü., Mert, C., & Öztürk, Ö. (2003). MM 106 anacı üzerine aşılı elma çeşitlerinin Görükle koşullarındaki verim ve kalite özelliklerinin incelenmesi II. Uludağ Üniversitesi Ziraat Fakültesi Dergisi, 17(2), 57-65. TÜİK. (2016). Türkiye İstatistik Kurumu, Tarım İstatistikleri. https, //biruni.tuik.gov.tr/bitkiselapp/bitkisel.zul Erişim tarihi: 06 Ekim 2018. Yaşasın, A. S., Burak, M., Akçay, M.E., Türkeli, Y., & Büyükyılmaz, M. (2006). Marmara Bölgesi için ümitvar elma çeşitleri - V. Bahçe, 35(1-2), 75-82. Yelmen, H. (2007). Doğu Akdeniz Bölgesinde farklı soğuklama yöntemleri kullanılarak olasılıklı soğuklama süre haritalarının çıkarılması (Yayımlanmamış yüksek lisans tezi). Çukurova Üniversitesi, Fen Bilimleri Enstitüsü, Adana, 119s. 267
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In vitro–in silico correlation of three-dimensional turbulent flows in an idealized mouth-throat model
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PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY RESEARCH ARTICLE In vitro–in silico correlation of three- dimensional turbulent flows in an idealized mouth-throat model Eliram NofID1¤a, Saurabh Bhardwaj1¤b, Pantelis Koullapis2, Ron Bessler1, Stavros Kassinos2, Josue´ SznitmanID1* Eliram NofID1¤a, Saurabh Bhardwaj1¤b, Pantelis Koullapis2, Ron Bessler1, Stavros Kassinos2, Josue´ SznitmanID1* 1 Department of Biomedical Engineering, Technion—Israel Institute of Technology, Haifa, Israel, 2 Computational Sciences Laboratory (UCY-CompSci), Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus 1 Department of Biomedical Engineering, Technion—Israel Institute of Technology, Haifa, Israel, 2 Computational Sciences Laboratory (UCY-CompSci), Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤a Current address: Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America ¤b Current address: Department of Biomedical Engineering, Pennsylvania State University, State College, Pennsylvania, United States of America * sznitman@bm.technion.ac.il a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤a Current address: Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America ¤b Current address: Department of Biomedical Engineering, Pennsylvania State University, State College, Pennsylvania, United States of America * sznitman@bm.technion.ac.il Editor: Alison L. Marsden, Stanford University, UNITED STATES Received: September 5, 2022 Accepted: February 8, 2023 Published: March 23, 2023 Received: September 5, 2022 Accepted: February 8, 2023 Published: March 23, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pcbi.1010537 Copyright: © 2023 Nof 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. OPEN ACCESS There exists an ongoing need to improve the validity and accuracy of computational fluid dynamics (CFD) simulations of turbulent airflows in the extra-thoracic and upper airways. Yet, a knowledge gap remains in providing experimentally-resolved 3D flow benchmarks with sufficient data density and completeness for useful comparison with widely-employed numerical schemes. Motivated by such shortcomings, the present work details to the best of our knowledge the first attempt to deliver in vitro–in silico correlations of 3D respiratory air- flows in a generalized mouth-throat model and thereby assess the performance of Large Eddy Simulations (LES) and Reynolds-Averaged Numerical Simulations (RANS). Numeri- cal predictions are compared against 3D volumetric flow measurements using Tomographic Particle Image Velocimetry (TPIV) at three steady inhalation flowrates varying from shallow to deep inhalation conditions. We find that a RANS k-ω SST model adequately predicts velocity flow patterns for Reynolds numbers spanning 1’500 to 7’000, supporting results in close proximity to a more computationally-expensive LES model. Yet, RANS significantly underestimates turbulent kinetic energy (TKE), thus underlining the advantages of LES as a higher-order turbulence modeling scheme. In an effort to bridge future endevours across respiratory research disciplines, we provide end users with the present in vitro–in silico cor- relation data for improved predictive CFD models towards inhalation therapy and therapeu- tic or toxic dosimetry endpoints. Citation: Nof E, Bhardwaj S, Koullapis P, Bessler R, Kassinos S, Sznitman J (2023) In vitro–in silico correlation of three-dimensional turbulent flows in an idealized mouth-throat model. PLoS Comput Biol 19(3): e1010537. https://doi.org/10.1371/ journal.pcbi.1010537 Editor: Alison L. Marsden, Stanford University, UNITED STATES a link to a repository: https://doi.org/10.6084/m9. figshare.20239125. a link to a repository: https://doi.org/10.6084/m9. figshare.20239125. a link to a repository: https://doi.org/10.6084/m9. figshare.20239125. resources, fluid dynamics (CFD) simulations are widely sought to predict deposition out- comes but often lack detailed experimental data to first validate the three-dimensional (3D) flow structures anticipated to arise in the upper respiratory tract. In an effort to rec- oncile such data scarcity, we deliver experimental-numerical correlations of 3D respira- tory airflows in an idealized 3D printed mouth-throat model against two widely- established numerical schemes with varying computational costs, namely coarse RANS and finer LES technique. Our time-resolved 3D flow data underline the complexity of these physiological inhalation flows, and discuss advantages and drawbacks of the differ- ent numerical techniques. With an outlook on future respiratory applications geared towards broad preclinical inhaled aerosol deposition studies, our open source data are made available for future benchmark comparisons for a broad range of end users in the respiratory research community. Funding: This work was supported by the Israel Science Foundation (ISF) (grant no. 1840/21 to JS), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 677772 to JS) as well as the PBC Fellowship Program by the Israeli council for supporting SB with partial funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: N/A. PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Author summary The dispersion and ensuing deposition of inhaled airborne particulate matter in the lungs are strongly influenced by the dynamics of turbulent respiratory airflows in the mouth- throat region during inhalation. To cirumvent costly in vitro experimental measurement Data Availability Statement: Relevant data are within the manuscript and its Supporting information files, and in addition we have provided 1 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 Introduction Respiratory airflow characteristics are known to strongly influence the transport and deposi- tion of inhaled aerosols in the human airways. The intricate geometry of the extra-thoracic that includes bends, expansions and constrictions leads to transition to turbulent airflows in the pharynx, larynx and trachea with Reynolds numbers on the order of several thousands (i.e., 2’000–10’000), depending on inhalation regimes [1–4]. Detailed modeling of these com- plex flows is required to determine the fate of particle-laden airflows and ensuing deposition patterns towards predicting for example pulmonary dosimetry [5] or the dispersion of air- borne pathogen in the lungs [6, 7]. p g g In recent years, computational fluid dynamics (CFD) have spearheaded advances to over- come some of the prohibitive costs of in vitro experimental campaigns. Numerical methods solving the Reynolds-Averaged Navier-Stokes (RANS) equations are the most widely adopted for modeling non-laminar flows due to their lower computational cost. RANS typically involves applying a turbulence model to a stationary solution at steady breathing conditions [8]. Coupled with the low-Reynolds number (LRN) k–ω turbulence model (e.g. k–ω SST), RANS is often used to predict laminar–transitional–turbulent flows in the respiratory tract [9, 10]. While RANS simulations are popular, they often come short of determining model con- stants, making clinical relevance and comparison with other studies challenging [11–15]. Ria- zuddin et al. [16] used a k-ω SST turbulence model to investigate breathing in a nasal cavity, demonstrating the model’s accuracy for modeling flows with unfavourable pressure gradients via good agreement with experimental and numerical data. Ma et al. simulated airflows and aerosol transport in patient-derived human airways with a k- model, showing good agree- ment with coarse (i.e., regionally averaged) in vivo deposition data [17]. Stapleton et al. simi- larly studied aerosol deposition using a k- model in an idealized mouth–throat validated with in vitro regional deposition measurements from gamma scintigraphy [18]. The authors found good agreement for laminar but not turbulent conditions, suggesting that particle deposition may be sensitive to pressure drop and flow recirculation. Longest et al. investigated local air- way aerosol deposition using different variants of the k-ω turbulence model [19], finding best agreement with in vitro deposition patterns using the low Reynolds number (LRN) approxi- mation. Despite such popularity, most RANS studies have used (sub-)regionally averaged deposition metrics for experimental validation, whereas more accurate and spatially-resolved deposition models would first require experimental validation of the underlying turbulent flows. Introduction In recent years, computational fluid dynamics (CFD) have spearheaded advances to over- come some of the prohibitive costs of in vitro experimental campaigns. Numerical methods solving the Reynolds-Averaged Navier-Stokes (RANS) equations are the most widely adopted for modeling non-laminar flows due to their lower computational cost. RANS typically involves applying a turbulence model to a stationary solution at steady breathing conditions [8]. Coupled with the low-Reynolds number (LRN) k–ω turbulence model (e.g. k–ω SST), 2 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model As RANS modelling is not sufficiently accurate to capture small-scale fluctuations in turbu- lent flows in extra-thoracic airways, a more adequate choice lies in Large Eddy Simulations (LES). There, only the smallest flow scales containing a small fraction of the kinetic energy are discarded, thereby retaining significantly more features of the underlying turbulence physics compared with RANS [20, 21]. Lin et al. [20] were among the first to use LES in assessing the effect of laryngeal jet-induced turbulence on airflow characteristics and tracheal wall shear stress. Their study revealed that turbulence generated by the laryngeal jet can significantly affect the downstream flow patterns, highlighting the importance of including the extrathor- acic airways in a model. Choi et al. [2] performed LES in two CT-derived upper airway models to examine the effect of inter-subject variabilities on the overall flow characteristics, finding that the glottis constriction ratio and the curvature and shape of the airways have significant effect on the generated flows. More recently, Koullapis et al. used LES to investigate inlet flow conditions in a CT-reconstructed geometry of the human airways [21] where flow field differ- ences largely dissipated just a short distance downstream of the mouth inlet. Furthermore, increasing the inhalation flowrate from sedentary to active breathing conditions left the mean flow field structures largely unaffected. Recent LES-based deposition studies have established the trustworthiness of the method using generalized geometries [22, 23] meanwhile constant gains in computing power have made LES more affordable. However, the underlying compu- tational expense of LES is still considerably higher than RANS, precluding its accessibility for broader use. Furthermore, imaging modalities are generating larger and more intricate patient geometries, requiring first the validation of simpler models for practical and patient-specific applications. Experimental method We briefly describe the experimental setup (Figs 1 and S1. A closed-loop perfusion system comprising a centrifugal pump, 15 l reservoir tank and digital flow rate sensor, supplies water/glycerol (58:42 mass ratio with a density of ρf = 1’150 kg/m3 and a dynamic viscosity of μf = 9.66 × 10−3 kg/m-s) through the phantom model (fabricated using previously described methods [34, 35]). We measure the three-dimensional, three-component (3D-3C) velocity fields in the phantom model using a high-speed, tomographic particle image veloci- metry setup (TPIV) (LaVision GmbH, Germany) consisting of four CMOS cameras (Fas- tcam Mini UX100, 1’280 x 1’024 pixel, 12 bit, Photron USA, Inc.) equipped with 100 mm focal length lenses (Zeiss Milvus, Germany). Volume illumination is provided by a 70 mJ dual-head Nd:YLF laser (DM30–527DH, Photonics Industries, USA) and continuous image acquisition was conducted using a frame straddling technique at a fixed 1’250 frames per second (fps), where the time separation between laser pulses was set to 25, 40 and 65 μs, respectively for the high, mid and low Re cases investigated. The field of view (FOV) spans 18.5 × 45 × 9 mm (x-y-z). These experimental settings were carefully optimized to ensure that individual seeded particles correspond to 3 to 5 pixels imaged in the instantaneous images so to avoid any peak-locking effects for PIV while maximum particle displacements betwee consecutive images range between 3 and 8 pixels depending on the flow case. The laser beam is introduced through the side of the model and shaped into a thick slab by an optical arrangement consisting of a beam expander and cylindrical lens, followed by a knife edge aperture (Fig 1a); the latter is commonly used in PIV to reduce light reflections from regions void of tracer particles, contributing amongst other to the rectangularity of the processed vector maps relative to the actual elliptical shape of the experimental model (see results). Details on the TPIV methodology, including refractive index matching and scaling following dynamic similarity can be found in our previous work [35], where raw images and TPIV pro- cessing are performed with Davis 10 (LaVision GmbH, Germany) and further analyzed in Matlab (Mathworks Inc., USA). Briefly, red fluorescent polystyrene particles (PS-FluoRed, microParticles GmbH, Germany) are seeded and act as flow tracers where optical filters are fitted to each camera lens to reduce non-fluorescent light reflection thereby increasing signal- to-noise (SNR) ratio. Geometry and flow conditions The oral airway is based on a simplified elliptic model extending from the mouth through the larynx and previously used in aerosol inhalation studies [31]. This elliptic mouth-throat (MT) model generalizes a patient-specific geometry derived from a healthy adult’s computed tomog- raphy (CT) scan [32]. Comparisons between in vitro measurements and in silico simulations are carried out at three distinct Reynolds numbers, namely Re = 1’500, 4’500 and 7’000 based on the inlet mouth diameter of the model. Values of Re correspond to steady inhalation air flowrates of approximately 11.5, 32.8 and 52 l/min; such inhalation conditions are comparable to seden- tary, light and heavy exercise conditions, respectively. Moreover, the two latter inhalation flowrates are specifically relevant for pulmonary drug delivery via a dry powder inhaler (DPI) [33]. Introduction To this end, a European Cooperation in Science and Technology (COST) action pub- lished a much needed benchmark case, known as the Siminhale benchmark [23], comparing several numerical schemes in an idealized airway geometry spanning mouth to the fourth bronchial generation at an inhalation flowrate of 60 l/min. In silico predictions were vali- dated against 2D particle image velocimetry (PIV) measurements [24] with good agreement between PIV and LES, with a slight over-prediction of turbulent kinetic energies (TKE) in the simulations. The experimental and numerical data in [24], along with two additional LES and a RANS datasets, are currently part of a common publicly accessible ERCOFTAC data- base providing best practice advice for setting up a computational fluid-particle dynamics (CFPD) model of the human upper airways with available validation data. However, the PIV data are limited to six orthogonal 2D planes and the laryngeal constriction, known to strongly modulate the inlet flow [25], was not captured due to obstruction from optical access. Several studies have measured flow in the laryngeal site using planar PIV alone [26, 27], while others employed numerical methods without comparable experimental measure- ments. Jayaraju et al. validated their LES simulations in a mouth-throat model with planar PIV, but measured only at the mid-sagittal plane [22]. In turn, a knowledge gap still remains in providing an experimental benchmark with sufficient data density and completeness for useful comparison to numerical schemes. Recently, volumetric flow measurement tools have been leveraged to study respiratory flows in 3D with tomographic particle image velocimetry (TPIV) and magnetic resonance velocimetry (MRV) [28, 29]. Notably, Kenjeres and Tijn validated their RANS and LES simula- tions with available 3D MRV data [28] in an identical upper airway geometry [30] but the use of a patient-specific model limits validations for future comparative studies. Motivated by these ongoing shortcomings, the present work details the first attempt to deliver in vitro–in silico flow correlations in a generalized mouth-throat model capturing 3D airflow patterns. RANS and LES predictions are compared against 3D TPIV flow measurements at three steady inhalation flowrates varying from shallow to deep inhalation conditions. We share our model 3 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model geometry and TPIV measurement data https://doi.org/10.6084/m9.figshare.20239125 as open- source material towards future benchmark references. Experimental method The mean particle diameter dp = 10 μm and particle density ρp = 1’050 kg/ 4 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Workstation Flowrate sensor Centrifugal pump Inlet Thermometer Outlet Ball valves Cylindrical lens Beam expander High speed cameras 15 liter reservoir °C Nd:YLF laser Knife edges Phantom model Experimental setup pharynx oral cavity larnyx trachea Phantom model 10 m/s PIV 1 mm (c) (b) (a) Fig 1. Model geometry and tomographic particle image velocimetry (TPIV) setup. (a) Experimental setup consisting of a laser, optical equipment, flow system, four high-speed cameras and a phantom idealized mouth-throat model. (b) The phantom model is illustrated schematically in three-dimensional view with inlet and outlet ports marked. Note that the laryngeal constriction is illuminated by laser light, maximizing spatial resolution in the specific region of interest (ROI). (c) A representative vector field is plotted along the mid-sagittal plane, following TPIV processing algorithms. Workstation Flowrate sensor Centrifugal pump Inlet Thermometer Outlet Ball valves Cylindrical lens Beam expander High speed cameras 15 liter reservoir °C Nd:YLF laser Knife edges Phantom model Experimental setup (a) Flowrate sensor Centrifugal pump Inlet Thermometer Outlet Ball valves High speed cameras 15 liter reservoir °C fe es Phantom model Experimental setup Experimental setup High speed cameras Ball valves Flowrate sensor 10 m/s PIV 1 mm (c) pharynx oral cavity larnyx trachea Phantom model ( (b) (c) (b) Phantom model Phantom model Fig 1. Model geometry and tomographic particle image velocimetry (TPIV) setup. (a) Experimental setup consisting of a laser, optical equipment, flow system, four high-speed cameras and a phantom idealized mouth-throat model. (b) The phantom model is illustrated schematically in three-dimensional view with inlet and outlet ports marked. Note that the laryngeal constriction is illuminated by laser light, maximizing spatial resolution in the specific region of interest (ROI). (c) A representative vector field is plotted along the mid-sagittal plane, following TPIV processing algorithms. https://doi.org/10.1371/journal.pcbi.1010537.g001 https://doi.org/10.1371/journal.pcbi.1010537.g001 m3 yield a corresponding particle relaxation time ts ¼ d2 prp=18mf of 0.6 μs and equivalent to a particle Stokes number much smaller than unity. Concurrently, particle drift due to buoyancy effects resulting from density differences between the working fluid and the particle, are largely negligible as the (buoyant) terminal velocity is estimated to be ut = 5.7 × 10−7 m/s (i.e. Numerical methods For the CFD simulations, we introdude the governing equations and computational methodol- ogy used to describe the motion of air based on the Eulerian approach. The governing equa- tions for incompressible fluid flow are composed of the Navier-Stokes’ (momentum) and continuity equations. Two distinct numerical methodologies (RANS and LES) are adopted to model turbulent flows in the mouth-throat geometry. RANS details. A commercial software (ANSYS Fluent, ANSYS Inc.) was used to perform the transient flow simulations using a RANS approach in which mass and momentum (i.e. Navier-Stokes) conservation equations are solved numerically by using the finite volume method (FVM) in the 3D domain (see S1 Text. for RANS equations). Turbulent flow phenom- ena were modeled using the Shear Stress Transport (SST) k-ω model with Low Reynolds Num- ber (LRN) correction; considered the most suitable RANS model for predicting low turbulence flow in the respiratory system, in particular with limited available computational resources. The popular SST k-ω turbulence model [36, 37] is a two-equation eddy-viscosity model. The use of a k-ω formulation in the inner parts of the boundary layer renders the model directly usable all the way down to the wall through the viscous sub-layer. Hence the SST k-ω model can be used as a Low-Re turbulence model without any extra damping func- tions. The SST formulation also switches to a k- behaviour in the free-stream and thus avoids the common k-ω problem whereby the model is too sensitive to the inlet free-stream turbu- lence properties. Furthermore, the SST k-ω model is often credited for its good behaviour in adverse pressure gradients and separated flows [3, 38]. Note that the SST k-ω model is acknowledged to produce slightly high turbulence levels in regions with large normal strains, e.g. stagnation regions and regions with strong acceleration. However, this tendency is much less pronounced than with a normal k- model [39, 40]. The computational geometry was discretized in ICEM (ANSYS Inc., Canonsburg, PA) using tetrahedral elements with prism layers at the walls. The final mesh was transformed into a polyhedral mesh in Fluent (ANSYS, Inc.). A rigorous mesh convergence study was first car- ried out (i.e. ranging from 2M to 6M tetrahedral cells) to eventually select the final mesh of *900’000 polyhedral cells (converted from *2.4M tetrahedral cells), with up to 10 prism lay- ers for near-wall refinements (see S2 Fig). Experimental method ut ¼ gd2 pðrf rpÞ=18mf), compared with characteristic velocities of the flow on the order of Oð1Þ m/s at the mouth inlet. m3 yield a corresponding particle relaxation time ts ¼ d2 prp=18mf of 0.6 μs and equivalent to a particle Stokes number much smaller than unity. Concurrently, particle drift due to buoyancy effects resulting from density differences between the working fluid and the particle, are largely negligible as the (buoyant) terminal velocity is estimated to be ut = 5.7 × 10−7 m/s (i.e. ut ¼ gd2 pðrf rpÞ=18mf), compared with characteristic velocities of the flow on the order of Oð1Þ m/s at the mouth inlet. 5 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 Numerical methods For each Re condition, a fully-developed parabolic velocity profile is provided at the inlet surface whereas outflow condition is prescribed at outlet surface and guaranteeing no-slip velocity conditions at the walls. A second-order implicit scheme is used for the transient formulation, with a time step of 10−3 s to ensure good accu- racy. To decrease numerical diffusion in the unstructured three-dimensional mesh, a second- order upwind approach is utilized to discretize the advection terms. A segregated solver is then used to solve the resultant system of equations. The SIMPLE algorithm is used to solve the gov- erning equations by coupling velocity and pressure. LES details. Large Eddy Simulations (LES) are performed using the dynamic version of the Smagorinsky-Lilly subgrid scale model [41] in order to examine the unsteady flow in the upper airways geometry. Previous studies have shown that this model performs well in transi- tional flows in the human airways [24]. The airflow is described by the filtered set of incom- pressible Navier-Stokes equations. In order to generate appropriate inlet velocity conditions for the CFD model, a mapped inlet (or recycling) boundary condition is used [42]. To apply this boundary condition, the pipe at the inlet is extended by a length equal to ten times its diameter. The pipe section is ini- tially fed with an instantaneous turbulent velocity field generated in a separate pipe flow LES. During the simulation, the velocity field from the mid-plane of the pipe domain is mapped to the inlet boundary. Scaling of the velocities is applied to enforce the specified bulk flow rate. In 6 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Fig 2. Large Eddy Simulation (LES) grid sensitivity analysis (Re = 7’000). Comparison of 1D profiles of normalized time-averaged velocity magnitudes (first column) and turbulent kinetic energy (second column), normalized by the mean inlet velocity uin, are presented at three stations (see inset; top right panel) along the larynx (A-A’), glottis (B-B’) and upper trachea (C-C’), respectively, for two grid resolutions: (i) coarse (9.2M cells) and (ii) fine (25M cells). Fig 2. Large Eddy Simulation (LES) grid sensitivity analysis (Re = 7’000). https://doi.org/10.1371/journal.pcbi.1010537.g002 Results and discussion Glottal jet characteristics Flow through the human larynx, i.e. the passageway connecting the mouth to the respiratory airways, is modulated by a valve-like constriction known as the glottis. During inhalation, this constriction forms a jet of air that extends into the trachea known as the glottal or laryn- geal jet. The glottal jet embodies the most dominant flow feature in the upper airways and has been studied extensively in the production of voice and speech [44] as well as in its role in mixing and dissipating boluses of inhaled aerosols [3, 20, 45]. By plotting the velocity mag- nitude contours for the highest flowrate case (Re = 7’000), we observe the characteristic structure clearly, namely a high velocity flow region extending within and downstream of the constriction and dissipating further downstream (Fig 3a–c). In our idealized geometry, we observe a mostly symmetric glottal jet, while asymmetries would be expected in more realistic patient-specific anatomies; a phenomenon also known as glottal jet skewing [44]. We briefly note that typically, limited velocity data are experimentally resolved in the vicinity of the model’s walls (Fig 3a and 3d); this is a well-known limitation of PIV techniques and generally due to the decreasing concentration or loss of tracer particles at the walls, in con- junction with significant flow gradients resulting from the no-slip condition (and possibly wall reflections) [46]. The glottal constriction has been known to represent a source of turbulent flow, despite the relatively low Reynolds numbers (O(103)) [10]. Shear flows such as the glottal free jet can reduce the critical Reynolds threshold and nevertheless generate turbulent kinetic energy (TKE); a key characteristic associated with the formation of eddies and other coherent flow structures used in classical turbulence analyses. In the examined cases, airflow enters the mouth in the laminar regime at the low flow rate case (Re = 1’500). However, low levels of tur- bulence develop downstream due to geometrical effects such as bends and constrictions. In contrast, both for the intermediate and high flow rate cases (Re = 4’500 and Re = 7’000), air- flow now enters the mouth under turbulent flow conditions. In Fig 3d–f, we plot mean flow TKE contours along the mid-sagittal plane for the three modalities (TPIV, RANS and LES) at the higher Reynolds number case (Re = 7’000). While the velocity magnitude contours agree rather closely between all three modalities, we observe discrepences between TKE results. Numerical methods Comparison of 1D profiles of normalized time-averaged velocity magnitudes (first column) and turbulent kinetic energy (second column), normalized by the mean inlet velocity uin, are presented at three stations (see inset; top right panel) along the larynx (A-A’), glottis (B-B’) and upper trachea (C-C’), respectively, for two grid resolutions: (i) coarse (9.2M cells) and (ii) fine (25M cells) https://doi.org/10.1371/journal.pcbi.1010537.g002 this manner, turbulent flow is sustained in the extended pipe section, and a turbulent velocity profile enters the mouth inlet. At the outlet of the model (lower trachea), uniform pressure is prescribed. A no-slip velocity condition is imposed on the airway walls. The governing equations are discretized using a finite volume method and solved using OpenFOAM, an open-source CFD code [43]. The scheme is second-order accurate in both space and time. To ensure numerical stability the final time step used is 5 × 10−7s. A total of 9.2M cells was used to have sufficient grid resolution for LES, based on a grid sensitivity analy- sis (see S3 Fig). Specifically, normalised time-averaged velocity and turbulent kinetic energy (TKE) predictions for two grid resolutions, namely coarse (9.2M cells) and fine (25M cells), were compared at an inlet Re = 7’000. The comparisons of 1D velocity magnitude profiles at three stations along the larynx (A-A’), glottis (B-B’) and upper trachea (C-C’) are shown in Fig 2. We find very good agreement between the two grid resolutions, ensuring the adequacy of the selected resolution for LES. 7 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Results and discussion Glottal jet characteristics Firstly, maximum values of TKE differ in the downstream wake of the jet, at the shear interface with the resting fluid; this observation was previously reported for example by Lin et al. [20] in a patient-specific geometry and concurrently by Das et al. using the same identicl idealized mouth-throat geometry [3]. For all three modalities, we observe a common feature with the presence of a thin streak of maximum TKE values originating at the glottic that gradually widens and dissipiates down- stream into the trachea. In both experiments and RANS, the shape appears to be in strong agreement whereas for the LES the peak TKE streak is more underlined. However, the RANS simulation reports more than *60% lower peak TKE values relative to both TPIV and LES, while a background base level of TKE upstream of the glottis is resolved in the TPIV measure- ment (i.e. 1–2 m/s) yet absent in RANS (i.e. baseline of near zero TKE) and much lower in LES (i.e. <1 m/s). It is known that differences in TKE hold potential ramifications towards predic- tions of inhaled aerosols dispersed in the lungs [8]. Notably, the significantly lower TKE levels obtained with RANS are acknowledged to lead to overpredictions of particle deposition when only the (time-averaged) RANS velocity field is used [23, 47]. Indeed, in regions where there is significant large-scale anisotropy in turbulence, turbulent dispersion plays an important role in particle transport and tends to decrease deposition. Hence, RANS are typically used together with a turbulent dispersion model to provide improved deposition predictions. In PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 8 / 16 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Fig 3. Comparison of flow characteristics between tomographic PIV (TPIV), Reynolds Averaged Navier Stokes (RANS) and Large Eddy Simulation (LES) for the high flowrate case (Re = 7’000). Top row: Velocity magnitude contours along the mid-sagittal plane shown along with several orthogonal transverse planes to illustrate 3D characteristics of the flow. Bottom row: Turbulent kinetic energy (TKE) contours are plotted on the mid-sagittal plane. Results are generally in good agreement with strong similarity but derivative TKE values reveal more subtle differences. Specifically, RANS underestimates the peak TKE values near the jet’s wake while the TPIV data introduce background noise. https://doi.org/10.1371/journal.pcbi.1010537.g003 Fig 3. Secondary flows In a next step, we compare ensuing secondary flows downstream of the glottal constriction. Fig 5 plots velocity magnitude contours and velocity vectors for each of the three modalities and flow rates, resulting in a 3 by 3 matrix. Similarly, as seen along the mid-sagittal plane (Fig 4), the basic flow features are largely invariant with respect to Re variation, whereas flow mag- nitudes vary as anticipated with higher Re number. Here, we observe a pair of counter-rotating vortices classically refered to as Dean vortices, originating from the curvature of the laryngeal geometry. The Dean number is defined as Dn = Re ffiffiffiffiffiffiffiffiffiffiffi D=2r p , where r denotes the curvature radius and D the cross-sectional diameter of the airway. In our analysis, the Dean number changes only with Re because of the statically defined geometry (i.e., r and D are constants). Therefore our measurements of increasingly stronger Dean vortex flow correlate with increased Re, as plotted in Fig 5. We note here that the cross-section in 2D appears rectangular, in contrast to the circular cross-sections in the numerical simulations (RANS and LES); a consequence of the volume illumination technique which involves knife-edges to form the laser light into a prism (see Fig 1, and subsequent post- processing masking steps that are performed in 2D. Results and discussion Glottal jet characteristics With such differences accounted for, we nevertheless consitently 10 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model observe the common laryngeal jet structure described above and present across all flowrates, underlining how the jet phenomenon scales consistently. In the low flowrate case (Re = 1’500) plotted in Fig 4a–c, we observe the weakest jet, with peak velocity values of *2 m/s and a *1 m/s upstream and downstream flow, with near zero counter flow at the shear interface. For the intermediate flow rate (Re = 4’500) plotted in Fig 4d–f, we observe the same jet structure, with higher peak velocity values (*5 m/s) and the same near zero counter flow to the right of the shear interface. Lastly, we plot the high flow rate case (Re = 7’000) in Fig 4g–i, with the highest peak velocity values at >9 m/s. We note that the near zero counter flow to the right of the shear interface is nearly identical between the mid and high Re cases, and slightly lower in the low Re case. While the three modalities agree well for the low Re case, we observe in the mid and high Re flows that the RANS simulations does not capture the zero-flow interface as well, which also explains the lower TKE values discussed earlier in Fig 3e, as a common source of energy for turbulent velocity fluctuations lies in the presence of shear in the mean flow. Results and discussion Glottal jet characteristics Comparison of flow characteristics between tomographic PIV (TPIV), Reynolds Averaged Navier Stokes (RANS) and Large Eddy Simulation (LES) for the high flowrate case (Re = 7’000). Top row: Velocity magnitude contours along the mid-sagittal plane shown along with several orthogonal transverse planes to illustrate 3D characteristics of the flow. Bottom row: Turbulent kinetic energy (TKE) contours are plotted on the mid-sagittal plane. Results are generally in good agreement with strong similarity but derivative TKE values Fig 3. Comparison of flow characteristics between tomographic PIV (TPIV), Reynolds Averaged Navier Stokes (RANS) and Large Eddy Simulation (LES) for the high flowrate case (Re = 7’000). Top row: Velocity magnitude contours along the mid-sagittal plane shown along with several orthogonal transverse planes to illustrate 3D characteristics of the flow. Bottom row: Turbulent kinetic energy (TKE) contours are plotted on the mid-sagittal plane. Results are generally in good agreement with strong similarity but derivative TKE values reveal more subtle differences. Specifically, RANS underestimates the peak TKE values near the jet’s wake while the TPIV data introduce background noise https://doi.org/10.1371/journal.pcbi.1010537.g003 contrast, since LES resolve the large scale eddies, these do not need an additional dispersion model when addressing particle-laden flows [48]. Next, we compare measurements for the low (Re = 1’500) and intermediate (Re = 4’500) flow rate cases, plotted as velocity magnitude contours overlaid with velocity vectors in Fig 4. 9 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Fig 4. Flow characterization of the laryngeal jet variation on the mid-sagittal plane in the region of interest (ROI). Results are presented as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively. https://doi.org/10.1371/journal.pcbi.1010537.g004 Fig 4. Flow characterization of the laryngeal jet variation on the mid-sagittal plane in the region of interest (ROI). Results are presented as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively. https://doi.org/10.1371/journal.pcbi.1010537.g004 https://doi.org/10.1371/journal.pcbi.1010537.g004 We briefly note that a small size deviation in the width of the experimental model relative to the numerical ones shown (i.e. a deviation on the order of <5%), likely introduced by the volume calibration step and the physical compliance of the silicone phantom model itself (and also seen in Fig 3d–f). One-dimensional velocity curves In a final step, we compare detailed flow characteristics between the experimental and numeri- cal approaches via the simultaneous plotting of 1D velocity magnitude curves. To this end, we first identify the mid-sagittal plane that bisects the geometry (see dashed-dot line in Fig 6a) and the region imaged via TPIV experiments (see Figs 1 and S1). Positioned in this orienta- tion, the mouth inlet is viewed in the normal direction, with a smaller isometric view given as a reference. Four transverse lines (labeled A through D) spanning the mid-sagittal plane are chosen for plotting 1D velocity curves, as shown in Fig 6b. Good agreement is observed between all three modalities (i.e, TPIV, RANS and LES) for each of the four lines, with variations slightly more pronounced in Line A due to the shorter y-axis range relative to Lines B-D. We observe that TPIV and LES velocity magnitude curves along line A are very similar (i.e. LES exceeds the TPIV values by a maximum of 1–2%), char- acterized by asymmetric twin peaks, with the higher peak on the left side of the dimensionless x-axis. The RANS curve, by contrast, features a more symmetric pair of peaks and deviates from the TPIV measurements by <4%. For lines B-D, excellent agreement (<1% deviation) is observed between all modalities. 11 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model C l i Fig 5. Flow characterization of the laryngeal jet with Reynolds variation on a transverse plane bisecting the jet’s wake. Results are presented as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively and exemplifies reconstructed flow streamlines in the 2D cut plane (see Line C in Fig 6a for the location of the cut plane). https://doi.org/10.1371/journal.pcbi.1010537.g005 Fig 5. Flow characterization of the laryngeal jet with Reynolds variation on a transverse plane bisecting the jet’s wake. Results are presented as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively and exemplifies reconstructed flow streamlines in the 2D cut plane (see Line C in Fig 6a for the location of the cut plane). https://doi.org/10.1371/journal.pcbi.1010537.g005 https://doi.org/10.1371/journal.pcbi.1010537.g005 Conclusion The present work has been motivated by the ongoing need for experimentally-resolved 3D flow data to improve the valididty and accuracy of computational fluid dynamics (CFD) simu- lations resolving turbulent airflows in the upper and extra-thoracic airways towards various Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 12 / 16 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model |u| (m/s) |u| (m/s) |u| (m/s) |u| (m/s) 2.5 2.3 2.9 2.7 3.1 3.3 0 2 4 6 8 10 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 non-dimensional axial position 0 1 0 1 0 1 0 1 TPIV RANS LES Line A Line B Line C Line D (a) (b) Line A Line B Line C Line D Midsagittal plane Isometric view 10 mm 5 mm Fig 6. 1-dimensional velocity magnitude curves for Re = 7’000 case. (a): Schematic of the full mouth-throat model geometry (see isometric view) used in the fabrication of the experimental phantom and computational mesh. The area illuminated via laser (Region of Interest) and imaged using tomographic particle image velocimetry (TPIV) is noted, along with four lines (labeled A-D) spanning the mid-sagittal plane. (b) Comparison of 1D velocity profiles along the anotated Lines A-D. https //doi org/10 1371/jo rnal pcbi 1010537 g006 |u| (m/s) |u| (m/s) |u| (m/s) |u| (m/s) 2.5 2.3 2.9 2.7 3.1 3.3 0 2 4 6 8 10 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 non-dimensional axial position 0 1 0 1 0 1 0 1 TPIV RANS LES Line A Line B Line C Line D (b) (a) ( Line A Line B Line C Line D Midsagittal plane Isometric view 10 mm 5 mm Fig 6. 1-dimensional velocity magnitude curves for Re = 7’000 case. (a): Schematic of the full mouth-throat model geometry (see isometric view) used in the fabrication of the experimental phantom and computational mesh. The area illuminated via laser (Region of Interest) and imaged using tomographic particle image velocimetry (TPIV) is noted, along with four lines (labeled A-D) spanning the mid-sagittal plane. (b) Comparison of 1D velocity profiles along the anotated Lines A-D. https://doi.org/10.1371/journal.pcbi.1010537.g006 https://doi.org/10.1371/journal.pcbi.1010537.g006 https://doi.org/10.1371/journal.pcbi.1010537.g006 inhalation therapy and therapeutic or toxic dosimetry applications. Conclusion To the best of our knowl- edge, the findings presented herein are the first detailed 3D in vitro–in silico correlations of respiratory airflows in a benchmark anatomical mouth-throat model. We find that a RANS k- ω SST model adequately predicts velocity flow patterns for Re numbers spanning 1’500 to 7’000, supporting results in close proximity to a more computationally-costly LES model. Yet, RANS significantly underestimates turbulent kinetic energy (TKE), thus demonstrating the superiority of LES as a higher-order turbulence modeling scheme. With a keen eye on end- user applications across various respiratory disciplines, researchers can leverage such valida- tion data in conjunction with open-access files (see SM) for improved predictive CFD models. Supporting information S1 Fig. Tomographic particle image velocimetry (TPIV) experimental setup. A: Photo- graphed in the lab B: a schematic illustration. (EPS) Author Contributions Data curation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Formal analysis: Saurabh Bhardwaj, Pantelis Koullapis. Investigation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman. Methodology: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Ron Bessler. Project administration: Eliram Nof, Josue´ Sznitman. Resources: Stavros Kassinos. Supervision: Stavros Kassinos, Josue´ Sznitman. Validation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Validation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Visualization: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Writing – original draft: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman. Writing – review & editing: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Stavros Kassi- nos, Josue´ Sznitman. Writing – review & editing: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Stavros Kassi- nos, Josue´ Sznitman. S3 Fig. Large eddy simulation (LES) mesh refinement and model scheme comparison. (EPS) 13 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537 March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model S1 Data. Mouth-throat geometry STL file. (STL) S2 Data. Modified mouth-throat geometry file used in 3D printing the mold for fabricat- ing the silicon phantom. (STL) S1 Data. Mouth-throat geometry STL file. (STL) S2 Data. Modified mouth-throat geometry file used in 3D printing the mold for fabricat- ing the silicon phantom. (STL) Author Contributions Conceptualization: Eliram Nof, Josue´ Sznitman. Data curation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Formal analysis: Saurabh Bhardwaj, Pantelis Koullapis. Funding acquisition: Josue´ Sznitman. Investigation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman. Methodology: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Ron Bessler. Project administration: Eliram Nof, Josue´ Sznitman. Resources: Stavros Kassinos. Supervision: Stavros Kassinos, Josue´ Sznitman. Validation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Visualization: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Writing – original draft: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman Writing – review & editing: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Stavros Kass nos, Josue´ Sznitman. References 2008; 41:2681–2688. https://doi.org/10.1016/j.jbiomech.2008.06.018 PMID: 18667205 13. Krause F, Wenk A, Lacor C, Kreyling WG, Mo¨ller W, Verbanck S. Numerical and experimental study on the deposition of nanoparticles in an extrathoracic oral airway model. J Aerosol Sci. 2013; 57:131–143. https://doi.org/10.1016/j.jaerosci.2012.11.004 14. Zhu Z, C CZ, Zhang L. Experimental and numerical investigation on inspiration and expiration flows in a three-generation human lung airway model at two flow rates. Respiratory Physiology & Neurobiology. 2019; 262:40–48. https://doi.org/10.1016/j.resp.2019.01.012 15. Qi S, Zhang B, Yue Y, Shen J, Teng Y, Qian W, et al. Airflow in tracheobronchial tree of subjects with tracheal bronchus simulated using CT image based models and CFD method. 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Koullapis P, Kassinos SC, Muela J, Perez-Segarra C, Rigola J, Lehmkuhl O, et al. Regional aerosol deposition in the human airways: The SimInhale benchmark case and a critical assessment of in silico methods. European Journal of Pharmaceutical Sciences. 2018; 113:77–94. https://doi.org/10.1016/j. ejps.2017.09.003 PMID: 28890203 24. References 1. Shinneeb AM, Pollard A. Identification of vortical structures inside the human pharynx/larynx region from POD-reconstructed velocity fields. Experiments in Fluids. 2012; 53:353–367. https://doi.org/10. 1007/s00348-012-1293-5 2. Choi J, Tawhai MH, Hoffman EA, Lin CL. On intra- and intersubject variabilities of airflow in the human lungs. Physics of Fluids. 2009; 21(10):101901. https://doi.org/10.1063/1.3247170 PMID: 19901999 3. Das P, Nof E, Amirav I, Kassinos SC, Sznitman J. Targeting inhaled aerosol delivery to upper airways in children: Insight from computational fluid dynamics (CFD). 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https://openalex.org/W2626932481
https://www.nature.com/articles/oncsis201749.pdf
English
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TIP30 regulates lipid metabolism in hepatocellular carcinoma by regulating SREBP1 through the Akt/mTOR signaling pathway
Oncogenesis
2,017
cc-by
7,395
1PLA General Hospital Cancer Center Key Lab, PLA Postgraduate School of Medicine, Beijing, China; 2Molecular Pathology Laboratory, College of Basic Medicine, Inner Mongolia Medical University, Hohhot, China; 3Department of Orthopedics, PLA General Hospital, Beijing, China; 4International Joint Cancer Institute, The Second Military Medical University, Shanghai, China; 5Changhai Hospital, The Second Military Medical University, Shanghai, China and 6Shanghai University Of Medicine & Health Sciences, Shanghai, China. Correspondence: Dr W Jing, Changhai Hospital, The Second Military Medical University, Changhai Road, Shanghai 200433, China. E-mail: jingwei7777@163.com or Professor J Zhao, International Joint Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shanghai 200433, China. E-mail: zhaojian@smmu.edu.cn 7These authors contributed equally to this work. Received 24 October 2016; revised 18 March 2017; accepted 2 May 2017 TIP30 regulates lipid metabolism in hepatocellular carcinoma by regulating SREBP1 through the Akt/mTOR signaling F Yin1,7, G Sharen1,2,7, F Yuan3,7, Y Peng1, R Chen4, X Zhou5, H Wei1, B Li4, W Jing5 and J Zhao1,4,6 Lipid reprogramming has been considered as a crucial characteristic in hepatocellular carcinoma (HCC) initiation and progression. However, detailed molecular mechanisms have yet to be clearly defined. Here, we examined the effects of tumor suppressor TIP30 on the regulation of HCC lipid metabolism. We found that decreased TIP30 expression leads to elevated fatty acid synthesis and enhanced levels of lipogenic enzymes SCD and FASN in HCC cells. Moreover, SREBP1 is one of the key transcription factors regulating liver lipid metabolism, and TIP30 deficiency significantly increased SREBP1 expression and nuclear accumulation. Small interfering RNAs targeting SREBP1 could reverse fatty acid synthesis induced by TIP30 deficiency. Furthermore, downregulating TIP30 activated the Akt/mTOR signaling pathway to upregulate SREBP1 expression, which promoted lipid metabolism by activating gene transcription of lipogenesis, including fasn and scd. We also showed that TIP30 deficiency-regulated lipid metabolism promoted proliferation of HCC cells. Clinically, our data revealed that TIP30 expression significantly correlated with SREBP1 in patients with HCC and that a combination of TIP30 and SREBP1 is a powerful predictor of HCC prognosis. Together, our data suggested a novel function of TIP30 in HCC progression and indicate that TIP30 regulation of SREBP1 may represent a novel target for HCC treatment. Oncogenesis (2017) 6, e347; doi:10.1038/oncsis.2017.49; published online 12 June 2017 OPEN OPEN OPEN Citation: Oncogenesis (2017) 6, e347; doi:10.1038/oncsis.2017.49 www.nature.com/oncsis 1PLA General Hospital Cancer Center Key Lab, PLA Postgraduate School of Medicine, Beijing, China; 2Molecular Pathology Laboratory, College of Basic Medicine, Inner Mongolia Medical University, Hohhot, China; 3Department of Orthopedics, PLA General Hospital, Beijing, China; 4International Joint Cancer Institute, The Second Military Medical University, Shanghai, China; 5Changhai Hospital, The Second Military Medical University, Shanghai, China and 6Shanghai University Of Medicine & Health Sciences, Shanghai, China. Correspondence: Dr W Jing, Changhai Hospital, The Second Military Medical University, Changhai Road, Shanghai 200433, China. E mail: jingwei7777@163 com INTRODUCTION metastatic human variant cells and less metastatic classic cells.6 Subsequently, TIP30 was found to be downregulated in various tumors and is considered to be a tumor suppressor due to its pro-apoptotic activity and its anti-metastatic and anti-angiogenic capacities.7–11 Our previous research reported that TIP30-regulated tumor metastasis and chemoresistance in various cancers.12–18 Moreover, we also found downregulated TIP30 induces epithelial– mesenchymal transition in HCC and pancreatic cancer.19,20 The morbidity and mortality of hepatocellular carcinoma (HCC) ranks top5 and top3 respectively among common malignant tumors worldwide. The poor outcomes of HCC patients are mainly due to high recurrence of HCC after surgery and resistance to chemotherapy.1 Meanwhile, sorafenib was used as first line targeted drugs in treating advanced HCC, which can only prolongs the survival period of less than 3 months.2 Consequently, to elaborate the pathogenesis and progression of HCC and to develop new therapeutic strategies seem extremely crucial. y Considering the crucial role of lipid metabolic reprogramming in cancer development, identifying new molecules and pathways that are involved in this process is vital. Recently, TIP30 has been preliminarily revealed to affect fatty acid storage and oxidation in hepatocytes21 and we will extensively investigate the role of TIP30 in lipid metabolism deregulation of HCC. We indicate decreased TIP30 promotes lipid metabolism via Akt/mTOR/SREBP1 signaling and that the combination of TIP30 and SREBP1 is an effective predictor for HCC prognosis. Major risk factors of HCC are viral hepatitis, exposure to hepatotoxins and alcohol abuse, whereas recent clinic and epidemiology researches indicate nonalcoholic fatty liver disease (NAFLD) increases HCC incidence.3 Recently, metabolic reprogramming, especially lipid metabolism alteration, is considered to be the initiating factor of tumor occurrence and progression. Continuous de novo cholesterogenesis and lipogenesis are frequently activated in tumors for providing extra lipids and lipid precursors during rapid cell proliferation.4 Several key enzymes have been identified to promote de novo lipid synthesis, including fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD) and acetyl-CoA carboxylase (ACC).5 However, detailed mechanisms of abnormal lipid metabolism have not yet been comprehensively identified during HCC progression. nt Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shanghai 200433, China. International Joint Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shangh mmu.edu.cn E mail: jingwei7777@163.com or Professor J Zhao, International Joint Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shanghai 200433, China. E-mail: zhaojian@smmu edu cn RESULTS TIP30 is a negative regulator of lipid metabolism in HCC cells To determine whether TIP30 regulates lipid metabolism of HCC cells, microarray analysis was firstly applied for comparing gene expression profiles of HCC-LM3 infected with shNon or shTip30. 837 genes were differentially expressed upon TIP30 knockdown (fold change ⩾2, Po0.05). Through GO and KEGG enrichment y TIP30, namely HTATIP2 or CC3, was firstly discovered in small cell lung carcinoma, using differential analyses between highly TIP30 regulates lipid metabolism in HCC F Yin et al TIP30 regulates lipid metabolism in HCC 2 analysis, these genes were enriched in several biological pathways. The results indicated that genes that significantly correlated to TIP30 expression were involved in fatty acid metabolism (Supplementary Figure 1A), which suggested the important role of TIP30 involved in lipid metabolism regulation. Furthermore, effects of TIP30 on cellular lipid levels were explored in HCC-LM3, SMMC-7721 and HepG2 cell lines using the lipophilic dye BODIPY 493/503. We employed lentiviruses to knockdown or overexpress TIP30 expression in HCC cell lines, and the lentiviral infection efficiency is showed in Figure 1d. We observed that silencing TIP30 led to increased levels of neutral lipid staining in HCC-LM3 and SMMC-7721 cells, whereas the staining was significantly decreased in TIP30-overexpressed cells (Figure 1a). Similar results were obtained when the lipid accumulation inducer oleate was added to the culture medium (Supplementary Figure 1B). Consistently, intracellular triglyceride levels were further determined to support the negative effects of TIP30 on lipid metabolism regulation (Figure 1b). Downregulating TIP30 enhances SREBP1 expression by activating the Akt/mTOR signaling pathway Loss of TIP30 can activate EGFR/AKT signaling of human lung adenocarcinoma and mammary cancer.25,26 We have previously confirmed that TIP30 deficiency can activate AKT signaling in HCC and laryngeal carcinoma.16,20 Previous studies have indicated activation of AKT/mTOR signaling had critical effects on lipid metabolism regulation.27,28 Here, we find downregulation of TIP30 expression activates AKT and causes elevated mTOR phosphoryla- tion in HCC-LM3 cells, whereas up-regulating TIP30 expression reduced AKT and mTOR phosphorylation levels (Figure 3a). Additionally, we found that blockade of AKT by its inhibitor, MK-2206, dramatically attenuated the TIP30 deficiency-induced up-regulation of p-mTOR and SREBP1 levels (Figure 3b). Moreover, MK-2206 could reverse the upregulated FASN and SCD expression in TIP30-deficient HCC-LM3 cells (Figure 3c). Intracellular triglycer- ide levels and the intensity of BODIPY staining were also reduced after MK-2206 treatment (Figures 3d and e). SREBP1 is essential for TIP30 deficiency-mediated lipogenesis- promoting effects The previous analysis showed that SREBPs (sterol regulatory element-binding proteins) were critical transcription factors that control lipogenesis and lipid uptake.22 SREBPs firstly located in endoplasmic reticulum membrane, which was considered as its inactive precursors. Once the sterol levels drop, SREBPs transferred from endoplasmic reticulum to Golgi apparatus, where mature forms were released by proteases (site-1 and site-2). Thereafter, mature SREBPs entered nucleus to bind SRE-containing gene promoters to induce transcription.23 Emerging evidence indicates that SREBP1 is a crucial linkage of oncogenic signaling transduc- tion and cancer metabolism.24 To elucidate the molecular mechanisms of the TIP30 deficiency-mediated upregulation of lipogenic enzymes, cellular SREBP1 levels were examined. qRT–PCR and western blot analyses revealed that SREBP1 mRNA levels were significantly increased after TIP30 knockdown (Figures 1c and 2a). Additionally, immunostaining showed nuclear accumulation of SREBP1 with TIP30 deficiency (Figure 2b; Supplementary Figure 2B). Meanwhile, overexpression of TIP30 leaded to adverse effects on SREBP1 levels in HCC cells (Figures 1c and 2a,b). SREBP1 expression was reduced in TIP30 knockdown cells which TIP30 are re-expressed in (Supplementary Figure 2A). We then used small interfering RNAs (siRNAs) targeting SREBP1 to explore whether TIP30 deficiency upregulated lipogenic enzyme expression through SREBP1. SREBP1 depletion of TIP30-deficient HCC-LM3 and SMMC-7721 cells leaded to decreased expression of FASN and SCD (Figure 2c). Moreover, intracellular triglyceride levels and the intensity of BODIPY staining were remarkably reduced by siSREBP1 in TIP30-deficient HCC cells (Figure 2d; Supplementary Figure 2C). These results demonstrate that SREBP1 is involved in TIP30 deficiency related lipogenesis-promoting effects. Decreased TIP30 is associated with elevated SREBP1 levels in HCC samples, and combinational biomarkers provide powerful prognostic value for HCC patients To better understand the correlation between TIP30 and SREBP1 expression, immunohistochemical staining of 80 clinical HCC samples was performed. SREBP1 protein levels in HCC tissues negatively correlated with TIP30 expression (r = −0.473, Po0.001) (Figures 5a and b), suggesting that SREBP1 may be upregulated by TIP30 deficiency in HCC. We then measured TIP30 and SREBP1 mRNA levels in 30 HCC tissue samples investigated to provide further support for our research. Results showed the negative association(r = −0.37, P = 0.039) between TIP30 and SREBP1 mRNA levels (Figure 5c). Using NCBI GEO databases to analyze the HCC sample array (GEO dataset accession GSE36376),29 we also found the negative correlation of TIP30 and SREBP1 in 240 HCC samples (r = −0.24, Po0.001), showing the same tendency as our results (Supplementary Figure 3). RESULTS These data show that AKT/mTOR/SREBP1 signaling is required for TIP30 to regulate lipid metabolism in HCC cells. p g g To well explain the mechanisms of TIP30 regulating de novo lipid synthesis, we analyzed lipogenesis-related enzymes (FASN, SCD, and ACC) levels of HCC cells with different TIP30 expressions. Results demonstrated both the mRNA and protein levels of SCD and FASN notablely elevated in TIP30-depleted cells, whereas overexpression of TIP30 reduced SCD and FASN levels comparing to control (Figures 1c and d). However, ACC expression remained unchanged with TIP30 knockdown or overexpression (Figure 1c). As fatty acid oxidation (FAO) played an important role in lipid metabolism reprogramming in several types of cancer, we also analyzed the levels of critical FAO-related factors, including CPT1A and ACOX1. However, TIP30 exerted no effects on these two oxidative enzymes in HCC cells (Figure 1c). Taken together, our research indicates TIP30 can regulate de novo fatty acid synthesis of HCC cells. Decreased TIP30 promotes the proliferation of HCC cells via SREBP1-related lipid metabolism in vitro and in vivo Given important effects of lipid metabolism on tumor progression, we examined whether TIP30-regulated HCC cell growth via SREBP1- mediated lipid metabolism. As shown in Figure 4a, depletion of SREBP1 signaling with siRNA inhibited TIP30 deficiency-induced enhanced HCC-LM3 and SMMC-7721 cell growth. Using colony- forming assays, we found that decreasing the TIP30 deficiency- induced HCC cell colony formation was also dependent on SREBP1 (Figure 4b). To better understand the role of lipid metabolism reprogramming in TIP30-regulated HCC growth in vivo, xenograft studies were performed. We established stable HCC-LM3 cell lines with both TIP30 and SREBP1 deficiency and the corresponding cells were subcutaneously injected into 4 weeks old BALB/c nude mice. Results showed decreased SREBP1 significantly abolished the accelerated tumor growth of TIP30-deficient HCC-LM3 cells (Figures 4c and d). Interestingly, the levels of triglycerides were increased in the tumor tissues with reduced TIP30 expression, whereas silencing SREBP1 reversed this effect (Figure 4e), further supporting the conclusion that lipid metabolism deregulation contributed to HCC cell growth. Meanwhile, immunohistochemical staining showed tumors originated from TIP30-silenced HCC-LM3 cells exhibited increased SREBP1, SCD and FASN expression (Figure 4f), which was coincident with results obtained in HCC cell lines in vitro. Taken together, our results suggest that TIP30 can modulate SREBP1-related lipid metabolism, which contributes to tumor growth in HCC. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis- promoting effects SREBP1 is essential for TIP30 deficiency-mediated lipogenesis- promoting effects Meanwhile, patients with low TIP30 Oncogenesis (2017), 1 – 9 Figure 1. TIP30 negatively regulates lipid metabolism of hepatocellular cancer cell lines. (a) HCC cells (HCC-LM3, SMMC-7721 and HepG2) we infected with lentivirus to knockdown or overexpress TIP30. BODIPY 493/503 (green staining) was used to stain neutral lipids of each cell. DA (blue staining) was used to stain nuclear. (magnification, × 200). For quantification of the mean integrated optical density (IOD) of BODIP staining, Image J software was applied and values were analyzed with unpaired t-test. (b) Intracellular triglyceride levels were detected in eac cell, with or without lipid accumulation inducer, oleate (0.05 mM), added in the culture medium. (c) qRT–PCR methods were applied to te mRNA levels of lipogenic enzymes (ACC, FASN, SCD and SREBP1) and fatty acid oxidation enzymes (ACOX1 and CPT1A) in the indicated cel (d) Western blot was applied to detect the SCD and FASN expression in each cell. Protein levels of TIP30 were also detected to determine th efficiency of lentivirus infection. *Po0.05. TIP30 regulates lipid metabolism in HCC F Yin et al TIP30 regulates lipid metabolism in HCC F Yin et al Figure 1. TIP30 negatively regulates lipid metabolism of hepatocellular cancer cell lines. (a) HCC cells (HCC-LM3, SMMC-7721 and HepG2) were infected with lentivirus to knockdown or overexpress TIP30. BODIPY 493/503 (green staining) was used to stain neutral lipids of each cell. DAPI (blue staining) was used to stain nuclear. (magnification, × 200). For quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed with unpaired t-test. (b) Intracellular triglyceride levels were detected in each cell, with or without lipid accumulation inducer, oleate (0.05 mM), added in the culture medium. (c) qRT–PCR methods were applied to test mRNA levels of lipogenic enzymes (ACC, FASN, SCD and SREBP1) and fatty acid oxidation enzymes (ACOX1 and CPT1A) in the indicated cells. (d) Western blot was applied to detect the SCD and FASN expression in each cell. Protein levels of TIP30 were also detected to determine the efficiency of lentivirus infection. *Po0.05. Oncogenesis (2017), 1 – 9 TIP30 regulates lipid metabolism in HCC F Yin et al l d h h l l h b d h fi d d d igure 2. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-promoting effects. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis- promoting effects (a) Western blot was performed for detecting cytosolic precursor (P) and nucleic mature (M) forms of SREBP1 in indicated HCC cells lysates. (b) Results of immunofluorescence analysis were howed in indicated HCC cells. Red staining represented SREBP1 protein. DAPI (blue staining) was used to stain nuclear. (magnification, × 200 For quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed with unpaired t-test. (c) Indicated protein levels were detected in HCC-LM3-shTip30 and SMMC-7721-shTip30 cells transfected with si-SREBP si-SREBPl-1 or si-SREBPl-2) or si-NC using western blot. (d) Intracellular triglyceride levels were detected in cells as decreased in c. *Po0.05 F Yin et al 4 Figure 2. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-promoting effects. (a) Western blot was performed for detecting cytosolic precursor (P) and nucleic mature (M) forms of SREBP1 in indicated HCC cells lysates. (b) Results of immunofluorescence analysis were showed in indicated HCC cells. Red staining represented SREBP1 protein. DAPI (blue staining) was used to stain nuclear. (magnification, × 200) For quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed with unpaired t-test. (c) Indicated protein levels were detected in HCC-LM3-shTip30 and SMMC-7721-shTip30 cells transfected with si-SREBPl (si-SREBPl-1 or si-SREBPl-2) or si-NC using western blot. (d) Intracellular triglyceride levels were detected in cells as decreased in c. *Po0.05. TIP30 was first discovered as a metastasis suppressor in 1997 and as a tumor suppressor in 2003.6,7 The tumor suppressor function of TIP30 has been extensively demonstrated in various types of human tumors, including HCC. TIP30 exerts its tumor-suppressive role by influencing cell apoptosis, growth, metastasis and angiogenesis.32 Recently, TIP30 has also been confirmed to regulate the metabolic adaptation to glucose limitation of HeLa cells, which contributes to tumor metastasis and aggressiveness.33 For the first time, our study demonstrated that TIP30 is a negative regulator of lipid metabolism in HCC. We also demonstrated that decreased TIP30 may facilitate lipid metabolism through the AKT/mTOR/SREBP1 signaling pathway to promote tumor growth in HCC. levels and high SREBP1 levels exhibited the poorest recurrence- free survival (RFS) as well as overall survival (OS), indicating that combinational detection of the two molecules may have a powerful prognostic value (Figures 5d and e). Oncogenesis (2017), 1 – 9 DISCUSSION chemoresistance and self-renewal of laryngeal carcinoma.16 Additionally, downregulation of TIP30 could activate AKT to regulate the levels of epithelial–mesenchymal transition related transcription factors in HCC-LM3 cells.20 mTOR activation by Akt contributes to regulation of de novo lipogenesis. Through up-regulating SREBP1 transcription, processing and nucleic accumulation, mTOR signaling senses nutrients for growth and accelerates de novo lipogenesis.42 In particular, Calvisi has reported that AKT-mTORC1 signaling- induced lipogenesis accelerated HCC development from transcrip- tional and post-transcriptional aspects, including downregulation of FASN ubiquitination and interruption of SREBPs degradation.40 Consistently, in our study, SREBP1 was revealed to be upregulated by TIP30 deficiency-mediated Akt/mTOR activation. Meanwhile, CD147 has been reported to form a complex with integrinβ to activate PI3K/Akt pathway and then reprogram lipid metabolism through Akt/mTOR/SREBP1 signaling in HCC.43,44 As a cancer- associated biomarker for detection and an effective target for treatment, CD147 also forms complexes with CD44 and EGFR to induces EGFR downstream signaling (ERK, pSTAT3) in breast cancer and pancreatic cancer.45,46 Also, loss of TIP30 can improve EGFR activity in various tumors and Tip30 knockout in primary hepatocytes of mouse leads to trapping of EGF-EGFR complex, which contributes to prolonged EGFR signaling.25,26,41,47 Consider- ing both TIP30 and CD147 could regulate EGFR related signaling, it would be interesting to figure out whether downregulated TIP30 expression may synergistically act with increased CD147 expression in HCC in future research. cells during their rapid growth and proliferation.34 Thus, several key lipogenic enzymes are activated to increase de novo lipogenesis of cancer cells. SREBP1-regulated downstream lipogenic enzymes (FASN and SCD), have been confirmed to be elevated in various tumors.35,36 Consistently, our results demon- strated that TIP30 deficiency could promote the lipid synthesis of HCC cells via the up-regulation of FASN and SCD. We also found that the mRNA levels of two oxidative enzymes (CPT1A and ACOX1) were not affected by TIP30 in HCC cells. However, a recent report preliminarily suggested that TIP30-regulated fatty acid oxidations in normal hepatocytes by evaluating the CO2 production of cells labeled with [14C] palmitate.21 Considering the different cell lines and research methods applied in these data, the role of TIP30 in fatty acid oxidations of HCC needs further evaluation from transcriptional and post-transcriptional regulation. As the main regulator of hepatic lipogenesis, SREBP1 is highly activated in cancers and activates the fatty acid pathway in human HCC cell lines. DISCUSSION Increasing evidences showed lipid metabolism was a key player in tumor growth, metastasis and resistance to therapies. As the main metabolic organ, liver is crucial for carrying out lipid metabolism, and aberrant activation of lipogenesis has been considered as an oncogenic event in human HCC.30,31 In the present research, we evaluated whether TIP30 participates in abnormal lipid metabolism of HCC. Increasing evidences showed lipid metabolism was a key player in tumor growth, metastasis and resistance to therapies. As the main metabolic organ, liver is crucial for carrying out lipid metabolism, and aberrant activation of lipogenesis has been considered as an oncogenic event in human HCC.30,31 In the present research, we evaluated whether TIP30 participates in abnormal lipid metabolism of HCC. Normal tissues often utilize circulating lipids, while more than 90% of fatty acids are produced from de novo synthesis in tumors TIP30 regulates lipid metabolism in HCC F Yin et al gure 3. Downregulating TIP30 enhanced SREBP1 expression through activating Akt/mTOR signaling. (a) Western blotting showed p-AKT and mTOR levels of HCC-LM3 cells infected with shNon, shTip30, LV-Non and LV-Tip30, respectively. (b) HCC-LM3-shNon and HCC-LM3-shTip30 ere treated with AKT inhibitor MK-2206 (0.5 μM) for 24 h, indicated protein expressions were analyzed using western blot. (c) MK-2206 .5 μM) was added in HCC-LM3-shTip30 cells. After 24 h of the treatment, mRNA levels of TIP30 and lipogenic enzymes were tested using RT–PCR. BODIPY 493/503 staining (magnification, × 200) (d) and intracellular triglyceride levels (e) were performed in cells as decreased in c. or quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed th unpaired t-test. *Po0.05. 5 Figure 3. Downregulating TIP30 enhanced SREBP1 expression through activating Akt/mTOR signaling. (a) Western blotting showed p-AKT and p-mTOR levels of HCC-LM3 cells infected with shNon, shTip30, LV-Non and LV-Tip30, respectively. (b) HCC-LM3-shNon and HCC-LM3-shTip30 were treated with AKT inhibitor MK-2206 (0.5 μM) for 24 h, indicated protein expressions were analyzed using western blot. (c) MK-2206 (0.5 μM) was added in HCC-LM3-shTip30 cells. After 24 h of the treatment, mRNA levels of TIP30 and lipogenic enzymes were tested using qRT–PCR. BODIPY 493/503 staining (magnification, × 200) (d) and intracellular triglyceride levels (e) were performed in cells as decreased in c. For quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed with unpaired t-test. *Po0.05. DISCUSSION Genetic or pharmacological inhibition of SREBP1 resulted in cell growth arrest and decreased cell proliferation.24 Recent study reported that inhibition of de novo lipid biosynthesis by suppressing the SREBP pathway prevented HCC progression.37 Our present data also confirmed that decreased TIP30 could promote HCC cell growth via SREBP1-related lipid metabolism in vitro and in vivo, which was also responsible for elevated FASN and SCD expression induced by TIP30 deficiency. Several experimental models have revealed critical effects of Akt on lipogenesis regulation. It has been recently found that liver tumors induced by AKT/c-Met displayed increased lipogenesis and genetic deletion of the main lipogenic enzyme, FASN, suppressed the in vivo hepatocarcinogenesis driven by AKT and c-Met oncogenes.38,39 Another research reported that excessive activation of AKT in mice liver accelerated fatty acid synthesis as well as tumor development.40 In human lung adenocarcinoma, breast tumors and glioma, p-AKT and p-ERK1/2 were upregulated by TIP30 deficiency.25,26,41 We previously revealed that loss of TIP30 activated AKT/GSK-3β/β-catenin signaling, which was vital to growth, In addition, we confirmed that TIP30 expression was negatively associated with SREBP1 expression in clinical HCC samples. TIP30 is an important prognostic predictor for various cancers.19,48,49 Upregulated SREBP1 associated with a poor prognosis of HCC patients.50 Remarkably, when the combined effects of TIP30 and SREBP1 were evaluated, the sensitivity for survival analysis of HCC patients was improved. In summary, we linked TIP30 to lipid metabolism through SREBP1 in HCC, which revealed alternative mechanisms under- lying TIP30-induced growth regulation in hepatocellular Oncogenesis (2017), 1 – 9 TIP30 regulates lipid metabolism in HCC F Yin et al Decreased TIP30 promotes HCC cells growth via modulating SREBP1-related lipid metabolism. (a) Cell proliferation was -shNon and HCC-LM3-shTip30 transfected with si-SREBP1 (si-SREBPl-1 or si-SREBPl-2) or si-NC using MTS. Similar metho in SMMC-7721 cells. (b) Clone formation was generated in cells as decreased in a. (c) Pictures showed the tumors dissec e, which were transplanted with HCC-LM3 cells infected with shNon, shTip30, or shTip30 and shSrebp1. (d) Average weight o uated in each group. (e) Levels of triglycerides were individually measured in tumor tissues of each group using a tissue tri (f) Tumors derived from nude mice were immunostained for TIP30, SREBP1, SCD and FASN (magnification, × 200). *Po0 F Yin et al 6 Figure 4. Decreased TIP30 promotes HCC cells growth via modulating SREBP1-related lipid metabolism. DISCUSSION After the concentration of RNA was tested, reverse transcription PCR was done as the PrimeScript RT reagent Kit (Takara Bio, Tokyo, Japan) guided. SYBR Green-based real-time PCR was operated on a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and Gapdh was detected for endogenous control. Primer sequences are listed in Supplementary Table 1. BODIPY staining HCC cells were fixed for 15 min using 4% paraformaldehyde. Thereafter, cells were washed then dyed by BODIPY 493/503 (10 μg/ml) (Thermo Fisher Scientific, Waltham, MA, USA) at 37 ℃for 15 min.Then, the nucleus was counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) for 10 min. We captured images using a fluorescence microscope (Olympus, Lake Success, NY, USA). DISCUSSION (a) Cell proliferation was tested in HCC-LM3-shNon and HCC-LM3-shTip30 transfected with si-SREBP1 (si-SREBPl-1 or si-SREBPl-2) or si-NC using MTS. Similar methods were repeated in SMMC-7721 cells. (b) Clone formation was generated in cells as decreased in a. (c) Pictures showed the tumors dissected from nude mice, which were transplanted with HCC-LM3 cells infected with shNon, shTip30, or shTip30 and shSrebp1. (d) Average weight of tumors was evaluated in each group. (e) Levels of triglycerides were individually measured in tumor tissues of each group using a tissue triglyceride assay kit. (f) Tumors derived from nude mice were immunostained for TIP30, SREBP1, SCD and FASN (magnification, × 200). *Po0.05. Oncogenesis (2017), 1 – 9 TIP30 regulates lipid metabolism in HCC F Yin et al Figure 5. TIP30 expression negatively associates with SREBP1 level in tumor tissues of HCC patients and the combinational biomarkers provide powerful prognostic value for HCC. (a) TIP30 and SREBP1 were detected by immunostaining in 80 HCC samples. Representative pictures of immunostaining were shown for two patients. (magnification, × 200). (b) The correlation between TIP30 and SREBP1 expression was analyzed in HCC tissues according to the scores of immunohistochemistry staining. (c) Scatter plot showed correlations between TIP30 and SREBP1 mRNA level in tumors of 30 HCC patients investigated. Recurrence-free survival rates and overall survival rates were analyzed between TIP30-high expression group and TIP30-low expression group (d), as well as between four subgroups (TIP30low/SREBP1low; TIP30low/SREBP1high; TIP30high/SREBP1low; TIP30high/SREBP1high) (e). F Yin et al 7 Figure 5. TIP30 expression negatively associates with SREBP1 level in tumor tissues of HCC patients and the combinational biomarkers provide powerful prognostic value for HCC. (a) TIP30 and SREBP1 were detected by immunostaining in 80 HCC samples. Representative pictures of immunostaining were shown for two patients. (magnification, × 200). (b) The correlation between TIP30 and SREBP1 expression was analyzed in HCC tissues according to the scores of immunohistochemistry staining. (c) Scatter plot showed correlations between TIP30 and SREBP1 mRNA level in tumors of 30 HCC patients investigated. Recurrence-free survival rates and overall survival rates were analyzed between TIP30-high expression group and TIP30-low expression group (d), as well as between four subgroups (TIP30low/SREBP1low; TIP30low/SREBP1high; TIP30high/SREBP1low; TIP30high/SREBP1high) (e). carcinoma. Meanwhile, we also expanded the novel function of TIP30 in HCC metabolism regulation, which should be extensively studied in other types of tumors. RNA extraction and real-time quantitative PCR NucleoSpin RNA kit (Macherey-Nagel, Germany) was used for total RNA extraction. MATERIALS AND METHODS Cell lines, antibodies and reagents Cell lines, antibodies and reagents HCC-LM3 was supported by the Liver Cancer Institute of Zhong Shan Hospital (Shanghai, China). HepG2 was available from American Type Culture Collection. SMMC-7721 was acquired from Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China). Cell lines have been tested for mycoplasma contamination and were cultured at 37 °C in a humidified condition with 5% CO2. The Dulbecco’s modified Eagle’s medium (DMEM) was used for cell culture, supplemented with 10% fetal bovine serum. Details of primary antibodies and their sources are as follows: TIP30 (produced by our lab as previously reported);17 p-mTOR (Ser2448), p-AKT (Ser473), mTOR, AKT, FASN and GAPDH (Cell Signaling Technology, Danvers, USA); SCD and SREBP1 (Abcam, Cambridge, USA). Additionally, we purchased horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse) from Santa Cruz Biotechnology (SCBT, CA, USA). MK-2206 was from Selleck Chemicals (Houston, TX, USA). Oleate was acquired from Sigma (St Louis, MO, USA). TG measurements The cellular TG content was tested using a Triglyceride Quantification Kit (Abcam, Cambridge, UK) with detailed experimental procedure provided in the kit. Oncogenesis (2017), 1 – 9 Immunofluorescence staining Lentivirus and small interfering RNA HCC cells were infected with lentivirus expressing Tip30 cDNA or shRNA targeting Tip30 as literature described.17 A siRNA targeting SREBP1 were designed by GenePharma (Shanghai, China). Briefly, the following sequences were used: for SREBP1-homo-523 (si-SREBP1-1), sense 5′-GCUCCUCUCUUGAAGCCUUTT-3′ and antisense 5′-AAGGCUUCAAG- AGAGGAGCTT-3′; for SREBP1-homo-1403 (si-SREBP1-2), sense 5′-GCA- ACACAGCAACCAGAAATT-3′ and antisense 5′-UUUCUG GUUGCUGUG- UUGCTT-3′. HCC cells were incubated on 24-well plates for 24 h and then were fixed using paraformaldehyde with the final concentration 4%. Triton X-100 was prepared in PBS for the final concentration 0.2% and then was used for permeabilization.10% BSA/PBS was used as blocking buffer. For immunofluorescence staining, primary antibodies were added for incubation for 24 h on a shaker setting at 4 °C. After washing three times, a goat anti-rabbit Alexa Fluor 555 antibody (Thermo Fisher Scientific) was mixed and for 1 h incubation. Thereafter, cells were counterstained using DAPI (Invitrogen). All matched samples were photographed (control and test) using confocal laser scanning microscopy (FLUOVIEW FV-1000). Protein extraction and western blotting Proliferation detection and colony-formation assays Statistical analysis in hepatocellular carcinoma. Oncogene 2015; 34: 1420–1431. 21 Liao BM, Raddatz K, Zhong L, Parker BL, Raftery MJ, Schmitz-Peiffer C. Proteomic analysis of livers from fat-fed mice deficient in either PKCdelta or PKCepsilon identifies Htatip2 as a regulator of lipid metabolism. Proteomics 2014; 14: 2578–2587. We repeated in vitro experiments in triplicate. SPSS software (version 16.0, Chicago, IL, USA) was used for statistical analysis. Pearson chi-square test and Student’s t-test were applied for analysis of dichotomous variables and continuous variables, respectively. Correlations of two variables were determined using Spearman rank test. Survival analyses of investigated patients were achieved using Kaplan–Meier analysis with log-rank test. When more than two data sets were analyzed, variance analysis was performed. The results are showed as the mean± s.e.m. The above statistical analyses were all two-sided with Po0.05 deemed statistically significant. 22 Horton JD. Sterol regulatory element-binding proteins: transcriptional activators of lipid synthesis. Biochem Soc Trans 2002; 30: 1091–1095. 23 Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002; 109: 1125–1131. 24 Guo D, Bell EH, Mischel P, Chakravarti A. Targeting SREBP-1-driven lipid metabolism to treat cancer. Curr Pharm Design 2014; 20: 2619–2626. 25 Li A, Zhang C, Gao S, Chen F, Yang C, Luo R et al. TIP30 loss enhances cytoplasmic and nuclear EGFR signaling and promotes lung adenocarcinogenesis in mice. Oncogene 2013; 32: 2273–2281. Tumor xenograft mouse model 11 NicAmhlaoibh R, Shtivelman E. Metastasis suppressor CC3 inhibits angiogenic properties of tumor cells in vitro. Oncogene 2001; 20: 270–275. Animal studies are authorized by medical ethics committee of PLA General Hospital. Male Balb/c nude mice (4 weeks old) are randomly allocated into three groups (6 mice/group) and the number of mice is determined according to prior experience of in vivo studies in our laboratory. We subcutaneously inject 5 × 106 indicated cells into each mouse. Investigators were not blinded for the animal studies. During the experiment, mice were monitored and euthanized for histopathology examination after cell inoculation for 28 days. Then, the tumor weight and their triglycerides levels were measured. 12 Zhao J, Lu B, Xu H, Tong X, Wu G, Zhang X et al. Thirty-kilodalton Tat-interacting protein suppresses tumor metastasis by inhibition of osteopontin transcription in human hepatocellular carcinoma. Hepatology 2008; 48: 265–275. 13 Zhao J, Ni H, Ma Y, Dong L, Dai J, Zhao F et al. TIP30/CC3 expression in breast carcinoma: relation to metastasis, clinicopathologic parameters, and P53 expression. Hum Pathol 2007; 38: 293–298. 14 Zhao J, Zhang X, Shi M, Xu H, Jin J, Ni H et al. TIP30 inhibits growth of HCC cell lines and inhibits HCC xenografts in mice in combination with 5-FU. Hepatology 2006; 44: 205–215. 15 Zhao J, Chen J, Lu B, Dong L, Wang H, Bi C et al. TIP30 induces apoptosis under oxidative stress through stabilization of p53 messenger RNA in human hepato- cellular carcinoma. Cancer Res 2008; 68: 4133–4141. Patients, immunohistochemistry and scoring Samples of 80 patients who had radical resection of HCC were collected from 2003 to 2007 at Guangxi Cancer Hospital (Nanning, China). Radical surgery was defined as previously reported.20 Informed consent authorized by Ethics Committee of Guangxi Cancer Hospital was acquired from patients when specimen collection was performed. Supplementary Table 2 showed clinicopathological features of the above patients. All the patients were monitored for recurrence every 1–6 months after the curative resection depending on the post-operative time. Immunohistochemistry of clinical samples were performed as previously reported.17,20 Two experienced pathologists independently evaluated the staining scores. According to the staining intensity and distribution, immunostaining scores were semiquanti- tatively estimated.17 Immunohistochemical scores of ⩽4 and scores of ⩾5 were classified as low and high expression, respectively. 16 Zhu M, Yin F, Yang L, Chen S, Chen R, Zhou X et al. The authors declare no conflict of interest. The authors declare no conflict of interest. 26 Zhang C, Mori M, Gao S, Li A, Hoshino I, Aupperlee MD et al. Tip30 deletion in MMTV-Neu mice leads to enhanced EGFR signaling and development of estrogen receptor-positive and progesterone receptor-negative mammary tumors. Cancer Res 2010; 70: 10224–10233. Microarray analysis HCC-LM3 cell line was infected with shTip30 or control lentivirus. After 7 days, TRIzol (Thermo Fisher Scientific) was used to extract cellular RNA, which was then purified by an RNeasy kit (Qiagen, Hilden, Germany). NimbleGen Gene Expression Microarray was applied in microarray analysis. Axon GenePix 4000B microarray scanner was used for scanning and raw data were extracted by NimbleScan software 2.5. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes (fold change ⩾2, Po0.05) were performed for predicting biologic effects of TIP30. Such analysis was achieved by the ClueGo plugin of Cytoscape (Software version 3.2.3, INSERM UMRS1138, Paris, France), which is a functional annotation way to evaluate over-representation of functional categories in interested genetic sets.51 Enrichment analysis was performed via functional annotation chart and annotation clustering options, which was limited to GO terms and KEGG pathways in ‘Biologic Process’ categories. Functional annotation was deemed significant with P-valueo0.05, using Fisher’s exact test. multicenter prospective study. Hepatology 2016; 63: 827–838. 4 Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, Lisanti MP. 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ACKNOWLEDGEMENTS This work is supported in part by grants from Ministry of Science and Technology of China “973” and “863” programs (2014AA021103, 2014AA020704), Beijing Nova Program (Z171100001117112), National Nature Science Foundation of China (81472281, 81502129, 81402424, 81472719, 81670573), Shanghai Key Laboratory of Cell Engineering (14DZ2272300), Shanghai Leading Academic Discipline Project (B905). 27 Porstmann T, Santos CR, Griffiths B, Cully M, Wu M, Leevers S et al. SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab 2008; 8: 224–236. 28 Bhat M, Sonenberg N, Gores GJ. The mTOR pathway in hepatic malignancies. Hepatology 2013; 58: 810–818. Oncogenesis (2017), 1 – 9 Oncogenesis (2017), 1 – 9 TIP30 regulates lipid metabolism in HCC F Yin et al 9 motif of integrin beta1 to modulate malignant properties of hepatoma cells. J Biol Chem 2012; 287: 4759–4772. 29 Lim HY, Sohn I, Deng S, Lee J, Jung SH, Mao M et al. 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CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness. J Biol Chem 2013; 288: 26089–26104. 32 Yu X, Li Z, Wu WK. TIP30: a novel tumor-suppressor gene. Oncol Res 2014; 22: 339–348. 33 Chen V, Shtivelman E. CC3/TIP30 regulates metabolic adaptation of tumor cells to glucose limitation. Cell Cycle 2010; 9: 4941–4953. 47 Zhang C, Li A, Zhang X, Xiao H. A novel TIP30 protein complex regulates EGF receptor signaling and endocytic degradation. J Biol Chem 2011; 286: 9373–9381. ACKNOWLEDGEMENTS 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/ 41 Hu Y, Chen F, Liu F, Liu X, Huang N, Cai X et al. Overexpression of TIP30 inhibits the growth and invasion of glioma cells. Mol Med Rep 2016; 13: 605–612. 42 Bakan I, Laplante M. Connecting mTORC1 signaling to SREBP-1 activation. Curr Opin Lipidol 2012; 23: 226–234. 43 Li Y, Wu J, Song F, Tang J, Wang SJ, Yu XL et al. Extracellular membrane-proximal domain of HAb18G/CD147 binds to metal ion-dependent adhesion site (MIDAS) © The Author(s) 2017 © The Author(s) 2017 Supplementary Information accompanies this paper on the Oncogenesis website (http://www.nature.c tary Information accompanies this paper on the Oncogenesis website (http://www.nature.com/oncsis) Oncogenesis (2017), 1 – 9 Oncogenesis (2017), 1 – 9
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Use of English Phrasal Verbs of Chinese Students Across Proficiency Levels: A Corpus-Based Analysis
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English phrasal verbs, Chinese learner corpus, proficiency level, single-word synonyms English phrasal verbs, Chinese learner corpus, proficiency level, single-word synonyms Use of English Phrasal Verbs of Chinese Students Across Proficiency Levels: A Corpus-Based Analysis Yuanyuan Wei Renmin University of China, China Abstract A number of studies have investigated phrasal verbs from various aspects (Liu, 2006; Yu, 2011; Liao & Fukuya, 2004). However, those studies lack generality, because only a couple of phrasal verbs were chosen as examples, or too much content prompt was offered in task response of L2 learners. The current study applies a corpus-based approach to investigate English phrasal verbs in free conversations from a Chinese learner corpus, SWECCL (Wen & Wang, 2008), which demonstrates Chinese students’ actual use of phrasal verbs. This study focuses on analysis of two phenomena: development of phrasal verb uses and avoidance of phrasal verbs across different proficiency levels in Chinese students’ oral English conversations. Results showed that students at higher proficiency level used more phrasal verbs in oral communication, and the progress of phrasal verb use was evident from middle proficiency level to advanced proficiency level. The study also found that phrasal verbs occurred less frequently overall in L2 learners’ oral conversations compared with frequency of their single-word synonyms. International Journal of TESOL Studies (2021) Vol. 3 (4) 25-41 https://doi.org/10.46451/ijts.2021.12.03 International Journal of TESOL Studies (2021) Vol. 3 (4) 25-41 https://doi.org/10.46451/ijts.2021.12.03 Address: School of Foreign Languages, University of Renmin University, Beijing, China Email: wei_yuanyuan23@163.com 2.1 Semantic and syntactical studies on phrasal verbs Relevant micro-linguistic studies demonstrated several special features of phrasal verbs (Zhao, 1984; Wang, 1993; Wang, 1997). Complicated multiple meanings and complex syntactical structures were two main concerns of those semantic and syntactic studies. On the one hand, phrasal verbs usually contain multiple meanings, of which literal meaning is at the core of semantic field, and metaphorical meanings are connected with it (Bolinger, 1971; Liu, 2006). On the other hand, phrasal verbs are syntactically complex because they can be transitive or intransitive. In a transitive construction, an object can be placed between a verb and a particle to construct a VOP or after a particle to be a VPO (Fan, 1995). Also, sometimes a prepositional phrase (PP) follows a particle to provide more information, such as fall out of the window (Han, 2018). In terms of their collocational association with other words, it is not fixed by regular rules, for example, map out a plan is proper but map out people is an inappropriate use. Most researchers are aware of syntactical and semantic difficulties in acquisition of verb-particle constructions, and phrasal verbs are pervasively used in English especially in many informal conversations (McArthur, Wang & Zhu, 1992). In order to tackle issues of acquiring phrasal verbs, researchers investigate phrasal verbs from a pedagogical perspective and emphasize conceptual meanings of them, and some progress was made. White (2012) encouraged denotation and metaphorical meaning of particles in phrasal verbs by asking them to draw pictures of them and proved this method was useful for acquisition. Ding and Yang (2016) introduced a new pedagogy of phrasal verbs’ acquisition—summing up figurative meanings of particles and generating verb patterns. For example, “at” means “attempt to”, so when it is used with a verb like “shoot”, the construction refers to “attempt to shoot somebody”. 1 Introduction Chinese students are prone to use high-frequency verbs repetitively, which leads to the lack of variety in language use. Native speakers also frequently use those verbs, but they often combine verbs with adverbs or prepositions to express various meanings. The combination which native speakers frequently use in their conversations is called phrasal verb (e.g., put on, take off). A phrasal verb is made up of a verb and a prepositional or adverbial particle (Waibel, 2007). Their meanings are not constituted through simply combining words’ meanings, and they can be replaced by single verbs. Appropriate use of phrasal verbs becomes a distinguishing feature of a proficient L2 learner. However, Chinese L2 learners are faced with challenges of acquirement of phrasal verbs, because verb-particle combinations are flexible rather than regulated by specific rules. Another difficulty is the polysemy of those phrasal verbs, a prevailing phenomenon in verb-particle combinations (Han, 2018). Due to those two major obstacles, Chinese students usually learn phrasal verbs through rote learning, International Journal of TESOL Studies 3 (4) 26 (4) which is extremely ineffective (Ding & Yang, 2016). While a considerable amount of literature has been published on semantic and syntactical analysis of phrasal verbs, and there has been little quantitative analysis which investigates Chinese college students’ phrasal verb use. Since empirical research on phrasal verbs is still not fully developed, corpora can be useful tools which contribute to demonstrating L2 learners’ performance on phrasal verb use. 2.2 Avoidance of phrasal verbs Semantic studies and error analysis on propositions usually focused on case study through the interpretation of cognitive theory. Thorough syntactical analysis of phrasal complex structure also heavily relied on giving examples (Zhao, 1984; Wang, 1993; Wang, 1997; Han, 2018). Another branch delves L2 learners’ preference for single-word verbs over phrasal verbs. Avoidance of verb-particle construction was confirmed by many studies. Dagut and Laufer (1985) conducted translation and multiple-choice tests. They concluded that Hebrew-speaking students used more single-word verbs over phrasal verbs and used more frequently their literal meanings over figurative meanings. Laufer and Eliasson (1993) studied advanced Swedish- speaking learners through similar assessments and found that cross-linguistic difference, specifically, the lack of phrasal verb structures in L1, mainly accounted for the underuse of this structure. Liu (2006) focused on assessing correct particle use of Chinese students through multiple choice questions and filling blanks and generalized three reasons why errors occurred when Chinese learners chose appropriate particles for phrasal verbs: (a) only considering literal meanings of phrasal verbs; (b) negative transfer of L1; (c) ignoring the context when students infer meanings of phrasal verbs. Controversially, Guo (2013) used multiple choice tests to count the frequency of phrasal verbs and single words and found that Chinese college students have preference for phrasal verbs over single-word verbs and underused phrasal verbs only under the condition of unfamiliarity with multiple meanings of them rather than interference of L1. With proficiency as a variable, studies with similar tests concluded that Chinese learners at middle level avoided 27 Yuanyuan Wei using phrasal verbs but used single-word verbs instead and students at middle level and advanced level avoid using phrasal verbs in figurative meanings (Liao & Fukuya, 2004; Zhang, 2007). However, those studies offered options for participants to choose. Therefore, the items used in tasks can be content prompts for participants, and those multiple-choice tasks do not mimic students’ language output in real life. Another potential problem of those studies was that researchers determined literal meanings and figurative meanings of the use of phrasal verbs. However, categories of phrasal verbs in figurative meanings and literal meanings are not completely distinguished like those researchers established in their studies, but a continuum with varying degrees of semantic transparency (Bolinger, 1971; Liu, 2006). In order to address those issues, corpora can be a helpful tool. 2.2 Avoidance of phrasal verbs Sung (2020) searched the frequencies of 150 most frequently used phrasal verbs and their single-word synonyms and concluded that frequency of underused phrasal verbs by L2 learners was statistically significantly lower than their single-word counterparts. Since only written data was taken into consideration, oral data may get different results and thus it is worth studying. The 150 phrasal verb list, S&AW PHaVE List (Liu & Myers, 2020), can be a useful reference which provides most common phrasal verbs and their meanings in both spoken and written registers. 2.3 L2 Leaners’ use of phrasal verbs across proficiency levels With the help of corpus method, various aspects of phrasal verbs can be analyzed effectively, such as collocational constraints of verb-particle combinations, multiplicity of phrasal verbs’ meanings, and frequent use of phrasal verbs among native speakers (Gardner & Davies, 2007). However, L2 learners’ use of phrasal verbs is not fully analyzed in corpus linguistics across proficiency levels. Regarding English proficiency, Chen explained why subjects in her research did not show avoidance of phrasal verbs, which was in conflict with Waibel (2007) who generated more frequent use of phrasal verbs by German students than L2 learners of other L1s. Furthermore, Yu (2011) focused on oral English performance of English majors and illuminated that the ability of using phrasal verbs developed with the improvement of English proficiency but it stopped developing from middle proficiency level to advanced proficiency level. However, since only the number of oral records was taken into consideration, and the text length of every record was also important to work as a basis for relative frequency, this research may lack accuracy in comparing performance of groups at different proficiency levels. Proficiency level, which works as an important variable in related corpus studies, can bring diversified findings to studies on phrasal verbs. Inspired by previous studies, the present study is concerned with actual performance of Chinese L2 learners on phrasal verb use. Corpora provide more scientific data than isolated linguistic examples, researchers’ intuitions or random group of phrasal verbs, because they deal with verb-particle combinations by utilizing frequency of occurrences in language calculated by computers and phrasal verb usage in real context (Gardner & Davies, 2007). Furthermore, unlike case studies which were frequently used to analyze Chinese students’ use (Ma & Shang, 2011; Yu, 2011), such as prepositions with frequent use, “in” “on” “over”, “come”, “do” “get” “give” “go” “make” “put” and “take” working as verbs in the frequency analysis, this paper adopted S&AW PHaVE List. The list was proposed by Liu and Myers (2020) based on COCA. It offered 150 phrasal verbs of most frequent use and their frequently-used meanings. Phrasal verb frequency difference among groups at different proficiency and frequency difference between phrasal verbs and single-word verbs were investigated in SWECCL to reveal Chinese learners’ performance. 3.2 Learner population Though there are a few combinations of verb + adverb or preposition in Chinese, such as “hui lai (come back)”, they differentiate from English phrasal verbs in that the amount is much smaller and most of them do not have any figurative meanings (Zhang, 2007). Because of cross-linguistic differences, Chinese learners find it challenging to appropriately use multiple meanings of English phrasal verbs. 3.1 Research questions The current study is intended to address the following two research questions: International Journal of TESOL Studies 3 (4) 28 (4) 1. How does frequency of phrasal verb use among Chinese L2 learners vary across different pro- ficiency levels? 1. How does frequency of phrasal verb use among Chinese L2 learners vary across different pro- ficiency levels? 1. How does frequency of phrasal verb use among Chinese L2 learners vary across different pro- ficiency levels? 2. How do Chinese students show avoidance of phrasal verbs in L2 oral communication? 2. How do Chinese students show avoidance of phrasal verbs in L2 oral communicatio 3.2 Learner population Learner population in this study was 1148 Chinese sophomores whose majors were English. Their speeches and free conversations were ranked by two professional scorers. And they were classified into four groups based on their ranks with rank 4 being the most proficient (Wang & Wen, 2007). In terms of similarities of phrasal verbs in Chinese and English, firstly, they are constructed by two components and one is complementary to the other. And they all work as predicates. Also, two components can be separable. (1) English: V + particle e.g., come over Chinese: V + particle e.g., guo lai come-PRS-DECL over-ADV (2) English: take a book out Chinese: na chu yi ben shu lai take-PRS-DECL one-CLF book-OBJ out-ADV Whereas, the difference is that a phrasal verb in English usually contains multiple meanings and some idiomatical meanings cannot be inferred easily through literal meaning of components. However, it is not the case in Chinese. For example, “come out” in English refers to “come out a book” or “come out from somewhere”. Phrasal verb use also undergoes negative influence of native language. Whereas, the difference is that a phrasal verb in English usually contains multiple meanings and some idiomatical meanings cannot be inferred easily through literal meaning of components. However, it is not the case in Chinese. For example, “come out” in English refers to “come out a book” or “come out from somewhere”. Phrasal verb use also undergoes negative influence of native language. For example, “eat completely”, “drink thoroughly”, “use completely” are translated directly from Chinese to English. However, “eat up”, “drink up”, “use up” are more frequently used among the native speakers (Wang, 1987). Though there are a few combinations of verb + adverb or preposition in Chinese, such as “hui lai (come back)”, they differentiate from English phrasal verbs in that the amount is much smaller and most of them do not have any figurative meanings (Zhang, 2007). Because of cross-linguistic differences, Chinese learners find it challenging to appropriately use multiple meanings of English phrasal verbs. Whereas, the difference is that a phrasal verb in English usually contains multiple meanings and some idiomatical meanings cannot be inferred easily through literal meaning of components However For example, “eat completely”, “drink thoroughly”, “use completely” are translated directly from Chinese to English. However, “eat up”, “drink up”, “use up” are more frequently used among the native speakers (Wang, 1987). 3.3 The corpora The Spoken and Written English Corpus of Chinese Learners (SWECCL) was employed for the investigation of the Chinese L2 learners’ use of phrasal verbs. SWECCL is a two-million-word corpus compiled by Nanjing University, Foreign Language Teaching and Research Press and Beijing Foreign Study University. SECCL is a part of the corpus which only compiles spoken data. The spoken data was collected from Test for English Majors-Band 4 (TEM-4) during 1996-2002. It contains 1148 transcripts of audio samples which are 1,460,042 words. All subjects were sophomore English majors from colleges around the country. They were asked to do three tasks: listening to and retelling a story, impromptu speech and conversation between two students. And their recordings were scored by two raters. Every task performance was given a rank, rank 1 referred to the worst performance and rank 4 meant the best performance. In this study, only 713 free conversations were chosen because they could reflect actual use of phrasal verbs in communication of daily life. For the convenience of research, this paper calculated words of each rank: 122306 words, 218430words, 27556 words, and 3922 words. 29 Yuanyuan Wei 3.4 The extraction of phrasal verbs and their single-word synonyms In order to address the issue which only focused on certain verbs and particles, this paper adopted S&AW PHaVE List (Liu & Myers, 2020) as a criterion to extract data in the learner corpus. The list generated the most common meanings of the most frequently used phrasal verbs in spoken and academic written English derived from COCA. Meanwhile, Wordnet (Princeton University, 2010) provided words related to phrasal verbs. Then words which matched the top two most common meanings of each phrasal verb in spoken register were determined as single-word synonyms. AntConc (Anthony, 2020) was used to do the searches, with verbs as search terms and particles as context words as shown in Figure 1. The searches for single-word synonyms were also conducted in AntConc, as shown in Figure 2. For parents to keep up to bring up their children is already very hard could be a concordance example of the phrasal verb, bring up. And but I think you know firstly the pay is good, you can to some extent raise your family was a concordance line of raise, one of the synonyms of bring up. Similar concordance examples like bring up their children and raise your family can also be found in COCA (Davies, 2008) as shown in Figure 3 and Figure 4. Those examples demonstrate that Chinese college students can use phrasal verbs and their single-word synonyms correctly in oral communications in English. Figure 1 Figure 1 Figure 1 An Example of Searching Phrasal Verbs Figure 2 An Example of Searching Single-Word Synonym An Example of Searching Phrasal Verbs An Example of Searching Phrasal Verbs An Example of Searching Phrasal Verbs Figure 2 An Example of Searching Single-Word Synonym An Example of Searching Single-Word Synonym An Example of Searching Single-Word Synonym International Journal of TESOL Studies 3 (4) 30 Figure 3 Concordance of the Phrasal Verb Bring up Searched in COCA Figure 3 Concordance of the Phrasal Verb Bring up Searched in COCA Concordance of the Phrasal Verb Bring up Searched in COCA Figure 4 gu e Concordance of Raise, the Synonym of Bring Up Searched in COCA Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each other, collection of data should take both usages into consideration. A maximum of five words between a verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven- word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because verbs have various inflectional forms, these forms were counted under the frequency of the original form, for example, goes, went, going were all grouped under go. Another challenge associated with data extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Concordance of Raise, the Synonym of Bring Up Searched in COCA Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each other, collection of data should take both usages into consideration. A maximum of five words between a verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven- word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because verbs have various inflectional forms, these forms were counted under the frequency of the original form, for example, goes, went, going were all grouped under go. Another challenge associated with data extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Concordance of Raise, the Synonym of Bring Up Searched in COCA Concordance of Raise, the Synonym of Bring Up Searched in COCA Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each other, collection of data should take both usages into consideration. A maximum of five words between a verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven- word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because verbs have various inflectional forms, these forms were counted under the frequency of the original form, for example, goes, went, going were all grouped under go. 4.1 Frequency difference among Chinese L2 learners at different proficiency levels Following extracting procedure, this paper firstly calculated absolute token frequency and relative frequency of 150 phrasal verbs in the task three, free conversation, of SECCL, the spoken corpus, as found in Appendix A. The frequency analysis of Chinese students’ phrasal verb use revealed a difference among rank 1, rank 2, rank3 and rank 4. Table 1 below demonstrated that relative token of rank 1 students was lower than rank 2 students, and rank 2 students had a lower relative token than rank 3 students. And rank 4 students had the highest relative token among students of four ranks. Table 1 Token Frequencies of Phrasal Verb in the Spoken Corpus of SWECCL Proficiency level Size Absolute token Relative token rank 1 122,306 181 147.99 rank 2 218,430 334 152.91 rank 3 27,556 47 170.56 rank 4 3,922 8 203.98 Note. Relative token frequency was calculated through the absolute token frequency divided by token size of each rank in the spoken corpus and then rounding it to the nearest tenth. Token Frequencies of Phrasal Verb in the Spoken Corpus of SWECCL P fi i l l Si Ab l t t k R l ti t k Token Frequencies of Phrasal Verb in the Spoken Corpus of SWECCL Note. Relative token frequency was calculated through the absolute token frequency divided by token size of each rank in the spoken corpus and then rounding it to the nearest tenth. Because word sizes of each rank are different, relative token frequencies of every phrasal verb used by students of four ranks rather than absolute token frequencies were used to compare difference. Non-para­ metric Kruskal-Wallis H test was conducted to compare difference of phrasal verb use frequency among four rank groups. And the Kruskal-Wallis H test found that there was a statistically significant difference on relative token frequencies among the different rank groups, χ2(3) = 51.751, p = .000, η²=0.082, with a mean rank frequency of 317.74 for rank 1 group, 349.77 for rank 2 group, 282.2 for rank 3 group and 252.29 for rank 4 group. The findings were in conflict with previous findings that phrasal verb use of students at rank 2, 3 and 4 was similar. Instead, statistically significant differences were found between rank 4 group and rank 3 group, rank 3 group and rank 2 group. Figure 4 All those exceptions were manually checked by the researcher and only phrasal verb structures were coded in this paper. Extracting single-word synonyms of phrasal verbs from AntConc also encountered great challenges that there were no complete synonyms. Those single word alternatives chosen from Wordnet contain multiple meanings. Therefore, in some cases, single-word verbs cannot replace their phrasal verb counterparts. For example, raise, as the single word alternative of bring up, also means increase, such as the concordance I think helping others can also raise our sense of, er, raise our sense of social life, then we can, er, then we can make ourselves to help others in SWECCL. In order to maintain correctness of results, every concordance was counted into frequency only when it was manually checked. Figure 4 Another challenge associated with data extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each other, collection of data should take both usages into consideration. A maximum of five words between a verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven- word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because verbs have various inflectional forms, these forms were counted under the frequency of the original form, for example, goes, went, going were all grouped under go. Another challenge associated with data extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each other, collection of data should take both usages into consideration. A maximum of five words between a verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven- word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because verbs have various inflectional forms, these forms were counted under the frequency of the original form, for example, goes, went, going were all grouped under go. Another challenge associated with data extraction was the similarity of form between verb-particle combinations and verb-preposition phrases 31 Yuanyuan Wei structures in the learner corpus and the research on phrasal verb analysis had to differentiate phrasal verbs from verb-preposition phrases structures (Kim & Baldwin, 2006). For example, take over works as a phrasal verb in he takes over the accounts, but it becomes a verb - preposition phrase in He takes the lamp over the table. All those exceptions were manually checked by the researcher and only phrasal verb structures were coded in this paper. structures in the learner corpus and the research on phrasal verb analysis had to differentiate phrasal verbs from verb-preposition phrases structures (Kim & Baldwin, 2006). For example, take over works as a phrasal verb in he takes over the accounts, but it becomes a verb - preposition phrase in He takes the lamp over the table. 4.1 Frequency difference among Chinese L2 learners at different proficiency levels It proved that capability of using phrasal verb in oral English conversation of Chinese college students was also improving from middle-level to advanced level. International Journal of TESOL Studies 3 (4) 32 Table 2 The Kruskal-Wallis H test: Ranks for Phrasal Verb Use’ Frequency of Different Proficiency Groups Ranks group N Mean Rank alt 1 150 317.74 2 150 349.77 3 150 282.2 4 150 252.29 Total 600 The Kruskal-Wallis H test: Ranks for Phrasal Verb Use’ Frequency of Different Proficiency Table 3 Pairwise Comparisons of Group for Phrasal Verb Use’ Frequency at Rank 1, 2, 3, 4 Sample 1-Sample 2 Test Statistic Std. Error Std. Test Statistic Sig. Adj. Sig.a 4.00-3.00 29.913 14.424 2.074 0.038 0.229 4.00-1.00 65.45 14.424 4.538 0 0 4.00-2.00 97.477 14.424 6.758 0 0 3.00-1.00 35.537 14.424 2.464 0.014 0.083 3.00-2.00 67.563 14.424 4.684 0 0 1.00-2.00 -32.027 14.424 -2.22 0.026 0.158 Each row tests the null hypothesis that the Sample 1 and Sample 2 distributions are the same. Asymptotic significances (2-sided tests) are displayed. The significance level is .05. a Significance values have been adjusted by the Bonferroni correction for multiple tests. Pairwise Comparisons of Group for Phrasal Verb Use’ Frequency at Rank 1, 2, 3, 4 4.2 Avoidance of phrasal verbs in L2 oral communication across proficiency levels In order to demonstrate phrasal verb avoidance in oral English, this study chose 10 phrasal verbs which were frequently used in free conversations compared with other 140 phrasal verbs. Meanwhile, if a complete synonym counterpart of a phrasal verb can be found in Wordnet was also taken into consideration when this paper chose phrasal verbs as typical examples. In the end, as Table 4 shows, 10 phrasal verbs and 20 single-word synonyms were picked out for analysis. And as Table 5 shows, phrasal verb use is less frequent than their single-word synonyms overall. Frequency of phrasal verb use at four separate rank groups are demonstrated in Appendix B. Ten Relatively Frequent-Used Phrasal Verbs in SWECCL and Their Single-Word Synonyms Phrasal verbs single-word synonyms bring up mention raise get in enter arrive go on happen continue build up develop enlarge get off escape leave Set up establish arrange take up start discuss get down lower begin get on board progress pick up collect learn Ten Relatively Frequent-Used Phrasal Verbs in SWECCL and Their Single-Word Synonym 33 Yuanyuan Wei Table 5 Frequencies of Phrasal Verbs and Single-Word Synonyms Phrasal verbs Absolute Frequency of phrasal verbs Absolute Frequency of single-word synonyms bring up 26 59 get in 19 92 go on 27 200 build up 7 119 get off 5 42 set up 7 19 take up 7 114 get down 6 33 get on 16 2 pick up 6 834 Then the analysis of phrasal verb use of Chinese college students at different proficiency levels was conducted. Because data was not normally distributed (skewness and kurtosis statistics were greater than ±2 when they divided by their respective errors), non-parametric independent samples test on relative frequency was adopted to assess frequency difference between phrasal verbs and single-word synonyms at each rank in the spoken corpus of SWECCL. The Wilcoxon Signed-Ranks test for rank 1 indicated that single-word synonyms (mean rank = 40.881) was rated more favorably than phrasal verbs (mean rank = 3.025), Z = 2.601, p = .009< .05, r = 0.58. The Wilcoxon Signed-Ranks test for rank 2 indicated that single-word synonyms (mean rank = 41.341) was rated more highly than phrasal verbs (mean rank = 3.388), Z = 2.701, p = .007< 0.05, r = 0.60. 4.2 Avoidance of phrasal verbs in L2 oral communication across proficiency levels The Wilcoxon Signed-Ranks test for rank 3 showed that single-word synonyms (mean rank = 36.653) was rated more highly than phrasal verbs (mean rank = 1.451), Z = 2.803, p = .005< .05, r = 0.63. Statistically significant differences were found for rank 1 group, rank 2 group and rank 3 group. The findings above showed phrasal verb avoidance in Chinese students’ oral English conversations at primary level and middle-level. However, at advanced level, no statistically significant difference was shown between the uses of phrasal verbs and single-word synonyms, the Wilcoxon Signed-Ranks test for rank 4 showed that single-word synonyms (mean rank = 28.047) was rated more highly than phrasal verbs (mean rank = 2.55), Z = 1.604, p = .109> .05, r = 0.36. Therefore, students at advanced level do not avoid using phrasal verbs. Table 6 Table 6 Descriptive Statistics of phrasal verb use and single-word synonym use for rank 1, 2, 3, 4 groups Descriptive Statistics for rank 1 N Minimum Maximum Mean Std. Error Std. Deviation Skewness Std. Error Kurtosis Std. Error Phrasal verbs 10 0.82 7.36 3.03 0.67904 2.15 1.05 0.69 0.41 1.33 Single- word 10 0 247.74 40.88 23.26 73.56 3.02 0.69 9.36 1.33 Valid N (listwise) 10 ve Statistics of phrasal verb use and single-word synonym use for rank 1, 2, 3, 4 groups Descriptive Statistics of phrasal verb use and single-word synonym use for rank 1, 2, 3, International Journal of TESOL Studies 3 (4) 34 Descriptive Statistics for rank 2 N Minimum Maximum Mean Std. Error Std. Deviation Skewness Std. Error Kurtosis Std. Error Phrasal verbs 10 0.92 8.24 3.39 0.97 3.07 0.98 0.69 -1.17 1.33 Single- word 10 0.46 219.29 41.34 20.63 65.23 2.72 0.69 7.84 1.33 Valid N (listwise) 10 Descriptive Statistics for rank 3 N Minimum Maximum Mean Std. Error Std. Deviation Skewness Std. Error Kurtosis Std. Error Phrasal verbs 10 0 7.25 1.45 0.80 2.54 1.66 0.69 2.03 1.33 Single- word 10 3.63 177.82 36.65 16.48 52.13 2.66 0.69 7.50 1.33 Valid N (listwise) 10 Descriptive Statistics for rank 4 N Minimum Maximum Mean Std. Error Std. Deviation Skewness Std. Error Kurtosis Std. Error Phrasal verbs 10 0 25.50 2.55 2.55 8.06 3.16 0.69 10.00 1.33 Single- word 10 0 178.48 28.05 18.41 58.20 2.37 0.69 5.59 1.33 Valid N (listwise) 10 5 Discussion The first question concerns phrasal verb use of Chinese L2 learners at different proficiency level in oral English. The corpus analysis identified frequency difference among four groups of students at different proficiency levels. The most proficient L2 learners used phrasal verbs most frequently, as the relative token frequency of phrasal verbs in rank 4 was found to be the highest among four ranks. And another finding, statistically significant differences between rank 2 and rank 3, rank 3 and rank 4, is inconsistent with previous research that Chinese students at middle level and advanced level did not show a striking difference in phrasal verb use (Yu, 2011). Consequently, this paper believes that phrasal verb use still develops when L2 learners at middle level are approaching advanced level. The second research question explores avoidance of phrasal verbs in L2 oral communication through a corpus approach. For phrasal verbs, their single-word synonyms convey similar meanings and can replace them in sentences. However, the frequency difference between them was statistically significant at lower proficiency level. And single words appeared more frequently in the learner corpus. The avoidance phenomenon did not show among advanced-level students. Why did Chinese students use less phrasal verbs than single-word synonyms in L2 daily communication? One reasonable explanation may be that they are not equipped with too much knowledge about phrasal verbs in oral English study 35 Yuanyuan Wei (Dagut & Laufer, 1985), although phrasal verbs are more colloquial and informal in style and they should be used more in oral English (Chen, 2013). Phrasal verbs are notoriously difficult for L2 learners, and because of its flexible and complex structure, teachers tend to refer it as chunks and require students to memorize it mechanically. Additionally, multiple meanings, especially figurative meanings are acquired by rote learning, actual contexts of using phrasal verbs are not provided in pedagogy. However, using phrasal verbs appropriately in spoken and written registers can make L2 learners more native- like, knowing the characteristics of them, such as structural variance and collocation with particles, is prerequisite to perform better in English oral communication (Gilquin, 2015). 6 Conclusion In conclusion, with the development of English proficiency, Chinese students gradually use phrasal verbs more frequently. And the phrasal verb use is still developing from middle level to advanced level. As for the reasons of avoiding using phrasal verbs in oral English communication, replacement of single- word synonyms should be taken into consideration because students at lower proficiency level use fewer phrasal verbs than their single-word synonyms. However, the statistically significant frequency difference does not show among advanced L2 learners. These findings bring light to future research on phrasal verbs, such as demonstrating use of phrasal verbs with corpus and quantitative method of phrasal verb analysis. The findings about relationship between development of English proficiency level and phrasal verbs use encourages L2 learners to acquire phrasal verbs in order to improve their English proficiency. Teachers are also required to adopt effective teaching pedagogy because of difficulties students are encountering during phrasal verb learning. There are some limitations in this paper. Firstly, Wordnet provided lists of words related to phrasal verbs, only two of them were chosen by the author. Also, the corpus used in this paper, Spoken English Corpus of Chinese Learners (SECCL), collected spoken data only from Chinese English majors, it may not represent Chinese L2 learners in general. Data collection is not flawless because phrasal verbs and their single-word counterparts cannot perfectly replace each other in terms of meaning, but every concordance of single-word synonyms had been manually checked before it was taken into account. When the meaning of a single-word synonym was different from the meaning of its phrasal verb, the concordance would be eliminated. Token frequency of 150 phrasal verbs in the spoken corpus of SWECCL Phrasal verbs Rank 1 Rank 2 Rank 3 Rank 4 Back up 0(0) 0(0) 0(0) 0(0) Blow up 0(0) 0(0) 0(0) 0(0) Break down 0(0) 0(0) 0(0) 0(0) Break off 0(0) 0(0) 0(0) 0(0) Break out 0(0) 0(0) 0(0) 0(0) Break up 0(0) 0(0) 0(0) 0(0) Bring about 0(0) 0(0) 0(0) 0(0) Bring back 0(0) 0(0) 1(3.62) 0(0) Bring down 0(0) 0(0) 0(0) 0(0) International Journal of TESOL Studies 3 (4) 36 Bring in 0(0) 0(0) 0(0) 0(0) Bring out 0(0) 0(0) 0(0) 0(0) Bring up 7(5.72) 17(7.78) 2(7.25) 0(0) Build up 5(4.09) 2(0.92) 0(0) 0(0) Call out 0(0) 0(0) 0(0) 0(0) Carry on 0(0) 2(0.92) 0(0) 0(0) Carry out 0(0) 4(1.83) 0(0) 0(0) Catch up 1(0.82) 2(0.92) 0(0) 0(0) Check out 0(0) 0(0) 0(0) 0(0) Clean up 0(0) 0(0) 0(0) 0(0) Close down 0(0) 0(0) 0(0) 0(0) Come about 0(0) 0(0) 0(0) 0(0) Come along 0(0) 0(0) 0(0) 0(0) Come around 0(0) 0(0) 0(0) 0(0) Come back 2(1.63) 16(7.33) 4(14.52) 0(0) Come down 0(0) 0(0) 0(0) 0(0) Come in 1(0.82) 0(0) 0(0) 0(0) Come off 0(0) 1(0.46) 0(0) 0(0) Come on 3(2.45) 15(6.87) 0(0) 1(25.5) Come out 0(0) 1(0.46) 0(0) 1(25.5) Come over 0(0) 0(0) 0(0) 0(0) Come through 0(0) 0(0) 0(0) 0(0) Come up 0(0) 2(0.92) 0(0) 0(0) Cut off 0(0) 0(0) 1(3.63) 0(0) End up 0(0) 0(0) 0(0) 0(0) Figure out 0(0) 2(0.92) 0(0) 0(0) Fill in 0(0) 1(0.46) 0(0) 0(0) Fill out 0(0) 0(0) 0(0) 0(0) Find out 2(1.63) 4(1.83) 0(0) 0(0) Follow up 0(0) 0(0) 0(0) 0(0) Get back 0(0) 5(2.29) 0(0) 0(0) Get down 1(0.82) 4(1.83) 0(0) 1(25.5) Get in 3(2.45) 16(7.33) 0(0) 0(0) Get off 1(0.82) 3(1.37) 1(3.63) 0(0) Get on 4(3.27) 3(1.37) 0(0) 0(0) Get out 2(1.64) 5(2.29) 0(0) 0(0) Get through 1(0.82) 0(0) 0(0) 0(0) Get up 0(0) 2(0.92) 1(3.63) 0(0) Give back 2(1.64) 5(2.29) 0(0) 0(0) Give in 0(0) 0(0) 0(0) 0(0) Give out 1(0.82) 0(0) 0(0) 0(0) Give up 4(3.27) 8(3.66) 2(7.26) 0(0) Go ahead 1(0.82) 1(0.46) 0(0) 0(0) Go along 1(0.82) 2(0.92) 1(3.63) 0(0) Go around 0(0) 2(0.92) 0(0) 0(0) 37 Yuanyuan Wei Go back 0(0) 4(1.83) 0(0) 0(0) Go down 0(0) 0(0) 0(0) 0(0) Go in 0(0) 1(0.46) 0(0) 0(0) Go off 1(0.82) 1(0.46) 1(3.63) 0(0) Go on 9(7.36) 18(8.24) 0(0) 0(0) Go out 32(26.16) 33(15.12) 2(7.26) 0(0) Go over 1(0.82) 0(0) 0(0) 0(0) Go through 0(0) 2(0.92) 0(0) 0(0) Go up 0(0) 2(0.92) 0(0) 0(0) Grow up 25(20.44) 16(7.33) 1(3.63) 0(0) Hand over 0(0) 0(0) 0(0) 0(0) Hang on 0(0) 0(0) 0(0) 0(0) Hang out 0(0) 0(0) 0(0) 0(0) Hang up 0(0) 0(0) 0(0) 0(0) Hold back 0(0) 0(0) 0(0) 0(0) Hold on 0(0) 0(0) 0(0) 0(0) Hold out 0(0) 0(0) 0(0) 0(0) Hold up 0(0) 0(0) 0(0) 0(0) Keep on 0(0) 0(0) 1(3.63) 0(0) Keep up 1(0.82) 1(0.46) 0(0) 0(0) Lay down 2(1.64) 0(0) 0(0) 0(0) Lay out 0(0) 0(0) 0(0) 0(0) Line up 0(0) 0(0) 0(0) 0(0) Look around 2(1.63) 0(0) 0(0) 0(0) Look back 0(0) 0(0) 0(0) 0(0) Look down 11(8.99) 11(5.04) 2(7.26) 0(0) Look out 1(0.82) 3(1.37) 0(0) 0(0) Look up 0(0) 0(0) 0(0) 0(0) Make out 0(0) 1(0.46) 0(0) 0(0) Make up 2(1.63) 5(2.29) 2(7.26) 0(0) Move back 0(0) 0(0) 0(0) 0(0) Move in 0(0) 0(0) 0(0) 0(0) Move on 0(0) 1(0.46) 0(0) 0(0) Move out 0(0) 2(0.92) 0(0) 0(0) Move up 0(0) 0(0) 0(0) 0(0) Open up 0(0) 0(0) 0(0) 0(0) Pass on 0(0) 0(0) 0(0) 0(0) Pay off 0(0) 1(0.46) 0(0) 2(50.6) Pick out 0(0) 1(0.46) 0(0) 0(0) Pick up 2(1.63) 2(0.92) 1(3.63) 1(25.5) Play out 0(0) 0(0) 0(0) 0(0) Point out 0(0) 3(1.37) 0(0) 0(0) Pull back 0(0) 0(0) 0(0) 0(0) Pull out 0(0) 1(0.46) 0(0) 0(0) Pull up 0(0) 0(0) 0(0) 0(0) International Journal of TESOL Studies 3 (4) 38 Put back 0(0) 0(0) 0(0) 0(0) Put down 0(0) 1(0.46) 0(0) 0(0) Put in 3(2.45) 9(4.12) 3(10.89) 0(0) Put off 0(0) 1(0.46) 0(0) 0(0) Put on 27(22.08) 52(23.81) 8(29.03) 0(0) Put out 0(0) 0(0) 0(0) 0(0) Put up 4(3.27) 0(0) 0(0) 0(0) Reach out 0(0) 0(0) 0(0) 0(0) Rule out 0(0) 0(0) 0(0) 0(0) Run out 0(0) 0(0) 0(0) 0(0) Send out 0(0) 1(0.46) 0(0) 0(0) Set about 0(0) 0(0) 0(0) 0(0) Set down 0(0) 0(0) 0(0) 0(0) Set off 0(0) 0(0) 0(0) 0(0) Set out 0(0) 0(0) 0(0) 0(0) Set up 3(2.45) 4(1.83) 0(0) 0(0) Settle down 0(0) 0(0) 0(0) 0(0) Show up 0(0) 1(0.46) 0(0) 0(0) Shut down 0(0) 0(0) 0(0) 0(0) Shut up 0(0) 1(0.46) 0(0) 0(0) Sit back 0(0) 0(0) 0(0) 0(0) Sit down 1(0.82) 0(0) 0(0) 0(0) Sit up 0(0) 0(0) 0(0) 0(0) Slow down 0(0) 0(0) 0(0) 0(0) Sort out 0(0) 0(0) 0(0) 0(0) Stand out 0(0) 0(0) 0(0) 0(0) Stand up 1(0.82) 2(0.92) 0(0) 0(0) Start out 0(0) 0(0) 0(0) 0(0) Step back 0(0) 0(0) 0(0) 0(0) Sum up 1(0.82) 0(0) 0(0) 0(0) Take back 0(0) 2(0.92) 0(0) 0(0) Take down 0(0) 0(0) 0(0) 0(0) Take in 2(1.64) 3(1.37) 1(3.63) 0(0) Take off 2(1.64) 6(2.75) 2(7.26) 1(25.5) Take on 0(0) 0(0) 0(0) 0(0) Take out 0(0) 0(0) 1(3.63) 0(0) Take over 1(0.82) 0(0) 0(0) 0(0) Take up 2(1.64) 5(2.29) 0(0) 0(0) Throw out 0(0) 0(0) 0(0) 0(0) Turn around 0(0) 0(0) 0(0) 0(0) Turn back 0(0) 0(0) 0(0) 1(25.5) Turn down 0(0) 0(0) 0(0) 0(0) Turn off 0(0) 0(0) 0(0) 0(0) Turn out 0(0) 0(0) 0(0) 0(0) Turn over 0(0) 0(0) 0(0) 0(0) 39 Yuanyuan Wei Turn up 0(0) 1(0.46) 0(0) 0(0) Wake up 0(0) 0(0) 0(0) 0(0) Walk out 0(0) 0(0) 0(0) 0(0) Wind up 0(0) 0(0) 0(0) 0(0) Work out 1(0.82) 2(0.92) 1(3.63) 0(0) Write down 0(0) 0(0) 0(0) 0(0) Note. Appendix B Frequencies of phrasal verbs and single-word synonyms Rank1 Rank2 Rank3 Rank4 phrasal verbs synonyms phrasal verbs synonyms phrasal verbs synonyms phrasal verbs synonyms bring up 7(5.72) 15(12.26) 17(7.78) 40(18.31) 2(7.25) 4(14.52) 0(0) 0(0) get in 3(2.45) 25(20.44) 16(7.33) 60(27.47) 0(0) 7(25.40) 0(0) 0(0) go on 9(7.36) 41(33.52) 18(8.24) 136(62.26) 0(0) 16(58.06) 0(0) 7(178.48) build up 5(4.09) 41(33.52) 2(0.92) 74(33.88) 0(0) 4(14.52) 0(0) 0 get off 1(0.82) 14(11.45) 3(1.37) 24(10.99) 1(3.63) 5(18.14) 0(0) 0 set up 3(2.45) 6(4.91) 4(1.83) 10(4.58) 0(0) 3(10.89) 0(0) 0 take up 2(1.64) 34(27.80) 5(2.29) 70(32.05) 0(0) 10(36.29) 0(0) 0 get down 1(0. 82) 21(17.17) 4(1.83) 9(4.12) 0(0) 2(7.26) 0(0) 1(25.50) get on 4(3.27) 0(0) 3(1.37) 1(0.46) 0(0) 1(3.63) 0(0) 0 pick up 2(1.63) 303(247.74) 2(0.92) 479(219.29) 1(3.63) 49(177.82) 1(25.5) 3(76.49) Note. Relative token frequency was calculated through the absolute token frequency divided by word size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Frequencies of phrasal verbs and single-word synonyms Note. Relative token frequency was calculated through the absolute token frequency divided by word size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Note. Relative token frequency was calculated through the absolute token frequency divided by word size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Token frequency of 150 phrasal verbs in the spoken corpus of SWECCL Relative token frequency was calculated through the absolute token frequency divided by word size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Note. Relative token frequency was calculated through the absolute token frequency divided by word size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both absolute and relative token frequencies are provided in this table with the latter presented in parentheses. References Anthony, L. (2020). AntConc (Version 3.5.9) [Computer Software]. Tokyo, Japan: Waseda University. https://www.laurenceanthony.net/software Anthony, L. (2020). AntConc (Version 3.5.9) [Computer Software]. Tokyo, Japan: Waseda University. https://www.laurenceanthony.net/software Bolinger, D. M. (1971). 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© 2012 Gloor-Juzi 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. Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Open Access Abstract Background: Dizziness and comorbid anxiety may cause severe disability of patients with vestibulopathy, but can be addressed effectively with rehabilitation. For an individually adapted treatment, a structured assessment is needed. The Vertigo Symptom Scale (VSS) with two subscales assessing vertigo symptoms (VSS-VER) and associated symptoms (VSS-AA) might be used for this purpose. As there was no validated VSS available in German, the aim of the study was the translation and cross-cultural adaptation in German (VSS-G) and the investigation of its reliability, internal and external validity. Methods: The VSS was translated into German according to recognized guidelines. Psychometric properties were tested on 52 healthy controls and 202 participants with vestibulopathy. Internal validity and reliability were investigated with factor analysis, Cronbach’s α and ICC estimations. Discriminant validity was analysed with the Mann–Whitney-U-Test between patients and controls and the ROC-Curve. Convergent validity was estimated with the correlation with the Hospital Anxiety Subscale (HADS-A), Dizziness Handicap Inventory (DHI) and frequency of dizziness. Results: Internal validity: factor analysis confirmed the structure of two subscales. Reliability: VSS-G: α = 0.904 and ICC (CI) =0.926 (0.826, 0.965). Discriminant validity: VSS-VER differentiate patients and controls ROC (CI) =0.99 (0.98, 1.00). Convergent validity: VSS-G correlates with DHI (r = 0.554) and frequency (T = 0.317). HADS-A correlates with VSS-AA (r = 0.452) but not with VSS-VER (r = 0.186). Conclusions: The VSS-G showed satisfactory psychometric properties to assess the severity of vertigo or vertigo- related symptoms. The VSS-VER can differentiate between healthy subjects and patients with vestibular disorders. The VSS-AA showed some screening properties with high sensitivity for patients with abnormal anxiety. The severity of symptoms and the perceived limitations in activities of daily life can be addressed effectively by in- dividually tailored vestibular rehabilitation based on ves- tibular habituation to movement [7-10], optionally combined with cognitive behavioural therapy [11-13]. A requirement for an individually tailored treatment is an assessment, which has to be carried out as thoroughly as possible. In order to focus on the symptoms associated with dizziness, the Vertigo Symptom Scale (VSS) was con- sidered to be a key instrument. The VSS consists of two subscales: 1) the Vertigo scale (VSS-VER) which assesses symptoms mainly associated with disorders of the vestibu- lar system and 2) the Anxiety and Autonomic symptom scale (VSS-AA) for the assessment of a group of generic symptoms which may be associated with autonomic Translation and validation of the vertigo symptom scale into German: A cultural adaption to a wider German-speaking population Thomas Gloor-Juzi1, Annette Kurre1, Dominik Straumann2 and Eling D de Bruin3* Background With a lifetime prevalence of 29.3 percent in the general German adult population, moderate to severe vertigo or dizziness is a frequent and often recurrent symptom [1]. Furthermore, as 80 percent of the patients reported se- vere limitations in daily activities [1], a considerable cur- tailing in health-related quality of life may be expected. Moderate to severe vertigo or dizziness can interrelate with psychiatric disorders, especially anxiety [2-6], which may increase disability [4]. * Correspondence: debruin@move.biol.ethz.ch 3Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich, Switzerland Full list of author information is available at the end of the article Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 2 of 13 arousal or somatic expressions of anxiety [14]. The ori- ginal VSS as well as a Spanish and a Swedish version showed acceptable psychometric properties [5,15]. How- ever, at the beginning of the study (Spring 2007) there was no validated VSS available in German. The author of the original VSS, L. Yardley [14] was contacted and she con- firmed that she had no knowledge of a currently on-going validation. Hence, she approved the German translation and cross-culturally adaptation of the VSS and its valid- ation. Thereby we intended to apply an approved method- ology guideline for the translation process [16,17] and to take in consideration existing cultural and language differ- ences between the German-speaking populations in Europe (e.g., Austria, Germany, Belgium, Switzerland, Italy) [18]. A German version of the VSS from Tschan et al. [19] that in the meantime was published did not spe- cifically comply with guidelines (e.g. use informed and un- informed translators and back-translators) and neither considered the possibility of cross-cultural differences, which might have biased their results. For this reason the primary aim of the study was the translation and the cross-cultural adaptation of the VSS into German for the German-speaking population of Switzerland and to inves- tigate its internal validity and reliability for patients with vestibulopathy. The secondary aim of the study was the investigation of the discriminant and convergent validity as parts of external validity determination. merger of both translations and in order to resolve discrep- ancies. Participants Th d The study participants were suffering from vertigo, dizzi- ness, or unsteadiness associated with a diagnosed ves- tibular disorder for at least one month. They had to be between 18 and 75 years old, capable of walking and in- dependently managing approximately 50% of their daily tasks and have good German language skills. The exclu- sion criteria comprised dizziness or unsteadiness exclu- sively due to cardiopulmonary diseases, musculoskeletal problems or neurologic disorders like severe paresis, spasticity, cerebellar ataxia, extrapyramidal diseases, or sensory loss. Furthermore, patients with diagnosed de- mentia, psychiatric disorders or blindness were excluded. Recruitment took place between July 2007 and July 2009 through the Interdisciplinary Centre for Vertigo & Bal- ance Disorders, Departments of ENT, Neurology and Psychiatry at the University Hospital Zurich. All patients who were referred to the department were asked to par- ticipate in this study. Those who met the criteria and submitted their signed written informed consent were included in the study. Healthy participants were mainly family members or friends of the authors and their col- leagues and were also included after submitting their signed written informed consent. The approval of the ethics committee of the Canton of Zurich was obtained in accordance with the Declaration of Helsinki. Translation and cross cultural adaptation of the VSS The VSS is a self-completed, 34-item questionnaire (19 items for VSS-VER and 15 for VSS-AA). The frequency of the symptoms is rated on a Likert-scale: 0 points: “never”, 1 point: “a few times (1–3 times a year)”, 2 points: “several times (4–12 times a year)”, 3 points: “quite often (on average, more than once a month)” and 4 points: “very often (on average more than once a week)”. The total score aims to figure out the latent di- mension of severity of dizziness and ranges from 0 points (no symptoms) to 136 points (severe vertigo). The translation and cross-cultural adaptation into German was performed in six steps, according to the inter- national guidelines for self-reported measures published by the American Association of Orthopaedic Surgeons Out- come Committee [16] and additional information about requirements for translators from Wild et al. [17]. First step: two independent German VSS-translations were requested in order to reflect possible ambiguous wording. Two native German-speaking translators with excellent English lan- guage skills translated independently the VSS into German. Background Third step: back translation of the previously obtained German version into English by two independent native English speaking translators with German language skills, both translators were withheld of the original version of the VSS, again one person was aware of the concept the other not. This step was necessary in order to bring out un- clear wording or cultural peculiarities in symptom descrip- tion and to help to assure a consistent translation of the content of each item. Fourth step: an expert committee consisting of the four translators, the methodologist and two health and language professionals produced the pre- final version of the VSS. Prior to this meeting; Lucy Yardley (author of the original VSS) compared the original VSS with both back translations. Her comments were also con- sidered in the discussion. Fifth step: the pre-final VSS-G was pilot tested according to the recommendations of Wild [17] in a group of fourteen patients who fulfilled the inclu- sion/exclusion criteria below. Sixth step: The transcription of the patient interviews and the documents of the preced- ing steps were analysed in the final stage and the final ver- sion of the VSS-G was written. Since cross-cultural differences exist between German-speaking countries [18] and both Swiss and German patients frequent our hospital, special consideration was given to specifically include both German and Swiss cultural background translators and physiotherapists in our research team. Results After the translation and pilot testing process of a pref- inal version, the VSS-G was tested with 202 participants with vertigo or dizziness and 52 healthy subjects (Table 1: characteristics of participants). Measures The Hospital Anxiety and Depression Scale (HADS) is a 14-item questionnaire measuring psychological distress. It is divided into two subscales assessing non-somatic symp- toms of anxiety and depression [20]. Although the whole questionnaire was submitted to the participants, only the results of the anxiety subscale are reported here. The items are rated from 0 to 3 points and the score ranges from 0 (no sign of anxiety) to 21 (maximum level of anxiety). The validity of the HADS has been demonstrated by a large number of patient groups. The psychometric properties of the German version are good and the recommended cut- off score for screening abnormal anxiety is eleven [21]. External validity determination Discriminant validity was analysed by comparing the me- dian scores of VSS-G, VSS-VER and VSS-AA between a group of 52 healthy subjects and the three diagnostic sub- groups: “1=peripheral vestibular disorder, 2=central vestibular disorder and 3= multifactorial/multisensory dis- orders”. Significant differences between the groups were investigated with the Mann–Whitney-U-Test for independ- ent groups. If any significant difference between the groups was found the ROC curve and the Youden Index [31] were carried out. The Mann–Whitney-U-Test was further used to investigate the ability of the VSS-AA median score to dif- ferentiate abnormal anxiety as defined by the HADS-A (score≥11). xThree items of the University of California Los Angeles-Dizziness Questionnaire UCLA-DQ [24] were used to rate the overall perceived frequency of dizziness (UCLA-DQ1), its intensity (UCLA-DQ2) and the impact of dizziness on daily activities (UCLA-DQ3). The pro- blems were rated on a scale ranging from 1 (lowest level of the severity of the problem) to 5 (highest level). Fur- thermore the patients had to rate their level of disability induced by dizziness as no disability (0 points), mild (1), moderate (2) or severe (3 points). Convergent validity of the VSS-G and its subscales was analysed using the Spearman’s correlations to investigate the association of VSS-G, VSS-VER and VSS-AA with HADS-A and DHI. Kendall’s Tau rank correlation was performed to analyse the associations of the VSS with the estimated level of disability and the 3 items of the UCLA-DQ: 1) frequency, 2) intensity and 3) impact of dizziness on daily activities. The postal data collection procedure for the reliability and validity investigation are summarized in Figure 1. The test-retest reliability analysis was carried out on 40 complete pairs of questionnaires [25]. The data collec- tion procedure was controlled on a daily basis. The qual- ity was assured by contacting the patients by phone in order to clear up ambiguous responses, to fill gaps in the questionnaires or to remind them to return the questionnaires. All the analysis was performed on IBM PASW Statistics (formerly SPSS Statistics) version 18 software. Participants Th d Moreover, as recommended by Wild [17], one translator was aware of the measured concept of the VSS (health pro- fessional), the other not. Second step: meeting of both of the translators and an observer to keep record of the Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 3 of 13 statistical analysis, ceiling or floor effects in the VSS-G or the subscales were checked in the study population. Reliability determination Cronbach's α coefficient was calculated to investigate the internal consistency of the VSS-G and both subscales. For psychometric scales Cronbach’s α > 0.8 is generally recom- mended [26]. The Cronbach's α coefficient was also esti- mated with each item excluded, where the difference between α-total and α-with-the-item-deleted should not be greater than 0.1 [27]. The corrected item-total correlations (CI-TC) were calculated to investigate the strength of rela- tionship between a single item and the other items in each of the scales. CI-TC should range between 0.20 and 0.40 [28], with the minimal recommended value of 0.2 [26]. The intra-tester reliability was calculated with the two-way random model with single measurement intraclass correl- ation coefficient (ICC2,1 absolute agreement type) [29,30]. The Dizziness Handicap Inventory (DHI) [22] is a 25-item questionnaire designed to evaluate the precipi- tating physical factors associated with dizziness and unsteadiness as well as the functional and emotional consequences of vestibular disease[22]. The items were rated with “yes” (4 points), “sometimes” (2 points) and “no” (0 points). The total score ranges from zero (no disability) to 100 (severe disability). The original ver- sion demonstrated good face validity, internal consistency and test-retest reliability in a population with different aetiologies of dizziness and unsteadiness [22]. A valid German version is available [23]. Translation and pilot testing of the prefinal translation of the VSS-G However the expert committee in the fourth step of the process reached consensus for each item and the patients (in the pilot testing) were able to recognise and associated the described symptoms with their vestibular problems. Compared to the German VSS version that was published during our data gathering process [19], we found differences in the way sentences were constructed. This could be due to cross-cultural dif- ferences between the German-speaking populations in Germany and Switzerland [18]. However, the wording used to describe the symptoms was essentially the same. For the pilot testing (cognitive debriefing) of the pre- final version, 14 patients (8 male) with vestibular disorders (8 peripheral (57%), 5 central vestibular disorder (37%) and 1 multifactorial (7%) causes of dizziness could be included. The mean age (SD) was 60.5 (14.13) years, the mean scores of the DHI (SD) = 46 (20.0), the VSS = 49 (25) and the subscales VSS-VER = 24 (14), VSS-AA = 25 (13). headed, swimmy, giddy or walk properly for the items of the VSS-VER. There were also some discussion needed to adapt descriptions of symptoms of the VSS-AA like: about to black out, tingling, prickling, spots before the eyes, heart pounding, soreness in muscles, stomach churning, hot or cold spells (not to confound with symp- toms of menopause). However the expert committee in the fourth step of the process reached consensus for each item and the patients (in the pilot testing) were able to recognise and associated the described symptoms with their vestibular problems. Compared to the German VSS version that was published during our data gathering process [19], we found differences in the way sentences were constructed. This could be due to cross-cultural dif- ferences between the German-speaking populations in Germany and Switzerland [18]. However, the wording used to describe the symptoms was essentially the same. The participants estimated the matching of the contents of the VSS items with their symptoms at 92.2 percent (SD 6.9). One outlier (more than 3 standard deviations differ- ences to the mean), who could not cite any additional problem or symptom the VSS was not accounting for, was excluded from the calculation. Fatigue and weariness were the most cited problems (3 from 14) which are not included in the VSS. Translation and pilot testing of the prefinal translation of the VSS-G According to the preceding studies [14,19], a principal component analysis (PCA) for two factors with Varimax rotation was performed, in order to compare the loadings of the items on both factors representing the structure of the subscales (VSS-VER and VSS-AA). Prior to the Throughout the translation and cross cultural adaptation process the translators encountered some difficulties to adapt definitions like: unsteadiness, actually fall, light- Page 4 of 13 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Inclusion: 52 healthy persons Inclusion: 202 persons with vertigo, dizziness or imbalance Baseline set of questionnaires adapted for healthy persons: VSS-G/ DHI-G/ HADS-G Baseline set of questionnaires: VSS1-G/ DHI-G/ HADS-G/ UCLA-DQ1/ UCLA-DQ2/UCLADQ3 socio-demographic data Discriminant Validity: • Pairwise U-Test between diagnostic groups and healthy participants • ROC-curve for VSS-VER After 1 week, a sample of persons assumed to be in stable health condition with respect of dizziness (n=58) received a second questionnaire (VSS2-G) Report of stable health condition Exclusion of reliability analysis no n= 18 yes n= 40 Test-Retest Reliability • ICC 2,1 Internal validity: • PCA with Varimax Rotation (2 factors) Internal Consistency: • Cronbach's α / α if item deleted • CI-TC Construct Validity: • Spearman’s Correlation coefficient • Kendall’s Tau correlation coefficient Figure 1 Flow-chart of data collection and analysis procedure. Inclusion: 202 persons with vertigo, dizziness or imbalance Baseline set of questionnaires: VSS1-G/ DHI-G/ HADS-G/ UCLA-DQ1 After 1 week, a sample of persons assumed to be in stable health condition with respect of dizziness (n=58) received a second questionnaire (VSS2-G) Internal validity: • PCA with Varimax Rotation (2 factors) Internal Consistency: • Cronbach's α / α if item deleted • CI-TC Construct Validity: • Spearman’s Correlation coefficient • Kendall’s Tau correlation coefficient Report of stable health condition Test-Retest Reliability • ICC 2,1 Exclusion of reliability analysis Figure 1 Flow-chart of data collection and analysis procedure. headed, swimmy, giddy or walk properly for the items of the VSS-VER. There were also some discussion needed to adapt descriptions of symptoms of the VSS-AA like: about to black out, tingling, prickling, spots before the eyes, heart pounding, soreness in muscles, stomach churning, hot or cold spells (not to confound with symp- toms of menopause). Translation and pilot testing of the prefinal translation of the VSS-G However, to the best of our know- ledge, even if both symptoms could appear with vestibular disorders, there is no association of fatigue as a symptom of dizziness described in literature. Therefore there was no item added to the VSS-G. The other cited symptoms were diarrhoea, inflammation of the pharynx (sensation of burning while swallowing), polyuria and incontinence, tickling and itching in the ears, hunger and panic. Some patients missed items with vertigo-causing activities (e.g. walking, head movements) or emotions like feeling helpless or alone, fear of becoming dependent or losing work. These propositions underpin the necessity to assess limitations in activity, participation and emotional distress but are beyond the scope of the VSS. Based on these results, the VSS-G can be considered to be complete. For the pilot testing (cognitive debriefing) of the pre- final version, 14 patients (8 male) with vestibular disorders (8 peripheral (57%), 5 central vestibular disorder (37%) and 1 multifactorial (7%) causes of dizziness could be included. The mean age (SD) was 60.5 (14.13) years, the mean scores of the DHI (SD) = 46 (20.0), the VSS = 49 (25) and the subscales VSS-VER = 24 (14), VSS-AA = 25 (13). Although most patients declared to easily understand the content of the VSS-G, the interviewers noticed Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 5 of 13 Page 5 of 13 Table 1 Characteristics of participants Participants with vestibular disorder (n=202) Subgroup for reliability testing (n=40) Healthy participants (n=52) Age in years: mean (SD) 50.0 (13.47) 50.7 (13.63) 46.7 (13.12) Gender: n women/men (% women): 124/78 (61.4) 23/17 (57.5) 28/24 (53.8) Independence or need of support for ADL/household: n (%) independent 162 (80.2) 33 (82.5) 52 (100.0) need of occasional support 27 (13.4) 6 (15.0) 0 need of regular weekly or daily support 13 (6.4) 1 (2.5) 0 Duration of dizziness: n (%) dizziness since ≥1 month, less than 6 mo 58 (28.7) 8 (20.0) dizziness since ≥6 mo up to 12 mo 27 (13.4) 7 (17.5) dizziness since > 12 mo 117 (57.9) 25 (62.5) Diagnosis: n (%) Unilateral peripheral vestibular disorder: 73 (36.14) 10 (25.00) unilat BPPV 26 (12.9) 3 (7.5) unilat. Morbus Meniere 27 (13.4) 4 (10.0) unilat. Internal validity determination Principal component analysis p p y Table 2 contains the results of the two factors extraction with a PCA and Varimax rotation and, for the sake of com- parison, the loadings of previous versions and translations of the VSS were also displayed [5,14,19]. Compared to the original VSS, 87% (13/15) of the items could be clearly associated to the VSS-AA scale. Indeed, sixteen items loaded> 0.5 on factor one and thirteen of these items were attributed to Yardley’s original VSS-AA subscale. The three remaining items were vertigo symptoms lasting for less than 2 minutes (1a, 7a, 18a). Their allocation to the VSS-VER subscale will be explained below. Two items (9, 13) showed a clearly higher loading on factor one than on factor two; these loadings were between 0.4 and 0.5. With high face validity, these two items were integrated in the VSS-AA subscale of VSS-G, so that the VSS-AA sub- scale was identical to the AA subscale of the original VSS. The VSS-VER subscale showed not such a clearly item matching. Only 47% (9/19) of the items were attributed to the VSS-VER based on their loadings >0.5 on factor two. Four items with loadings from 0.4 to 0.5 (1b, 1e, 7e, 15) were added to VSS-VER because this loading was clearly higher on factor two than factor one. Three items (4, 5, 11) showed unclear loadings either on factor one or two. They were allocated to VSS-VER for the following reason. Item Table 2 contains the results of the two factors extraction with a PCA and Varimax rotation and, for the sake of com- parison, the loadings of previous versions and translations of the VSS were also displayed [5,14,19]. Compared to the original VSS, 87% (13/15) of the items could be clearly associated to the VSS-AA scale. Indeed, sixteen items loaded> 0.5 on factor one and thirteen of these items were attributed to Yardley’s original VSS-AA subscale. The three remaining items were vertigo symptoms lasting for less than 2 minutes (1a, 7a, 18a). Their allocation to the VSS-VER subscale will be explained below. Two items (9, 13) showed a clearly higher loading on factor one than on factor two; these loadings were between 0.4 and 0.5. With high face validity, these two items were integrated in the VSS-AA subscale of VSS-G, so that the VSS-AA sub- scale was identical to the AA subscale of the original VSS. Translation and pilot testing of the prefinal translation of the VSS-G neuritis vestibularis 4 (2.0) 2 (5.0) others 16 (7.9) 1 (2.5) Bilateral peripheral vestibular disorder: 17 (8.4) 4 (10.0) bilat. BPPV 2 (1.0) 0 bilat. M. Meniere 1 (.5) 0 bilat. neuritis vestibularis 4 (2.0) 3 (7.5) others 10 (5.0) 1 (2.5) Central vestibular disorder: 73 (36.1) 18 (45.0) incomplete central compensation 7 (3.5) 2 (5.0) psycho-physical vertigo 17 (8.4) 2 (5.0) others (e.g. vest. Migraine) 49 (24.3) 14 (35.0) Multifactorial/multisensory vestibular disorder: 39 (19.3) 8 (20.0) Self percieved level of disability: n (%) little 56 (27.7) 7 (17.5) moderate 100 (49.5) 23 (57.5) severe 46 (22.8) 10 (25.0) UCLA 1: frequency of dizziness: n (%) rarely 23 (11.4) 4 (10.0) sometimes 91 (45.0) 19 (47.5) approximately half of the time 39 (19.3) 10 (25.0) usually 34 (16.8) 4 (10.0) always 15 (7.4) 3 (7.5) UCLA 2: Intensity of dizziness: n (%) very mild 10 (5.0) 2 (5.0) mild 23 (11.4) 4 (10.0) moderate 78 (38.6) 17 (42.5) moderately severe 73 (36.1) 13 (32.5) severe 18 (8.9) 4 (10.0) UCLA 3: dizziness induced limitations in activities or participation: n (%) Table 1 Characteristics of participants Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 6 of 13 Table 1 Characteristics of participants (Continued) no effect at all 15 (7.4) 3 (7.5) continuing all activities but allowance for dizziness 39 (19.3) 8 (20.0) continuing most of the activities 79 (39.1) 18 (45.0) continuing some of the activities 51 (25.2) 9 (22.5) unable to continue any of the activities 18 (8.9) 2 (5.0) Table 1 Characteristics of participants (Continued) 04 “falling over” showed inconsistent loading across the studies but was attributed to VSS-VER, due to its face val- idity as a possible symptom in acute vertigo attacks. Simi- larly, the items 05 “Nausea” and 11 “Vomiting” are two generic symptoms of the autonomous system and showed, as expected, somewhat higher loadings on factor one (VSS-AA). Nevertheless, because of their association to vestibular disorder, particularly acute vertigo attack, these symptoms were attributed to VSS-VER. Another issue was the clear loading on factor one (VSS-AA) of vestibular symptoms lasting less than 2 minutes (items 1a, 7a, 18a). Translation and pilot testing of the prefinal translation of the VSS-G This result seems to confirm a trend observed in other works (Table 2) [5,14,19] and raises the assumption whether this result represents the interface between vertigo and anxiety [4] and thus, could be interpreted as an exacer- bation of autonomic arousal leading to vertigo. among seven persons (50% of participants) difficulties to understand how to deal with the two-tiered questions 1, 7 and 18. These items were used to determine the fre- quency range of a vertigo symptom with different duration. The main problem seemed to be the two time-related con- cepts of symptom duration and frequency. These symptoms might appear or increase with activities (e.g. walking, head movements) and will last through the whole activity, which possibly is an additional confusing time-related aspect. A similar problem was also described by Holmberg for a Swedish version of the VSS [32], in order to counteract this problem we used a clear structure in the layout of these items , even so health professionals may take into account, that patients will need assistance for rating these items. Upon suggestion of some patients, the definitions of the scale-ranking were reported on top margin of each page of the questionnaire (Additional file 1). Due to the apparently high stability in face of factor extraction, particularly for the VSS-AA subscale and the already documented validity of the structure of the VSS in general [5,14,19], we suggest to keep Yardley’s original subscale structure for our VSS-G. Internal validity determination Principal component analysis The cumulative percentage of the scores of the VSS-G, VSS-VER and VSS-AA were analysed. There was no floor effect of the VSS-G and the subscales to be assumed in our sample because the respective scores of the 15 percent threshold [33] lays above the minimal possible score of zero: VSS-G = 16–17, VSS-VER = 6–7, VSS-AA = 5–6. The same holds true for the ceiling effect as the scores of the 85 percent threshold [33] lays clearly under the respective maximal score: VSS-G = 59–60 (maximal score = 136), VSS-VER = 32-33(max = 76), VSS- AA = 30 (max = 60). Reliability determination All the Cronbach’s α-coefficient estimations showed good internal consistency on scale-level (Table 3). This was also the case on item-level, as there were no differ- ences > 0.1 in Cronbach’s α-coefficient estimation in case the respective item was deleted (Additional file 2) and the CI-TC showed no value under 0.2. For test- retest reliability estimations, the mean time between the first and the second measurement was 5.5 days (SD 1.97 days). As shown in Table 3, all the ICC coefficients reached the recommended threshold of 0.75 [29]. The VSS-VER subscale showed not such a clearly item matching. Only 47% (9/19) of the items were attributed to the VSS-VER based on their loadings >0.5 on factor two. Four items with loadings from 0.4 to 0.5 (1b, 1e, 7e, 15) were added to VSS-VER because this loading was clearly higher on factor two than factor one. Three items (4, 5, 11) showed unclear loadings either on factor one or two. They were allocated to VSS-VER for the following reason. Item Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 7 of 13 Page 7 of 13 Table 2 PCA with Varimax Rotation: table for two factor loadings comparison Item Factor 1 Factor 2 CH Germany Mexic hosp UK hosp. Prim care (UK) Swiss Germany Mexic hosp UK hosp Prim care (UK 01. Things spinning/moving (V**): a. less than 2 min 0.55 0.35 0.46* 0.34 0.24 0.02 0.13 −0.07 0.21 −0.06 b. 2 to 20 min 0.27 0.08 0.12 0.13 0.14 0.47* 0.64 0.53 0.71 0.44* c. 20 min to 1 h 0.17 0.08 −0.03 −0.02 0.13 0.60 0.65 0.54 0.83 0.74 d. several hours −0.08 −0.01 −0.09 −0.08 −0.08 0.63 0.52 0.59 0.71 0.68 e. more than 12 h −0.02 0.25 −0.02 0.06 −0.03 0.43* 0.06 0.51 0.52 0.36 02. Heart/chest pain (A***) 0.52 0.52 0.67 0.52 0.52 −0.06 0.04 0.09 −0.14 −0.19 03. Hot or cold spells (A) 0.66 0.61 0.67 0.59 0.65 0.16 0.07 0.25 0.16 0.12 04. Falling over (V) 0.40* 0.42* 0.28 0.27 0.40* 0.24 0.20 0.44* 0.36 0.23 05. Nausea, feeling sick (V) 0.47* 0.50 0.58 0.40* 0.56 0.32 0.14 0.35 0.36 0.23 06. Muscle tension/sore (A) 0.63 0.68 0.81 0.67 0.57 0.08 0.14 0.01 0.01 0.18 07. Light-headed/giddy (V): a. Reliability determination less than 2 min 0.54 0.28 0.50 0.43* 0.28 0.05 0.17 −0.07 0.11 −0.06 b. 2 to 20 min 0.40 0.18 0.30 0.17 0.11 0.60 0.65 0.46* 0.70 0.53 c. 20 min to 1 h 0.21 0.08 0.13 0.18 0.08 0.73 0.77 0.65 0.70 0.74 d. several hours 0.11 0.10 0.08 0.04 0.12 0.70 0.56 0.71 0.73 0.69 e. more than 12 h 0.05 0.24 0.17 0.02 −0.09 0.45* 0.16 0.50 0.46* 0.55 08. Trembling, shivering (A) 0.56 0.63 0.67 0.56 0.53 0.15 0.08 0.27 0.02 0.16 09. Pressure in the ear (A) 0.41* 0.57 0.60 0.27 0.46* 0.06 0.12 0.16 0.24 0.09 10. Heart pounding (A) 0.54 0.59 0.58 0.66 0.64 0.10 −0.12 0.23 0.02 0.09 11. Vomiting (V) 0.22 0.15 0.30 0.01 0.26 0.12 0.22 0.28 0.32 0.38 12. Heavy feeling arms/legs (A) 0.58 0.74 0.72 0.59 0.70 0.24 0.04 0.04 0.04 0.02 13. Visual disturbances (A) 0.42* 0.71 0.72 0.58 0.61 0.11 0.14 0.16 0.10 0.18 14. Headache/pressure (A) 0.57 0.48* 0.71 0.52 0.58 0.11 0.18 0.23 0.06 0.11 15. Unable to stand/walk (V) 0.16 0.40* 0.38 0.27 0.54 0.42* 0.18 0.40* 0.35 0.35 16. Breathing difficulties (A) 0.63 0.69 0.62 0.53 0.72 0.05 −0.07 0.08 0.06 −0.02 17. Loss of concentration (A) 0.56 0.59 0.64 0.61 0.72 0.28 0.05 −0.03 0.05 0.15 18. Feeling unsteady (V): a. less than 2 min 0.60 0.27 0.54 0.42 0.24 0.22 0.22 0.13 0.13 −0.04 b. 2 to 20 min 0.39 0.12 0.28 0.13 0.10 0.64 0.64 0.62 0.71 0.58 c. 20 min to 1 h 0.28 0.02 0.17 0.06 0.13 0.75 0.76 0.66 0.74 0.77 d. several hours −0.00 −0.06 −0.12 −0.03 0.08 0.70 0.60 0.69 0.77 0.68 e. more than 12 h 0.03 0.25 −0.04 0.00 −0.06 0.51 0.29 0.60 0.61 0.49* 19. Tingling, prickling (A) 0.55 0.60 0.75 0.59 0.63 −0.00 0.05 0.00 −0.02 0.06 20. Pain in the lower back (A) 0.59 0.56 0.71 0.56 0.66 −0.04 −0.07 −0.08 −0.20 −0.09 21. Excessive sweating (A) 0.57 0.60 0.19 0.47 0.60 0.21 0.05 0.19 0.12 0.18 22. Feeling faint, black out (A) 0.51 0.61 0.02 0.45 0.60 0.19 0.12 0.26 0.30 0.06 The table is adapted from Tschan (Germany, Mainz) [19] and completed with the results of present study Switzerland, (CH) Interdisciplinary Centre for Vertigo & Balance Disorders, University Hospital Zurich. * Significant (2 tailed) at a <0.001 level. * Significant (2 tailed) at a <0.001 level. Significant (2 tailed) at a <0.001 level. VSS-G = VSS total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale. Mann–Whitney-U-test (U) 1) for comparison of median VSS-VER scores between healthy and diagnostic groups, 2) median VSS-AA scores between patients without and patients with anxiety (HADS-A ≥11). External validity determination distribution of median scores of VSS-AA showed a sig- nificant difference between people without and those with abnormal anxiety as defined by the HADS-A (Score≥ 11). However, the ROC-Curve showed very low specificity. Discriminant validity The Mann–Whitney-U-Test showed significant differences in the distribution of the median scores of the VSS-G, the VSS-VER (Figure 2) and VSS-AA between healthy subjects and persons with vestibular disorders (Table 3). Therefore, the areas under the ROC-Curve (CI) were calculated: VSS-G = 0.95 (0.92, 0.97), VSS-VER = 0.99 (0.98, 1.00) and VSS-AA = 0.84 (0.78, 0.89) (Figure 3). According to the Youden Index [31] the best relationship of 0.95 sensitivity and 1.00 specificity could be set at VSS-VER Score = 4.5 (or 0.24 item mean). Particularly the VSS-VER (Figure 2) was able to discriminate between healthy subjects and persons with vestibular disorders. But there was no skill to differentiate between the groups of diagnosis. The Reliability determination Factor loadings ≥0.5 are highlighted in bold face; * highest factor loading in analysis when ≥0.4 (but ≤0.5). As grey background is not acceptable, it was necessary to add the (V) or (S) definition for ich item; ** (V)= item attributed to VSS-VER; ***(A)= item attributed to VSS-AA. Table 2 PCA with Varimax Rotation: table for two factor loadings comparison Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 Page 8 of 13 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 8 of 13 Table 3 Internal validity, reliability, discriminant validity results Table 3 Internal validity, reliability, discriminant validity results VSS-G VSS-VER VSS-AA Cronbach's α [n = 202] .904 .859 .864 ICC (CI) [n = 40] .926 (.826/.965) .920 (.854/.957) .913 (.737/.963) Medians Healthy [n = 52] vs. peripheral vest disorder [n = 90] U 326.50 43.00 1046.50 Z −8.529* −9.782* −5.486 8* Medians Healthy [n = 52] vs. central vest. disorder [n = 73] U 153.500 22.000 467.500 Z −8.742* −9.467* −7.171* Medians Healthy [n = 52] vs. multifactorial disorder [n = 39] U 89.50 62.00 210.00 Z −7.421* −7.797* −6.456* No anxiety [n=166] vs. abnormal anxiety [n = 36] U 1776.50 2446.00 1427.00 Z −3.811* −1.706 −4.912* * Significant (2 tailed) at a <0.001 level. VSS-G = VSS total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale. Mann–Whitney-U-test (U) 1) for comparison of median VSS-VER scores between healthy and diagnostic groups, 2) median VSS-AA scores between patients without and patients with anxiety (HADS-A ≥11). Convergent validity In order to confirm the structure of the subscale regard- ing the possible interaction with anxiety, a different cor- relation of VSS-VER and VSS-AA with measurement of anxiety (HADS-A) was expected. According to the def- inition of the thresholds of Gill-Body [34], the VSS-AA correlated fairly with the HADS-A, while there was only low correlation between the VSS-VER and the HADS-A (Table 4). Furthermore, the VSS-G showed moderate correlation with self-perceived disability as measured by the DHI (Table 4). The Kendall Tau rank correlation Figure 2 Medians and interquartile range of VSS-VER subscores of the three diagnostic groups and healthy subjects. igure 2 Medians and interquartile range of VSS-VER subscores of the three diagnostic groups and healthy subject Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 9 of 13 Page 9 of 13 Figure 3 ROC-Curve for discrimination between dizzy patients and healthy subjects. igure 3 ROC-Curve for discrimination between dizzy patients and healthy subjects. vestibular disorders and to integrate the information in the treatment plan. coefficient (Table 5) showed a significant but only fair correlation between the VSS-G and the self-rated fre- quency of dizziness (UCLA-DQ1), this correlation could partly be displayed by the box plot of the VSS-G median scores against the UCLA-DQ1 (Figure 4). The VSS-G total score and both subscores correlated fairly with self-estimated (mild, moderate, severe) disability. The impact on daily activities correlated fairly with VSS-G and VSS-VER; however, it only correlated weakly with VSS-AA. Discussion The VSS was translated and cross-culturally adapted into German for a wider German speaking population of Switzerland. The original’s structure of the subscales could be confirmed and acceptable psychometric properties were found. The VSS-G may be adopted to collect the self-perceived symptoms of patients with Table 4 Spearman’s correlation coefficients Spearman's Rho n = 202 VSS-G VSS-VER VSS-AA HADS-A VSS-VER .875** VSS-AA .865** .549** HADS-A .369** .186** .452** DHI .554** .496** .464** .448** **. The correlation is significant on a 0,01 Level (two sided). VSS-G = VSS total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale, DHI = Dizziness Handicap Inventory total score, HADS-A = Hospital Anxiety and Depression Scale - Anxiety subscale). Table 4 Spearman’s correlation coefficients Table 4 Spearman’s correlation coefficients Spearman's Rho n = 202 VSS-G VSS-VER VSS-AA Table 4 Spearman’s correlation coefficients Spearman's Rho n = 202 VSS-G VSS-VER VSS-AA HADS Internal validity determination Reliability determination The VSS-G translation exhibits high internal consistency and acceptable test-retest reliability, similar to the original VSS and its subscales [14]. This indicates the ability of the translated instrument to reliably screen groups of patients. The aspect of agreement was not analysed in the study [35]; however, as the VSS was conceived to screen the fre- quency of the symptoms rather than to measure change, this was not deemed a compulsory psychometric character- istic. However, we admit that measuring change remains an important topic in vestibular rehabilitation. The short form of the VSS [36,37], for which acceptable limits of agreement were found in a Turkish translation [38], seems to be an ap- propriate instrument for this purpose. The findings of this study justify a German language translation for the short form VSS for the purpose of establishing an instrument capable of measuring change in symptoms. Discriminant validity y Particularly the VSS-VER subscale was able to discriminate between healthy subjects and people with dizziness. If the determined VSS-VER cut-off score would be applied, eleven participants out of 252 (4.5%) were misclassified as **. The correlation is significant on a 0,01 Level (two sided). VSS-G = VSS total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale, DHI = Dizziness Handicap Inventory total score, HADS-A = Hospital Anxiety and Depression Scale - Anxiety subscale). Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 10 of 13 Table 5 Kendall’s Tau rank correlation coefficients of VSS with self-estimated disability and 3 items of UCLA-DQ Kendall-Tau n = 202 VSS-G VSS-VER VSS-AA frequency intensity disability VSS-VER .711** VSS-AA .703** .397** frequency (UCLA-DQ1) .317** .262** .299** intensity (UCLA-DQ2) .227** .275** .108* .101 disability .400** .385** .321** .369** .469** impact on daily activities (UCLA-DQ3) .281** .309** .172** .133* .590** .508** ** correlation is significant on a 0,01 Level (two sided). * correlation is significant on a 0,05 Level (two sided). VSS-G = VSS-G total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale; UCLA-DQ1 = self-estimated frequency of dizziness, UCLA-DQ2 = self-estimated intensity of dizziness, UCLA-DQ3 = self-estimated impact of dizziness on daily activities; disability = self-rated mild, moderate, severe disability. ble 5 Kendall’s Tau rank correlation coefficients of VSS with self-estimated disability and 3 items o ation coefficients of VSS with self-estimated disability and 3 items of UCLA-DQ correlation is significant on a 0,05 Level (two sided). VSS-G = VSS-G total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale; UCLA-DQ1 = self-estimated frequency of dizziness, UCLA-DQ2 = self-estimated intensity of dizziness, UCLA-DQ3 = self-estimated impact of dizziness on daily activities; disability = self-rated mild, moderate, severe disability. false negatives. This may indicate that the vestibular symp- toms of these patients are very low, which may mislead to scrutinise the effectiveness of a vestibular rehabilitation. Therefore, particularly for these patients all the aspects of the health problems like comorbidities and limitations in activities of daily life and participation should be considered in the treatment plan. Anyway, further investigations were necessary to analyse the usefulness of this cut-off score prior to vestibular rehabilitation. The low discriminant properties for vestibular disorders of the VSS-AA may highlight the interface between vestibular dysfunction and disorders of autonomous system and anxiety. Discriminant validity In contrast the VSS-AA subscale seemed capable to screen for possible abnormal anxiety, but it seems fair to state that further re- search is needed on this discriminative property. research for a tool to measure the severity of vertigo symp- toms, which is supposed to be uncontaminated by anxiety [14]. On the other hand, the VSS-AA’s fair association with HADS-A and its discriminant property for abnormal anx- iety, may suggest that the symptoms of VSS-AA might be associated with an anxiety disorder. This may confer to the subscale a certain ability to screen for possible anxiety pro- blems without providing any other conclusive information about anxiety (e.g. aetiology of comorbidity). This property may be helpful in rehabilitation management to counsel the patients and refer them to health professionals experienced in psychological disorders for further assessment. The influence of the severity of the symptoms on limita- tions of daily activities was showed by correlations of the VSS-G with measurements of dizziness related disability in daily activities (DHI, UCLA-DQ3) and the self-estimated disability due to dizziness. These findings are comparable to the work of Tamber et al.[39], who showed an associ- ation between the Norwegian VSS short-form and the DHI; Limitations h The present study was not without limitations; the capacity of the VSS-VER subscale to differentiate between the designed diagnostic groups could not be reproduced like in other similar studies. Our group allocation hampered com- parison of the results unlike those in other studies with somewhat other classifications. However, it showed to be appropriate when targeting vestibular rehabilitation. Fur- thermore, this group allocation yielded quite unequal group sizes which, in turn, could have biased the results. There- fore, calculations were carried out with random selections of cases within the groups in order to balance the group sizes, and the described differences were maintained. The fact that in the meantime a German translation of the VSS was published [19] could be seen as a further limitation. However, although this translation by a German team was published during our data collection process, too late to in- fluence the generation of our version, our work cannot be outclassed for several reasons. Firstly, our study method- ology [16,17] took in consideration cultural and language differences between the German-speaking population of Switzerland [18]. Secondly, the broad group allocation in peripheral, central and multifactorial vestibular disorders was designed to validate the VSS-G for screening people with vertigo, dizziness or balance disorders prior to vestibu- lar rehabilitation. Thirdly, the convergent validity analysis carried out by Tschan and colleagues did not use any dizziness-specific measurement of disability. Further than disability, symptoms may also have some influence on health-related quality of life (HRQoL). This is an important issue, as HRQoL measurement figures out the dimension of the impact of dizziness on the patient’s participation to social life. 1) In order to meas- ure the outcome of vestibular rehabilitation Morris et al. [40,41] developed the Vestibular Rehabilitation Benefit Questionnaire (VRBQ) with a HRQoL subscale, which showed a moderate correlation to the VSS. However, this result should be interpreted cautiously, because there were VSS items included in this VRBQ subscale. Never- theless the scale seems useful for measuring changes in vestibular rehabilitation. 2) Yanik [38] translated and validated the VSS short-form and the Vertigo, Dizziness and Imbalance questionnaire (VDI) [42] (containing a HRQoL subscale) into Turkish, unfortunately there were no intercorrelations reported. 3) In her work, Tschan[19] analysed the correlations of the VSS with generic quality of life measurement (physical and mental health compo- nents of the SF-36). Conclusions The present German translation of the VSS shows satisfac- tory psychometric properties for the assessment of the self- perceived severity of the symptoms on a patient group level in a large geographic area of German-speaking countries. The high discriminant validity of the VSS-VER subscale allows it to be used for screening vestibular dysfunction and coordinate the treatment. The VSS-AA subscale is able to screen for symptoms which might refer to an anxiety disorder, which might be addressed additionally in the con- text of vestibular rehabilitation. As both scales measure frequency, it may be surprising that the plot of the VSS-G median scores to the frequency of dizziness (UCLA-DQ1, Figure 4) is not more distinct. The mismatch seems to appear with increasing frequency (Figure 4) and is probably due to the different structure of the instruments. As a matter of fact, in the VSS-G several item scores were added, while the UCLA-DQ1 is a single overall estimation of the frequency of dizziness. Possibly there are some confounders influencing frequency: 1) the described interaction of intensity [43] and/or 2) the non- negligible mutual influence between the symptoms of both subgroups (VSS-VER and VSS-AA). Further research is needed to illustrate the impact of all these possible interactions. Competing interests Competing interests The authors declare that there are no competing interests. Competing interests Convergent validity The low association of the VSS-VER with the HADS-A seems to imply that our finding might confirm Yardley’s Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Page 11 of 13 and Yardley who measured disability with the Vertigo Handicap Questionnaire [5,14]. Limitations h She found remarkably weaker corre- lations with HRQoL for the VSS-VER subscale than for the VSS-AA [19]. However, these results should be inter- preted cautiously because of a possible lack of relation- ship between the SF-36 measure and the salient areas of dizziness. Previously, Prieto et al. [42] recommended the VDI for more relevant and responsive measurements of health-related quality of life. However, current in-depth research on this instrument is lacking, which impedes the possible discussion about which instrument should be preferred in future trials. Nevertheless, the findings of Tschan underpin the influence of emotional or psycho- logical distress on generally perceived quality of life, which should also be considered in vestibular rehabilitation. Additional files Additional file 1: Final validated VSS-G version. Additional file 2: Reliability results on item level. Additional file 1: Final validated VSS-G version. Additional file 2: Reliability results on item level. Additional file 1: Final validated VSS-G version. Additional file 2: Reliability results on item level. 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Garson GD: Reliability Analysis.: ; http://faculty.chass.ncsu.edu/garson/PA765/reliab htm#rater; Copyright 1998, 2008 by G. David Garson. Last updated 3/29/08. py g y p 31. Youden WJ: Index for rating diagnostic tests. Cancer 1950, 3(1):32–35. 6. Godemann F, Linden M, Neu P, Heipp E, Dorr P: A prospective study on the course of anxiety after vestibular neuronitis. J Psychosom Res 2004, 56 (3):351–354. 32. Holmberg J, Karlberg M, Harlacher U, Magnusson M: Experience of handicap and anxiety in phobic postural vertigo. Acta Otolaryngol 2005, 125(3):270–275. 7. Cowand JL, Wrisley DM, Walker M, Strasnick B, Jacobson JT: Efficacy of vestibular rehabilitation. Otolaryngol Head Neck Surg 1998, 118(1):49–54. 33. McHorney CA, Tarlov AR: Individual-patient monitoring in clinical practice: are available health status surveys adequate? Qual Life Res 1995, 4(4):293–307. 8. Cohen HS, Kimball KT: Increased independence and decreased vertigo after vestibular rehabilitation. Otolaryngol Head Neck Surg 2003, 128(1):60–70. 9. Hansson E: Vestibular rehabilitation – For whom and how? A systematic review. Advances in Physiotherapy 2007, 9(3):106–116. 8. Received: 9 January 2012 Accepted: 26 June 2012 Published: 2 July 2012 Received: 9 January 2012 Accepted: 26 June 2012 Published: 2 July 2012 24. Honrubia V, Bell TS, Harris MR, Baloh RW, Fisher LM: Quantitative Evaluation of Dizziness Characteristics and Impact on Quality of Life. Am J Otol 1996, 17(4):595–602. 25. Donner A, Eliasziw M: Sample size requirements for reliability studies. Stat Med 1987, 6(4):441–448. Authors' contributions Further research is also needed to determine the ex- tent of usefulness of VSS in vestibular rehabilitation such as the determination of cut-off scores which pro- duce a relevant predictive evidence for rehabilitation success on the individual patient level. TG-J contributed to the design of the survey. He conducted the statistical analysis and wrote the manuscript. AK co-initiated the study and contributed to the design of the study and to the process of data collection and interpretation. She reassessed the article. DS contributed to the analysis of data and revised the article thoroughly for its content. As the co-director of the interdisciplinary centre for vertigo and balance disorders, he bore the Page 12 of 13 Page 12 of 13 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Author details 1Ph i h O Author details 1Physiotherapy Occupational Therapy, University Hospital Zurich, Zurich, Switzerland. 2Interdisciplinary Centre for Vertigo & Balance Disorders, Departments of ENT, Neurology & Psychiatry, University Hospital Zurich, Zurich, Switzerland. 3Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich, Switzerland. 22. Jacobson GP, Newman CW: The development of the Dizziness Handicap Inventory. Arch Otolaryngol Head Neck Surg 1990, 116(4):424–427. 1Physiotherapy Occupational Therapy, University Hospital Zurich, Zurich, Switzerland. 2Interdisciplinary Centre for Vertigo & Balance Disorders, Departments of ENT, Neurology & Psychiatry, University Hospital Zurich, 3 23. Kurre A, van Gool CJ, Bastiaenen CH, Gloor-Juzi T, Straumann D, de Bruin ED: Translation, cross-cultural adaptation and reliability of the german version of the dizziness handicap inventory. Otol Neurotol 2009, 30 (3):359–367. Zurich, Switzerland. 3Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich, Switzerland. References Clin Otolaryngol Allied Sci 1999, 24(4):286–293. 41. Morris AE, Lutman ME, Yardley L: Measuring outcome from vestibular rehabilitation, part II: refinement and validation of a new self-report measure. Int J Audiol 2009, 48(1):24–37. 41. Morris AE, Lutman ME, Yardley L: Measuring outcome from vestibular rehabilitation, part II: refinement and validation of a new self-report measure. Int J Audiol 2009, 48(1):24–37. y g 16. Beaton DE, Bombardier C, Guillemin F, Ferraz MB: Guidelines for the process of cross-cultural adaptation of self-report measures. Spine (Phila Pa 1976) 2000, 25(24):3186–3191. process of cross-cultural adaptation of self-report measures. Spine (Phila Pa 1976) 2000, 25(24):3186–3191. Page 13 of 13 Page 13 of 13 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7 http://www.biomedcentral.com/1472-6815/12/7 42. Prieto L, Santed R, Cobo E, Alonso J: A new measure for assessing the health-related quality of life of patients with vertigo, dizziness or imbalance: the VDI questionnaire. Qual Life Res 1999, 8(1–2):131–139. 43. Perez N, Martin E, Garcia-Tapia R: Dizziness: relating the severity of vertigo to the degree of handicap by measuring vestibular impairment. Otolaryngol Head Neck Surg 2003, 128(3):372–381. doi:10.1186/1472-6815-12-7 Cite this article as: Gloor-Juzi et al.: Translation and validation of the vertigo symptom scale into German: A cultural adaption to a wider German-speaking population. BMC Ear, Nose and Throat Disorders 2012 12:7. 42. Prieto L, Santed R, Cobo E, Alonso J: A new measure for assessing the health-related quality of life of patients with vertigo, dizziness or imbalance: the VDI questionnaire. Qual Life Res 1999, 8(1–2):131–139. 43. Perez N, Martin E, Garcia-Tapia R: Dizziness: relating the severity of vertigo to the degree of handicap by measuring vestibular impairment. Otolaryngol Head Neck Surg 2003, 128(3):372–381. doi:10.1186/1472-6815-12-7 Cite this article as: Gloor-Juzi et al.: Translation and validation of the vertigo symptom scale into German: A cultural adaption to a wider German-speaking population. BMC Ear, Nose and Throat Disorders 2012 12:7. doi:10.1186/1472-6815-12-7 Cite this article as: Gloor-Juzi et al.: Translation and validation of the vertigo symptom scale into German: A cultural adaption to a wider German-speaking population. BMC Ear, Nose and Throat Disorders 2012 12:7. 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Molecular characterisation of NPM1 and FLT3-ITD mutations in a central South African adult de novo acute myeloid leukaemia cohort
African journal of laboratory medicine
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African Journal of Laboratory Medicine ISSN: (Online) 2225-2010, (Print) 2225-2002 Page 1 of 6 Original Research Page 1 of 6 Original Research Page 1 of 6 Page 1 of 6 Dates: Keywords: acute myeloid leukaemia; AML; NPM1; FLT3-ITD; frequency; South Africa. Keywords: acute myeloid leukaemia; AML; NPM1; FLT3-ITD; frequency; South Africa How to cite this article: Kloppers JF, De Kock A, Cronjé J, Van Marle A-C. Molecular characterisation of NPM1 and FLT3-ITD mutations in a central South African adult de novo acute myeloid leukaemia cohort. Afr J Lab Med. 2021;10(1), a1363. https://doi. org/10.4102/ajlm.v10i1.1363 Molecular characterisation of NPM1 and FLT3-ITD mutations in a central South African adult de novo acute myeloid leukaemia cohort Authors: Jean F. Kloppers1,2 André de Kock1,2 Johané Cronjé1,2 Anne-Cecilia van Marle1,2 Affiliations: 1Department of Haematology and Cell Biology, School of Pathology, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa Background: Recognition of molecular abnormalities in acute myeloid leukaemia (AML) has improved our understanding of its biology. NPM1 and FLT3-ITD mutations are recurrent in AML and clinically significant. NPM1 mutations are associated with a favourable prognosis, while FLT3-ITD mutations are an independent poor prognostic factor in AML. Objective: This study described the prevalence and molecular characteristics of the NPM1 and FLT3-ITD mutations in a newly diagnosed AML patient cohort in central South Africa. Objective: This study described the prevalence and molecular characteristics of the NPM1 and FLT3-ITD mutations in a newly diagnosed AML patient cohort in central South Africa. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Affiliations: Affiliations: 1Department of Haematology and Cell Biology, School of Pathology, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa Methods: The study included 40 de novo AML patients. An NPM1 and FLT3-ITD multiplex polymerase chain reaction assay was optimised to screen patients for the respective mutations and were confirmed using Sanger sequencing. The prevalence of the NPM1 and FLT3-ITD mutations were determined, and mutation-specific characteristics were described in relation to patients’ demographic information and AML classifications. Methods: The study included 40 de novo AML patients. An NPM1 and FLT3-ITD multiplex polymerase chain reaction assay was optimised to screen patients for the respective mutations and were confirmed using Sanger sequencing. The prevalence of the NPM1 and FLT3-ITD mutations were determined, and mutation-specific characteristics were described in relation to patients’ demographic information and AML classifications. 2Department of Haematology and Cell Biology, Universitas Academic Unit, National Health Laboratory Services, Bloemfontein, South Africa Results: The patients’ median age was 38.5 years, with 77.5% (n = 31) of patients being self- proclaimed Black Africans. AML with recurrent genetic abnormalities was most prevalent (57.5%; n = 23), of which acute promyelocytic leukaemia (APL) was most common (40.0%; n = 16). None of the patients had the NPM1 mutation. FLT3-ITD was present in 37.5% (6/16) of APL patients and in one (20.0%) of five AML patients with a t(8;21) translocation. Most patients had an FLT3-ITD allele ratio of ≥ 50% and ITD lengths of > 39 bp. Conclusion: FLT3-ITD mutations were mainly found in APL cases at a similar prevalence as reported in the literature. High FLT3-ITD allele ratios and long ITD lengths predominated. No NPM1 mutations were detected. Open Access Introduction Over the past decade, the field of acute myeloid leukaemia (AML) diagnostics has shifted from a primarily clinicopathological assessment to an integrated approach, including morphology, immunophenotyping, cytogenetics, and molecular genetics.1,2 In an era of precision medicine, the use of molecular genetic data, in particular, has fundamentally shaped the diagnostic approach to patients with AML.3 Genetic profiling has led to more accurate diagnostic classification and improved risk stratification and identification of potential therapeutic targets.1 Acute myeloid leukaemia is a genetically heterogeneous disease.2 Recurrent structural chromosomal aberrations detected by conventional cytogenetics were previously considered the most relevant prognostic variable in addition to the patient’s age and performance status.2,3 Three main cytogenetic risk groups (low, intermediate, and high) were defined, with approximately 50% of patients having a normal karyotype and falling into the intermediate cytogenetic risk group.3,4 Copyright: © 2021. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. The recognition of additional molecular abnormalities, especially in cytogenetically normal AML (CN-AML), has helped to improve our understanding of the disease biology and better predict outcomes.3,4 Mutational analysis performed on diagnostic samples of AML patients enrolled in the Eastern Cooperative Oncology Group E1900 trial demonstrated somatic mutations in as many as 97.3% of these patients.5 The Cancer Genome Atlas Research Group6 has shown that, on average, patients with AML have 13 acquired mutations at the time of diagnosis across the entire genome. Two genes in which recurrent mutations, which often co-occur, with known clinical significance have been identified in patients with AML include the gene encoding nucleophosmin protein-1 (NPM1) and one of the signalling genes, FMS (Feline McDonough Sarcoma gene)-related tyrosine kinase 3 (FLT3).3,6 NPM1 mutations are associated with a favourable prognosis,3 while the FLT3- Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. http://www.ajlmonline.org http://www.ajlmonline.org Open Access Original Research Page 2 of 6 Page 2 of 6 internal tandem duplication (ITD) mutation is considered an independent poor prognostic factor in patients with AML.7 However, the co-occurrence of these two gene mutations modulates their prognostic impacts.3,7 Study population All adult patients (n = 40; coded FN1–FN40) that were diagnosed with de novo AML at the Universitas Academic Hospital in Bloemfontein, South Africa, during the study period (November 2018 – December 2019), and who were able to give written informed consent were included in this study. All AML patients were included regardless of cytogenetic findings. Patients’ demographic variables (age, sex, and self-proclaimed ethnic background) and disease- specific characteristics (AML classification according to the World Health Organization’s Classification of Tumours of Haemopoietic and Lymphoid Tissues, revised 4th edition16) were obtained from patients’ medical records. Based on the laboratory information records that were reviewed, none of the patients had any previous clonal myeloid disorder. Blood samples were obtained from each participant for this study during routine blood collection by the treating physician. Nucleophosmin is a nucleolar phosphoprotein that shuttles between the nucleus and the cytoplasm.3 Nucleophosmin protein-1 is thought to be involved in many cellular functions, including protein synthesis, DNA replication, and regulation of the cell cycle.3 The reported frequency of NPM1 mutations in patients with AML varies between 27% and 30% and is considered the most frequently identified molecular abnormality in CN-AML, occurring in 45% – 60% of cases.4,6,8,9 Four common mutations cause mutant NPM1 (one 4 bp duplication and three separate 4 bp insertions). These mutations are all restricted to exon 12, the region that encodes the C-terminus of NPM-1, and result in a frame shift.10 The FLT3 gene encodes a Class III receptor tyrosine kinase. Mutations in signalling genes such as FLT3, which lead to constitutive activation of the receptor tyrosine kinase, confer survival and proliferative advantage to cells.3,9 FMS-related tyrosine kinase 3 mutations occur in approximately one-third of patients with AML, and the ITD mutation is considered the second most common molecular abnormality in CN- AML (28% – 34% of cases).4,9 FMS-related tyrosine kinase 3-ITD mutations usually occur between exons 14 and 15 and can range between 3 bp and more than 400 bp in size.11 DNA extraction Genomic DNA was extracted from whole blood samples using the Wizard® Genomic DNA Purification Kit (Promega, Madison, Wisconsin, United States). DNA samples were quantified using the BioDrop™ µLITE instrument (BioDrop, Cambridge, United Kingdom). NPM1 and FLT3-ITD multiplex PCR optimisation The limited reports on the prevalence of the FLT3-ITD and NPM1 mutations in African AML populations indicate lower frequencies than reported internationally. In an Egyptian study that included 123 AML patients, FLT3-ITD and NPM1 mutations were detected in 17.9% and 19.5% of patients.12 Another study by Sofan et al. (2014) detected NPM1 mutations in 28% of CN-AML.13 Contrary to these findings, no NPM1 mutations were found in a study of 100 Sudanese AML patients.14 The frequency of NPM1 and FLT3-ITD mutations in a South African adult de novo AML cohort was 7.5% and 12%.15 Anonymised samples that were positive and negative for the NPM1 and FLT3-ITD mutations and formed part of an external quality control programme (generously donated by  the Department of Haematology at Charlotte Maxeke Johannesburg Academic Hospital, National Health Laboratory Service, Johannesburg, South Africa) were used to optimise the assay. Published primers, fluorescently labelled with hexadecimal colour and fluorescein amidites fluorophores17 were used in the assay (NPM1 Forward: 5’- GTT TCT TTT TTT TTT TTT CCA GGC TAT TCA AG- 3’; Reverse: 5’- HEX CAC GGT AGG GAA AGT TCT CAC TCT GC- 3’; and FLT3-ITD Forward: 5’FAM- AGCA ATT TAG GTA TGA AAG CCA GCTA- 3’; Reverse: 5’- CTT TCA GCA TTT TGA CGG CAA CC- 3’). Primers were evaluated for target-specificity using the Basic Local Alignment Search Tool (available at https://blast.ncbi.nlm.nih.gov/Blast.cgi) from the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, United States). This study investigated the presence and molecular characteristics of the NPM1 and FLT3-ITD mutations in a newly diagnosed AML population in central South Africa. The study further aimed to describe the NPM1 and FLT3-ITD mutations in relation to patient demographics and specific AML classifications. Sanger sequencing of NPM1 and FLT3-ITD amplicons NPM1 and FLT3-ITD control samples, as well as the amplicons from the 40 patient samples, were sequenced to confirm and validate the PCR results. Sequencing reactions for the NPM1 and FLT3-ITD mutations were prepared separately using primers as listed above (primers for sequencing were not fluorescently labelled). The PCR products were purified using the ExoSAP-IT® Express PCR Product Clean-up (Affymetrix, Santa Clara, California, United States) as per the manufacturer’s instructions. Sanger sequencing reactions were prepared using the BigDye™ Terminator version 3.1 kit (Applied Biosystems, Foster City, California, United States) as per the manufacturer’s instructions. Briefly, the sequencing reaction consisted of 2 µL Sequencing Reaction Mix (BigDye™ Terminator v3.1), 1  µL Sequencing Buffer (BigDye™ Terminator v3.1), 5 µL nuclease-free water, 1 µL forward primer (FLT3 or NPM1), 1  µL reverse primer (FLT3-ITD or NPM1) and 1 µL PCR product. The sequencing reaction was subjected to the following cycling conditions in a 2720 Thermal Cycler (Applied Biosystems, Foster City, California, United States): one cycle at 96 °C for 1 min, and 25 cycles at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Sequence reactions were cleaned up with the Zymo Research DNA Sequencing Clean- up Kit™ (Promega Madison, Wisconsin, United States) as per the manufacturer’s instructions. Sequence raw reads Ethical considerations gradient temperatures ranged between 65 °C and 70 °C. The subsequent PCR cycling conditions were one cycle at 95 °C for 10 min, 45 cycles of 95 °C for 20 s, 65 °C for 40 s, and 72 °C for 40 s, and a final step of one cycle at 72 °C for 32 min. Polymerase chain reaction products were subjected to capillary electrophoresis (3500 Genetic Analyser, Applied Biosystems, Foster City, California, United States). Briefly, 1  µL of each sample was mixed with 9.5 µL of Hi-Di™ Formamide (Applied Biosystems, Foster City, California, United States) and 0.5 µL GeneScan™ 600 LIZ™ size standard (Applied Biosystems, Foster City, California, United States). The optimal primer concentrations (5 μM) and DNA limit of detection were determined as part of the optimisation procedure. The NPM1 and FLT3-ITD multiplex PCR assay had a lower limit of detection of 0.8 ng/µL. Capillary electrophoretic results were analysed using GeneMapper version 6 software (Applied Biosystems, Foster City, California, United States). Ethical considerations A polymerase chain reaction (PCR) mixture was prepared with the GoTaq® DNA polymerase (Promega Madison, Wisconsin, United States) reagents. Components of the kit were manually mixed and the mixture consisted of 5 μl 5Х GoTaq Buffer, 2 μL of MgCl2, 0.5 μL of dNTPs, and 0.2 μL of GoTaq DNA polymerase (5 U/µL). Additionally, 1 μL of genomic DNA (100 ng/μL), 14.3 μL of nuclease-free water, and 1 μL each of the forward and reverse primers (5 μM) (for FLT3 and NPM1) were added to the PCR mixture. A temperature gradient experiment was used to determine the optimal PCR annealing temperature for the primers. The Approval for the study was obtained from the Health Sciences Research Ethics Committee from the University of  the Free State (study approval number: UFS- HSD2018/1174/2711) and the Free State Province Department of Health (study approval number: FS_201810_016). Written informed consent was obtained from all participants prior to sample collection. Samples were allocated study numbers to ensure patient confidentiality, and patient data were stored on password-protected devices, which were only accessible to the researchers. http://www.ajlmonline.org http://www.ajlmonline.org Open Access Page 3 of 6 Original Research Page 3 of 6 gradient temperatures ranged between 65 °C and 70 °C. The subsequent PCR cycling conditions were one cycle at 95 °C for 10 min, 45 cycles of 95 °C for 20 s, 65 °C for 40 s, and 72 °C for 40 s, and a final step of one cycle at 72 °C for 32 min. Polymerase chain reaction products were subjected to capillary electrophoresis (3500 Genetic Analyser, Applied Biosystems, Foster City, California, United States). Briefly, 1  µL of each sample was mixed with 9.5 µL of Hi-Di™ Formamide (Applied Biosystems, Foster City, California, United States) and 0.5 µL GeneScan™ 600 LIZ™ size standard (Applied Biosystems, Foster City, California, United States). The optimal primer concentrations (5 μM) and DNA limit of detection were determined as part of the optimisation procedure. The NPM1 and FLT3-ITD multiplex PCR assay had a lower limit of detection of 0.8 ng/µL. Capillary electrophoretic results were analysed using GeneMapper version 6 software (Applied Biosystems, Foster City, California, United States). calculated by determining the area under the curve for the mutant to wild-type alleles, expressed as a percentage. The respective positive and negative controls yielded the expected amplicon sizes upon evaluation of the capillary electrophoresis results (Figure 1). NPM1 and FLT3-ITD detection in acute myeloid leukaemia patients The optimised NPM1 and FLT3-ITD multiplex PCR was used to screen the 40 de novo AML patients for the respective mutations. Positive and negative controls, as well as a no- template control, were included in each subsequent run for quality control purposes. The NPM1 and FLT3-ITD wild- type amplicon sizes were expected to be 170 base pairs (bp) and 330 bp. The NPM1 positive samples were expected to have an amplicon at 170 bp for the wild-type and an additional amplicon at 174 bp. FMS-related tyrosine kinase 3-ITD positive samples were expected to have an amplicon at 330 bp and an additional amplicon larger than 330 bp. The allele ratio for the respective mutations in each patient was http://www.ajlmonline.org Open Access 50 000 25 000 RFU Fragment size in base pairs (bp) Fragment size in base pairs (bp) Fragment size in base pairs (bp) 0 0 120 240 360 480 600 a b c 0 120 240 360 480 600 50 000 25 000 0 RFU RFU 0 120 240 360 480 600 50 000 25 000 0 RFU, relative fluorescence units. Figure 1: Observed capillary electropherograms of the NPM1 and FLT3 control samples (February 2020, Bloemfontein, Free State, South Africa). (a) NPM1 positive control with an additional amplicon at 174 base pairs representing the 4 base pairs insertion, and wild-type FLT3-ITD at 330 base pairs. (b) FLT3-ITD positive control with an additional amplicon at 405 base pairs, representing the 75 base pairs internal tandem duplication and NPM1 wild-type at 170 base pairs. (c) NPM1 and FLT3-ITD negative control with expected amplicons at 170 base pairs and 330 base pairs. a a b RFU, relative fluorescence units. RFU, relative fluorescence units. Figure 1: Observed capillary electropherograms of the NPM1 and FLT3 control samples (February 2020, Bloemfontein, Free State, South Africa). (a) NPM1 positive control with an additional amplicon at 174 base pairs representing the 4 base pairs insertion, and wild-type FLT3-ITD at 330 base pairs. (b) FLT3-ITD positive control with an additional amplicon at 405 base pairs, representing the 75 base pairs internal tandem duplication and NPM1 wild-type at 170 base pairs. (c) NPM1 and FLT3-ITD negative control with expected amplicons at 170 base pairs and 330 base pairs. Data analysis AML, acute myeloid leukaemia; wt, wild-type; Δ, mutant; APL, acute promyelocytic leukaemia; ITD, internal tandem duplication; CN-AML, cytogenetically normal AML. AML, acute myeloid leukaemia; wt, wild-type; Δ, mutant; APL, acute promyelocytic leukaemia; ITD, internal tandem duplication; CN-AML, cytogenetically normal AML. Fragment analysis electropherograms (Figure 1) were generated using the GeneMapper v6 software (Applied Biosystems, Foster City, California, United States), and displayed the size of the alleles obtained for the target PCR fragments (Applied Biosystems, Foster City, California, United States). FLT3-ITD allelic ratios were determined by calculating the ratio of the area under the curve of the FLT3- ITD mutant allele to the FLT3 wild-type allele as displayed by GeneMapper. Table 2: Acute myeloid leukaemia patients positive for the FLT3-ITD mutation (August 2020, Bloemfontein, Free State, South Africa). Table 2: Acute myeloid leukaemia patients positive for the FLT3-ITD mutation (August 2020, Bloemfontein, Free State, South Africa). Patient AML classification Age (years) Sex Race ITD length (bp) Allele ratio (%) FN14 APL t(15; 17) 25 M ME 39 63.3 FN19 APL t(15; 17) 26 F B 27 48.4 FN20 APL t(15; 17) 19 M B 59 50.1 FN21 APL t(15; 17) 33 M B 44 94.9 FN26 APL t(15; 17) 59 F W 50 39.6 FN32 APL t(15; 17) 34 F B 19 79.5 FN36 t(8; 21) 20 M B 75 17.5 AML, acute myeloid leukaemia; APL, acute promyelocytic leukaemia; M, male; F, female; ME, mixed ethnicity; B, Black; W, White; ITD, internal tandem duplication; bp, base pairs. The sequence was analysed using the Sequencing Analysis Program version 5.3.1 (Applied Biosystems, Foster City, California, United States) and Chromas version 2.6.6 (Technelysium Pty Ltd, Brisbane, Australia). Tables were generated using Microsoft Word 2016 (Microsoft, Redmond, Washington, United States). Patients’ demographic data and AML classification were summarised in table format. Ethnicity, defined as either black, white or mixed race, was based on patients’ self-proclaimed identity and accordingly documented. AML, acute myeloid leukaemia; APL, acute promyelocytic leukaemia; M, male; F, female; ME, mixed ethnicity; B, Black; W, White; ITD, internal tandem duplication; bp, base pairs. duplication lengths of the FLT3 mutations observed in this study ranged between 19 bp and 75 bp, and the FLT3-ITD allele ratios ranged between 17.5% and 94.9%. Discussion Acute myeloid leukaemia is a genetically heterogeneous disease2 with numerous clinical and genetic factors influencing the final diagnosis. The recognition of additional molecular abnormalities, such as NPM1 and FLT3-ITD in AML, has ensured that the disease biology is better understood and has allowed for improved risk stratification to be included in the diagnostic algorithms.3,4 This is the first study to investigate the presence of NPM1 and FLT3-ITD in a central South African AML population. The NPM1 was absent in this study population, while the FLT3-ITD mutation predominated in the APL subtype. Furthermore, the majority of patients that were positive for the FLT3-ITD mutation had long ITD lengths and allelic ratios above 50%. NPM1 and FLT3-ITD detection in acute myeloid leukaemia patients Open Access http://www.ajlmonline.org Page 4 of 6 Page 4 of 6 Original Research Table 1: Classification of acute myeloid leukaemia patients’ NPM1 and FLT3-ITD genotypes (August 2020, Bloemfontein, Free State, South Africa). AML classification NPM1wt/ FLT3-ITDwt NPM1Δ/ FLT3-ITDΔ NPM1wt/ FLT3-ITDΔ NPM1Δ/ FLT3-ITDwt n % n % n % n % AML with recurrent genetic abnormalities 16 40.0 0 0 7 17.5 0 0  Translocation t(8; 21) 4 10.0 0 0 1 2.5 0 0  Inversion 16 1 2.5 0 0 0 0 0 0  Translocation t(9; 22) 1 2.5 0 0 0 0 0 0  APL translocation t(15;17) 10 25.0 0 0 6 15.0 0 0 AML with myelodysplasia- related changes 3 7.5 0 0 0 0 0 0 AML not otherwise specified (CN-AML) 14 35.0 0 0 0 0 0 0  Minimal differentiation 1 2.5 0 0 0 0 0 0  Without maturation 3 7.5 0 0 0 0 0 0  With maturation 6 15.0 0 0 0 0 0 0  Myelomonocytic 4 10.0 0 0 0 0 0 0 AML, acute myeloid leukaemia; wt, wild-type; Δ, mutant; APL, acute promyelocytic leukaemia; ITD, internal tandem duplication; CN-AML, cytogenetically normal AML. Table 1: Classification of acute myeloid leukaemia patients’ NPM1 and FLT3-ITD genotypes (August 2020, Bloemfontein, Free State, South Africa). were loaded onto the 3500 Genetic Analyser (Applied Biosystems, Foster City, California, United States). For comparison, sample sequences were aligned against NPM1 (GenBank accession: NG_016018.1) and FLT3-ITD (GenBank accession: NG_007066.1) reference sequences retrieved from the National Center for Biotechnology Information using the Local Pairwise Sequence Alignment Algorithm software (Available at https://embnet.vital-it.ch/software/ LALIGN_form.html). Sanger sequence analysis confirmed that the NPM1 positive control had a 4 bp insertion (bases: CATG), and the FLT3-ITD positive control had a 75 bp ITD. The mutation-negative controls were compared to the respective reference sequences, and were found to be 100% similar. Conclusion The FLT3-ITD mutation was mainly found in APL cases at a similar prevalence as reported in the literature. High FLT3- ITD allele ratios and long ITD lengths predominated. No NPM1 mutations were detected. The absence and lower frequency of NPM1 and FLT3-ITD mutations could possibly be attributed to the low median age at presentation and the majority of patients presenting with AML with recurrent genetic abnormalities. Acknowledgements The authors acknowledge the National Health Laboratory Service for funding the project, Ms Leandi du Plessis for technical assistance and Dr Daleen Struwig for assistance with the technical and editorial preparation of the manuscript. An interesting observation was the unusually high number of APL cases among our AML cohort, which, according to the revised 4th edition of the Classification of Tumours of Haemopoietic and Lymphoid Tissues, only accounts for 5% – 8% of AML cases.16 Notably, eight of our APL patients presented within a single month. Several studies, including a South African study, have alluded to the association between APL clustering and seasonality.25,26,27 With such a small study cohort, one can only speculate that seasonality may have contributed and warrants further investigation. Competing interests The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. Limitations This study was limited by a small sample population. In addition, the predominance of APL cases in our cohort may have contributed to a biased prevalence of the FLT3-ITD mutation. An association between phenotype and genotype was also not determined. Internal tandem duplication lengths of the FLT3 mutations observed in this study ranged between 19 bp and 75 bp and were found in the juxtamembrane domain of the gene. According to Liu et al.,23 longer ITD lengths (more than 39  bp) have been associated with a worse prognosis, the possible reason being that longer insertions in the juxtamembrane domain may cause more significant disruption of the domain’s auto-inhibitory function.23 In our study, 71.4% (5/7) of the patients with FLT3-ITD mutations had an ITD length of 39 bp or longer. Further investigations are needed to determine whether the high prevalence of FLT3-ITD mutations in our APL cohort confers a worse prognosis. This investigation would be justified considering that the majority (4 of 6) of APL patients with FLT3-ITD mutations had allele ratios above 50% and in other AML subgroups, allelic ratios above 50% are stratified as an adverse prognostic risk.24 Original Research considering that the frequency of NPM1 mutations increases proportionally with age.28 In addition, NPM1 mutations are more commonly associated with CN-AML,16 which accounted for only 35% (n = 14) of all cases in this study.4,9 The Cancer Genome Atlas Research Network reported that nearly 50% of all AML patients are CN-AML cases, and the observed prevalence of CN-AML in the current study was lower.6 The lower prevalence of CN-AML relative to AML with recurrent genetic abnormalities in our study, and, by association, the lower prevalence of these two mutations commonly linked with CN-AML, might be attributed to race, as Black patients were previously found to be more likely to have AML associated with cytogenetic abnormalities.18,29 self-proclaimed Black Africans, and there was no predominant disease presentation based on sex. Two other African studies found a similar age distribution and sex ratio.19,20 The FLT3-ITD mutation was only detected in patients with AML with recurrent genetic abnormalities, of which APL predominated. Compared to a reported frequency of FLT3- ITD mutations in APL of up to 40%,16 our results were not surprising. However, FLT3-ITD mutations in AML with a t(8;21) translocation, detected in one of our patients, is thought to be uncommon, occurring in less than 10% of AML cases.21 The reported frequency of FLT3-ITD mutations in CN-AML in African populations varies significantly from 11% in South African patients to up to 34.6% in an Egyptian cohort, which is more in line with data from high-income countries.9,15,22 The absence of FLT3-ITD in CN-AML patients in our study was unexpected, but could likely be attributed to the small study cohort. We recommend that the FLT3-ITD frequency should be investigated in a larger central South African CN-AML population. Results The AML patients (FN1 to FN40) were all successfully screened with the NPM1 and FLT3-ITD multiplex PCR assay. The AML patient cohort had a median age of 38.5 years (range 18–85 years) at presentation and the ratio of women to men was 1:1.2. The cohort included 31 black African patients, four white patients, and five patients of mixed ethnicity. Acute myeloid leukaemia subtypes in our patient cohort comprised of AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, and AML not otherwise specified (also referred to as CN-AML). Acute myeloid leukaemia with recurrent genetic abnormalities was most prevalent (57.5%; n = 23), of which acute promyelocytic leukaemia (APL) was most common (40.0%; n = 16). None of the patients had the NPM1 mutation. FMS-related tyrosine kinase 3-ITD was present in 37.5% (6/16) of APL patients and in one (20.0%) of five AML patients with a t(8; 21) translocation (Table 1). Patients with an FLT3-ITD mutation had a median age of 30.8 years at presentation, and the majority of patients were black Africans (n = 5/7) (Table 2). Internal tandem It was notable that the median age of our patient cohort was 38.5 years. The global median age at presentation for AML is 67 years18 and, in comparison, the current patient cohort presented with the disease at a considerably younger age. This finding was similar to a previous South African study, where adult AML patients presented with the disease at a median age of 41 years.15 Most patients in this study were http://www.ajlmonline.org Open Access Page 5 of 6 Page 5 of 6 Original Research References 19. Gmidène A, Sennana H, Wahchi I, et al. Cytogenetic profile of a large cohort of Tunisian de novo acute myeloid leukemia. Hematology. 2012;17(1):9–14. https:// doi.org/10.1179/102453312X13221316477417 1. Shanmugam V, Kim AS. Molecular versus morphological classifications of myeloproliferative neoplasms: You don’t know JAK! The Hematologist: ASH News and Reports [serial online]. 2019;16(1):12–13. Available from: https://pdfs. semanticscholar.org/5ac1/b04d4eed355331e2c6910a351efdc79b6c39.pdf?_ ga=2.24175993.2125433055.1594648374-906043641.1594648374 1. Shanmugam V, Kim AS. Molecular versus morphological classifications of myeloproliferative neoplasms: You don’t know JAK! The Hematologist: ASH News and Reports [serial online]. 2019;16(1):12–13. Available from: https://pdfs. semanticscholar.org/5ac1/b04d4eed355331e2c6910a351efdc79b6c39.pdf?_ ga=2.24175993.2125433055.1594648374-906043641.1594648374 20. Khoubila N, Bendari M, Hda N, et al. Cytogenetic profile of a representative cohort of young adults with de novo acute myéloblastic leukaemia in Morocco. Cancer Genet. 2019;238:1–9. https://doi.org/10.1016/j.cancergen.2019.06.010 2. Haferlach T, Schmidts I. The power and potential of integrated diagnostics in acute myeloid leukaemia. Br J Haematol. 2020;188(1):36–48. https://doi.org/10.1111/ bjh.16360 2. Haferlach T, Schmidts I. The power and potential of integrated diagnostics in acute myeloid leukaemia. Br J Haematol. 2020;188(1):36–48. https://doi.org/10.1111/ bjh.16360 21. Lin P, Chen L, Luthra R, Konoplev SN, Wang X, Medeiros J. Acute myeloid leukemia harboring t(8;21)(q22;q22): A heterogeneous disease with poor outcome in a subset of patients unrelated to secondary cytogenetic aberrations. Mod Pathol. 2008;21(8):1029–1036. https://doi.org/10.1038/modpathol.2008.92 3. Kansal R. Acute myeloid leukemia in the era of precision medicine: Recent advances in diagnostic classification and risk stratification. Cancer Biol Med. 2016;13(1):41–54. https://doi.org/10.20892/j.issn.2095-3941.2016.0001 22. Shamaa S, Laimon N, Aladle DA, et al. Prognostic implications of NPM1 mutations and FLT3 internal tandem duplications in Egyptian patients with cytogenetically normal acute myeloid leukemia. Hematology. 2014;19(1):22–30. https://doi.org/ 10.1179/1607845413Y.0000000085 4. Roloff GW, Griffiths EA. When to obtain genomic data in acute myeloid leukemia (AML) and which mutations matter. Blood Adv. 2018;2(21):3070–3080. https:// doi.org/10.1182/bloodadvances.2018020206 23. Liu SB, Dong HJ, Bao XB, et al. Impact of FLT3-ITD length on prognosis of acute myeloid leukemia. Haematologica. 2019;104(1):e9–e12. https://doi.org/10.3324/ haematol.2018.191809 5. Patel JP, Gönen M, Figueroa ME, et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med. 2012;366(12):1079–1089. https://doi.org/10.1056/NEJMoa1112304 24. Tallman MS, Wang ES, Altman JK, et al. Acute myeloid leukemia, version 3.2019, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2019;17(6):721–749. https://doi.org/10.6004/jnccn.2019.0028 6. Cancer Genome Atlas Research Network, Ley TJ, Miller C, et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368(22):2059–2074. https://doi.org/10.1056/NEJMoa1301689 25. Nunes Barroso KS, Lorand-Metze I, Pagnano KB, et al. Authors’ contributions J.F.K. was involved in the study design, assisted in performing the study, performed data analysis and contributed to writing the manuscript. A.-C.v.M. analysed clinical data and contributed to writing the manuscript. J.C. assisted in performing the study and critically evaluated the manuscript. A.d.K. was involved in the study design, supervised the study and critically evaluated the manuscript. All the authors approved the final version of the article prior to submission. Similar to a Sudanese study involving 100 AML patients,14 none of the patients in this study had an NPM1 mutation. However, NPM1 mutations were detected in 28.3% of Egyptian CN-AML patients and 7.5% of South African AML patients.13,15 The complete absence of the NPM1 mutation in this study could be attributed to the low median age at disease presentation in the study cohort, especially http://www.ajlmonline.org Open Access Page 6 of 6 Original Research Disclaimer 16. Arber DA, Brunning RD, Le Beau MM, et al. Acute myeloid leukaemia with recurrent genetic abnormalities. In: Swerdlow SH, Campo E, Harris NL, et al. editors. WHO classification of tumours of haematopoietic and lymphoid tissues. Revised 4th ed. Lyon: IARC, 2017; p. 129–170. The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. 17. Huang Q, Chen W, Gaal KK, et al. A rapid, one step assay for simultaneous detection of FLT3/ITD and NPM1 mutations in AML with normal cytogenetics Br J Haematol. 2008;142(3):489–492. https://doi.org/10.1111/j.1365-2141.2008.07205.x 18. National Cancer Institute. SEER Program. Cancer stat facts: Leukemia – Acute myeloid leukemia (AML) [homepage on the Internet]. Available from: https://seer. cancer.gov/statfacts/html/amyl.html Sources of support 12. El Gammal MM, Ebid GT, Madney YM, et al. Clinical effect of combined mutations in DNMT3A, FLT3-ITD, and NPM1 among Egyptian acute myeloid leukemia patients. Clin Lymphoma Myeloma Leuk. 2019;19(6):e281–e290. https://doi. org/10.1016/j.clml.2019.02.001 Funding was received from the National Health Laboratory Service Research Development Grant (GRANT004_94767). 13. Sofan MA, Elmasry SA, Salem DA, et al. NPM1 gene mutation in Egyptian patients with cytogenetically normal acute myeloid leukemia. Clin Lab. 2014;​60(11):​ 1813–1822. https://doi.org/10.7754/clin.lab.2014.140121 Data availability 14. Elzain E, Khalil HB. Frequency and prognostic value of NPM1 mutations in Sudanese acute myeloid leukemia patients. bioRxiv. 2020 (pre-print). https://doi. org/10.1101/2020.05.31.126334 Data are available from the corresponding author upon reasonable request. 15. Marshall RC, Tlagadi A, Bronze M, Kana V, Wiggil TM, Carmona SC. Lower frequency of NPM1 and FLT3-ITD mutations in a South African de novo AML cohort. Int J Lab Hematol. 2014;36(6):656–664. https://doi.org/10.1111/ ijlh.12204 References Evaluation of seasonality in the incidence of promyelocytic leukemia in Brazil. Blood. 2013;122 (21):5005. https://doi.org/10.1182/blood.V122.21.5005.5005 7. Chen F, Sun J, Yin C, et al. Impact of FLT3-ITD allele ratio and ITD length on therapeutic outcome in cytogenetically normal AML patients without NPM1 mutation. Bone Marrow Transplant. 2020;55(4):740–748. https://doi. org/10.1038/s41409-019-0721-z 26. Mohammed AS, Ali TH, Alwan AF. Seasonality in acute promyelocytic leukemia: Fact or myth? Iraqi J Hematol. 2020;9(2):113. https://doi.org/10.4103/ijh.ijh_27_20 8. Bhattacharyya J, Nath S, Saikia KK, et al. Prevalence and clinical significance of FLT3 and NPM1 mutations in acute myeloid leukaemia patients of Assam, India. Indian J Hematol Blood Transfus. 2018;34(1):32–42. https://doi.org/10.1007/ s12288-017-0821-0 27. Gilbert RD, Karabus CD, Mills AE. Acute promyelocytic leukemia. A childhood cluster. Cancer. 1987;59(5):933–935. https://doi.org/10.1002/1097- 0142(19870301)59:5%3C933::AID-CNCR2820590513%3E3.0.CO;2-R 9. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373(12):1136–1152. https://doi.org/10.1056/NEJMra1406184 28. Verhaak RG, Goudswaard CS, Van Putten W, et al. Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): Association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic expression significance. Blood. 2005;106(12):3747–3754. https://doi.org/10.1182/blood-2005-05-2168 10. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med. 2005;​ 352(3):254–266. https://doi.org/10.1056/NEJMoa041974 29. Sekeres MA, Peterson B, Dodge RK, et al. Differences in prognostic factors and outcomes in African Americans and whites with acute myeloid leukemia. Blood. 2004;103(11):4036–4042. https://doi.org/10.1182/blood-2003-09-3118 29. Sekeres MA, Peterson B, Dodge RK, et al. Differences in prognostic factors and outcomes in African Americans and whites with acute myeloid leukemia. Blood. 2004;103(11):4036–4042. https://doi.org/10.1182/blood-2003-09-3118 11. Bacher U, Kohlmann A, Haferlach C, Haferlach T. Gene expression profiling in acute myeloid leukaemia (AML). Best Pract Res Clin Haematol. 2009;22(2): 169–180. https://doi.org/10.1016/j.beha.2009.04.003 http://www.ajlmonline.org
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https://pure.eur.nl/ws/files/55041714/bmjopen_2020_044544.pdf
English
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Development of a core outcome set for congenital pulmonary airway malformations: study protocol of an international Delphi survey
BMJ open
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To cite: Hermelijn S, Kersten C, Mullassery D, et al. Development of a core outcome set for congenital pulmonary airway malformations: study protocol of an international Delphi survey. BMJ Open 2021;11:e044544. doi:10.1136/ bmjopen-2020-044544 Protocol Protocol Open access Development of a core outcome set for congenital pulmonary airway malformations: study protocol of an international Delphi survey Strength and limitations of this study After the core outcome set has been defined we intend to design an international randomised controlled trial: the COllaborative Neonatal NEtwork for the first CPAM Trial, which will be aimed at determining the optimal management of patients with asymptomatic CPAM ► ►Existing comprehensive literature reviews shall be used to inform initial outcome parameters instead of a systematic literature search as no randomised trials and very few prospective studies have yet been published regarding the outcome of patients with CPAM. ► ►Parents’ and patients’ views are not included in the protocol as the final core outcome set is specifical- ly intended for patients with asymptomatic CPAM, however their input will play an important role when designing future studies. ► ►Parents’ and patients’ views are not included in the protocol as the final core outcome set is specifical- ly intended for patients with asymptomatic CPAM, however their input will play an important role when designing future studies. Ethics and dissemination  Electronic informed consent will be obtained from all participants. Ethical approval is not required. After the core outcome set has been defined, we intend to design an international randomised controlled trial: the COllaborative Neonatal NEtwork for the first CPAM Trial, which will be aimed at determining the optimal management of patients with asymptomatic CPAM. For numbered affiliations see end of article. Correspondence to Dr Marco Schnater; ​j.​schnater@​erasmusmc.​nl © Author(s) (or their employer(s)) 2021. Re-­use permitted under CC BY. Published by BMJ. born with a CPAM are asymptomatic but others may show symptoms such as neonatal respiratory distress, persistent cough or recurrent lung infections in the first years of life.3 A worldwide lack of consensus exists on the optimal management and follow-­up of infants with an asymptomatic CPAM.4 5 Prospective studies on postnatal management are lacking, and cohort studies vary widely in the outcome measures they report.2 6 Development of a core outcome set for congenital pulmonary airway malformations: study protocol of an international Delphi survey Sergei Hermelijn  ‍ ‍ ,1 Casper Kersten,1 Dhanya Mullassery,2 Nagarajan Muthialu,2 Nazan Cobanoglu,3 Silvia Gartner,4 Pietro Bagolan,5 Carmen Mesas Burgos,6 Alberto Sgro,7 Stijn Heyman,8 Holger Till,9 Janne Suominen,10 Maarten Schurink,11 Liesbeth Desender,12 Paul Losty,13 Kjetil Ertresvag,14 Harm A W M Tiddens,15 Rene M H Wijnen,1 Marco Schnater,1 On behalf of the CONNECT study consortium COS development group, On behalf of the CONNECT study consortium COS development group ► ►Prepublication history for this paper is available online. To view these files, please visit the journal online (http://​dx.​doi.​ org/​10.​1136/​bmjopen-​2020-​ 044544). Strength and limitations of this study Introduction  A worldwide lack of consensus exists on the optimal management of asymptomatic congenital pulmonary airway malformation (CPAM) even though the incidence is increasing. Either a surgical resection is performed or a wait-­and-­see policy is employed, depending on the treating physician. Management is largely based on expert opinion and scientific evidence is scarce. Wide variations in outcome measures are seen between studies making comparison difficult thus highlighting the lack of universal consensus in outcome measures as well. We aim to define a core outcome set which will include the most important core outcome parameters for paediatric patients with an asymptomatic CPAM. ► ►The core outcome set is a disease-­specific collec- tion of the most important outcomes that will be es- tablished by consensus between key stakeholders. Participants will be an international group of special- ists with experience in the treatment of patients with congenital pulmonary airway malformation (CPAM), which will result in a universally approved synthesis of expert opinion. ► ►Prepublication history for this paper is available online. To view these files, please visit the journal online (http://​dx.​doi.​ org/​10.​1136/​bmjopen-​2020-​ 044544). ► ►This protocol describes an international online Delphi survey that should identify the most import- ant core outcome parameters including optimal tim- ing and modality to monitor paediatric patients with an asymptomatic CPAM. Received 09 September 2020 Revised 17 March 2021 Accepted 23 March 2021 Methods and analysis  This study will include a critical appraisal of the current literature followed by a three-­ stage Delphi process with two stakeholder groups. One surgical group including paediatric as well as thoracic surgeons, and a non-­surgeon group including paediatric pulmonologists, intensive care and neonatal specialists. All participants will score outcome parameters according to their level of importance and the most important parameters will be determined by consensus. Methods and analysis  This study will include a critical appraisal of the current literature followed by a three-­ stage Delphi process with two stakeholder groups. One surgical group including paediatric as well as thoracic surgeons, and a non-­surgeon group including paediatric pulmonologists, intensive care and neonatal specialists. All participants will score outcome parameters according to their level of importance and the most important parameters will be determined by consensus. Ethics and dissemination  Electronic informed consent will be obtained from all participants. Ethical approval is not required. Received 09 September 2020 Revised 17 March 2021 Accepted 23 March 2021 Study design Study design The COS will be developed in an online, three-­round, Delphi process, preceded by an appraisal of previously published literature. METHODS The COS development will follow the Core Outcome Set-­ STAndards for Development Recommendations12 and the Core Outcome Measures in Effectiveness Trials (COMET) handbook.11 This COS development study was registered with the COMET Initiative in May 2020 (http://www.​comet-​initiative.​org/​ studies/​details/​1570). The COS development will follow the Core Outcome Set-­ STAndards for Development Stakeholders and recruitment Paediatric surgeons and thoracic surgeons are the health- care professionals who are most frequently involved in the operative management of patients with CPAM. Initial consultation or follow-­up is variably done by paediatric surgeons, maternal-­fetal medicine specialists, neonatol- ogists, paediatricians or paediatric pulmonologists. We therefore decided to form two stakeholder groups: (1) surgeons (ie, paediatric and thoracic surgeons) and (2) non-­surgeons (eg, maternal-­fetal medicine special- ists, paediatricians, paediatric pulmonologists and neonatologists). Recommendations12 and the Core Outcome Measures in Effectiveness Trials (COMET) handbook.11 This COS development study was registered with the COMET Initiative in May 2020 (http://www.​comet-​initiative.​org/​ studies/​details/​1570). Open access up until the age of 18 years and will not include fetal outcome. Outcome parameters as well as their measure- ment instruments, and age at assessment will be included. An asymptomatic CPAM is either surgically resected or a wait-­and-­see policy is employed depending on physi- cian preference or local guidelines. Either way, cases are ideally discussed in a multidisciplinary team in which parental preferences are taken into account as well. To date, no definitive evidence exists on the optimal manage- ment, long-­term outcomes are still unknown and factors for predicting symptoms are still being investigated.6 The arguments for a wait-­and-­see policy are that the malfor- mation is originally benign, potentially regresses and in most cases remains asymptomatic.7 The arguments for surgical management include the risk of recurrent lung infections (which could make subsequent surgery more difficult), risk of acute respiratory distress, potential malignant transformation, parental anxiety and allowing for compensatory lung growth.8 9 Literature review A systematic literature review is recommended to yield an initial outcome set for the first round in the Delphi process.11 To date, no randomised studies have yet been done and other studies examining the management of asymptomatic CPAMs report a large variety in outcome parameters.6 A literature review will be done by the study management coordinators (SH, CK) informed by two literature reviews, previously published in a special issue of a paediatric surgery scientific journal, each covering opposing thoughts on the arguments for surgical manage- ment8 or a wait-­and-­see policy.7 In addition, we will scru- tinise a recent systematic review and meta-­analysis, which covers the risks associated with either surgical resection or a wait-­and-­see policy of asymptomatic CPAM.3 Existing definitions, measurement tools and common measure- ment time-­points for outcomes will be extracted, and formatted into appropriately phrased questions for use in the first round of the Delphi process. The indepen- dent coordinators will be blinded for participant identity during the process by means of a unique identification number and will ensure the Delphi process is performed according to the protocol. Consensus needs to be reached on outcome measures and their timing that can be applied in international studies aimed at identifying the optimal management of asymptomatic CPAM. A core outcome set (COS) is a disease-­specific collection of outcomes that have been identified by consensus between key stakeholders as being the most important in determining success of a treatment.10 Such consensus is often reached through a Delphi method in which stakeholders anonymously rate outcome measures according to their importance, in one or multiple rounds.11 We aim to develop a COS for patients with CPAM using the Delphi method as a tool for reaching consensus and present the study protocol for this. INTRODUCTION Congenital pulmonary airway malforma- tion (CPAM), formerly known as congenital cystic adenomatoid malformation, is the most common congenital lung abnormality.1 The incidence of CPAM has increased up to 4 per 10 000 births over the last years.2 Most infants Correspondence to Dr Marco Schnater; ​j.​schnater@​erasmusmc.​nl Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 1 Open access Delphi round 3 analysis and final COS development Delphi round 3 analysis and final COS development All outcome parameters meeting the criteria for consensus of inclusion by all participants will be included in the final COS. All other outcome parameters will be excluded. To achieve a COS that is feasible for clinical use in trials, we aim to include a maximum of 10 outcome parameters in the final COS. If the number of outcome parameters meeting the criteria for consensus of inclusion greatly exceeds this maximum number, we will only include the 10 outcomes with the highest level of consensus for the Delphi round 2 All participants who completed the first round will auto- matically be invited to participate in round 2. Per stakeholder group, the median scores assigned in the first round will be made known. This will allow partic- ipants to consider the views of the other participants in the stakeholder group. They will be invited to look at all items again and consider adjusting their own scores. Furthermore, they will be asked to score the newly added outcome parameters suggested in the first round. Sample size There is no consensus on the optimal sample size for a Delphi study11; recruitment will therefore be based on the prospective study for which it is primarily intended (CONNECT). For the CONNECT trial, we aim to include at least 12 international centres, and therefore set the minimum of participants in each stakeholder group in the final round at 12. To reduce bias, no more than two participants from a single centre can participate in a stakeholder group. To minimise attrition bias in consec- utive rounds, we aim to achieve that 75% of participants complete a round.11 13 Therefore, the minimum number of participants in each stakeholder group for round 1 will be 21, and 16 for round 2. We believe that these minimum numbers constitute a representative sample, consid- ering the rarity of the disease and the limited number of professionals with experience in managing this disease. Previous Delphi COS studies in paediatric surgery used similar numbers for investigating a more common disease such as appendicitis.14 15 Participants are invited to suggest additional outcome parameters stating: (1) the outcome parameter, (2) the measurement instrument and (3) the age at assessment. These additional outcome parameters will be scored in the second round. Delphi round 3 All participants who completed the first and second rounds will be invited to participate in the third round. The median score of their own stakeholder group and the score of the other stakeholder group will be presented to participants. This will allow participants to consider the views of the other stakeholder group before rescoring the outcomes. They will be invited to look at all remaining items again and consider adjusting their own scores. In addition, participants will be asked to identify a single outcome parameter, which is the most important for determining the treatment choice in patients with asymp- tomatic CPAM according to them. Delphi round 1 analysis Outcome parameters will be analysed separately for each stakeholder group (surgeons and non-­surgeons) and all parameters will be included in the second round of the Delphi process. The additional outcomes provided by participants will be reviewed to confirm they represent new outcomes. If confirmed, the item in question will be included in the second round as well. Delphi study Consensus All outcome parameters meeting consensus criteria for exclusion by all participants will be excluded from the third round. All other parameters will be included in the third round. Participants will be asked to score each outcome param- eter using the Grading of Recommendations, Assessment, Development and Evaluations scale.16 The 9-­point Likert scale will label 1–3 as ‘not important’, 4–6 as ‘important but not critical’ and 7–9 as ‘critical’. Consensus for inclu- sion is reached if ≥70% of participants rate the outcome parameter 7–9 and <15% rate it 1–3. Consensus for exclu- sion is reached if >70% participants rate the outcome 1–3 and <15% rate it 7–9. Outcomes not meeting these defi- nitions will be classified as ‘no consensus’.11 Open access “How important would you rate the following outcome parameter including measurement instrument and age in determining the best management of asymptomatic CPAM patients?” “How important would you rate the following outcome parameter including measurement instrument and age in determining the best management of asymptomatic CPAM patients?” Timeline Participants will be asked to complete each round of the Delphi process within 4 weeks. A weekly reminder email will be sent to those who have not yet completed the survey. Those failing to complete the questionnaire within the allocated 4 weeks will be excluded from next rounds. The deadline shall be extended if the projected minimum sample size has not been reached and those failing to complete the questionnaire shall be approached individually. Attrition bias Attrition bias will be assessed separately for each round of the Delphi process, and separately in each stakeholder group. In each group, the median score of every outcome will be compared using the Wilcoxon rank-­sum test, between those only completing the previous round and those completing the consecutive round as well.11 Delphi round 2 analysis Scope This protocol describes the Delphi method which shall define a COS for all asymptomatic patients who are either prenatally or postnatally diagnosed with a CPAM. Asymp- tomatic patients are defined as those who have no need for prolonged respiratory support (>24 hours) including supplemental oxygen and ventilation. The COS will include the most important outcome measures for patients with CPAM, regardless of the management. The COS may be used as a guideline for clinical follow-­up or in future research studies. After the COS has been defined, we intend to design a randomised controlled trial: the COllaborative Neonatal NEtwork for the first European CPAM Trial (CONNECT). This trial will be performed by the CONNECT study consortium and aims to iden- tify the optimal management of patients with asymptom- atic CPAM. The COS is intended for paediatric patients We will recruit study participants through an existing international network of paediatric surgeons and pulmo- nologists who have expressed interest in collaborating on the CONNECT trial. We will inform potential participants on the aims and procedures of the Delphi process and encourage them to enrol other specialists involved in the care of patients with CPAM in their own centres. Prior to the first round, those who have been found willing to participate will be sent an email explaining the aims and procedures of the Delphi process, and emphasising the importance of finishing each round within the allocated time. 2 Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 ORCID iD ORCID iD Sergei Hermelijn http://​orcid.​org/​0000-​0002-​7296-​9932 Ethics and dissemination Electronic informed consent will be obtained from all participants. Prior ethical approval for the Delphi study is not required. The final COS will be published in an international peer-­reviewed scientific journal and on the COMET Initiative website (https://www.​comet-​initiative.​ org/). Contributors  All authors contributed to the design of this protocol. SH, DM, NC, SG, PB, CMB, AS, SH, JS, MS, LD, KE, HT, RW and JMS initiated the project. The protocol was drafted by SH and CK. The protocol was critically reviewed by DM, NM, NC, SG, PB, CMB, AS, SH, HT, JS, MS, LD, PL, KE, HAWMT, RMHW and JMS. All authors contributed to the manuscript and read and approved the final manuscript. The CONNECT study consortium COS development group consists of all participants of the Delphi process. They have all read, refined and approved the final manuscript. Funding  The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-­for-­profit sectors. Open access Open access COS and report those excluded in this stage. The level of consensus will be determined by the median score of each outcome parameter in round 3. C. Wallis (Great Ormond Street Hospital, London, UK); D. Cox (Children’s Health Ireland at Crumlin, Dublin, Ireland); P. Bagolan, F. Morini (Bambino Gesu Pediatric Hospital, Rome, Italy); C. Mesas Burgos, P. Conner, E. Caffrey Osvald, C. Bitkover (Karolinska Institutet, Stockholm, Sweden); H. Decaluwe, M. Proesmans, M. Boon, J. Deprest (University Hospital Leuven, Leuven, Belgium); S. Gartner, A. Lain (Hospital Universitari Vall d Hebron, Barcelona, Spain); P.D. Losty, I. Sinha (Alder Hey Children’s Hospital, School Of Health and Life Science, University of Liverpool, Liverpool, UK); I. Yardley (Evelina London Children’s Hospital, London, UK); M. Singh (Birmingham Children’s Hospital, Birmingham, UK); L. Wessel, K. Zahn, T. Schaible (University Hospital Mannheim, Mannheim, Germany); N. Qvist (Odense Univeristy Hospital, Odense, Denmark); M. Zampoli (Red Cross Children’s Hospital, Cape Town, South Africa); G. Aksnes (Oslo University Hospital, Oslo, Norway); C.K. van der Ent, K.M. Winter-­de Groot (University Medical Centre Utrecht, Utrecht, The Netherlands); R. Peters (Royal Manchester Children’s Hospital, Manchester, UK); E. Hannon (Leeds Children’s Hospital, Leeds, UK); Q. Jöbsis, M. Bannier (Maastricht University Medical Centre, Maastricht, The Netherlands). The final COS will be a collection of the most important outcome parameters in patients with CPAM. The final COS will be annotated according to the outcome taxonomy, which was constructed to maximise future data harmonisation. Additionally, the final COS will be divided into the four core areas of the OMERACT filter: death, life impact, pathophysiological manifestations and resource use.10 REFERENCES 10Pediatric Surgery, University of Helsinki Children's Hospital, Helsinki, Finland 1 EUROCAT. Prevalence tables. 2007-2018. Available: http://www.​ eurocat-​network.​eu/​ACCESSPREVALENCEDATA/​PrevalenceTables [Accessed Jan 2019]. 11Pediatric Surgery, Radboud University Medical Centre Amalia Children's Hospital, Nijmegen, The Netherlands 12Pediatric Surgery, Ghent University Faculty of Medicine and Health Sciences, Gent, Belgium 2 Stocker LJ, Wellesley DG, Stanton MP, et al. The increasing incidence of foetal echogenic congenital lung malformations: an observational study. Prenat Diagn 2015;35:148–53. 2 Stocker LJ, Wellesley DG, Stanton MP, et al. The increasing incidence of foetal echogenic congenital lung malformations: an observational study. Prenat Diagn 2015;35:148–53. 13Pediatric Surgery, University of Liverpool, Liverpool, UK 14Pediatric Surgery, Oslo University Hospital, Oslo, Norway 3 Stanton M, Njere I, Ade-­Ajayi N, et al. Systematic review and meta-­ analysis of the postnatal management of congenital cystic lung lesions. J Pediatr Surg 2009;44:1027–33. 15Pediatric Pulmonology, Erasmus MC Sophia Children's Hospital, Rotterdam, The Netherlands 15Pediatric Pulmonology, Erasmus MC Sophia Children's Hospital, Rotterdam, The Netherlands 4 Lo AY-­S, Jones S. Lack of consensus among Canadian pediatric surgeons regarding the management of congenital cystic adenomatoid malformation of the lung. J Pediatr Surg 2008;43:797–9. Collaborators  The following people form the CONNECT study consortium COS development group: S.M. Hermelijn, C.M. Kersten, J.M. Schnater, R.M.H. Wijnen, H.A.W.M. Tiddens, S.C.M. Cochius-­den Otter (Sophia Children’s Hospital, Rotterdam, The Netherlands); J. Suominen, M. Pakarinen, L. Martelius (Helsinki Children’s Hospital, Helsinki, Finland); S. Heyman, D. Vervloessem (ZNA-­GZA Paola Children’s Hospital, Antwerp, Belgium); H. Steyaert (Queen Fabiola Children’s University Hospital, Brussels, Belgium); A. Sgro, P. Gamba (University Hospital Padua, Padua, Italy); M. Schurink, S. van der Heide, J. Roukema (Amalia Children’s Hospital, Nijmegen, The Netherlands); N. Rikkers-­Mutsaerts (Leiden University Medical Centre, Leiden, The Netherlands); S. Terheggen-­Lagro, S. de Beer, E. Haarman (Amsterdam University Medical Centre, Amsterdam, The Netherlands); H. Till, G. Singer (Medical University of Graz, Graz, Austria); M. Metzelder, P. Sezen (Medizinische Universität Wien, Vienna, Austria); L. Desender, H. Schaballie (Ghent University Hospital, Ghent, Belgium); N. Cobanoglu, G. Gollu (Ankara University, School of Medicine, Ankara, Turkey); M. Stanton (University Hospital Southampton, Southampton, UK); A. Bonnard (Hopital Universitaire Robert Debré, Paris, France); R. Sfeir (Hopital Jeanne de Flandre, Lille, France); N. Muthialu, D. Mullassery, 5 Morini F, Zani A, Conforti A, et al. Current Management of Congenital Pulmonary Airway Malformations: A “European Pediatric Surgeons’ Association” Survey. Eur J Pediatr Surg 2018;28:001–5. 5 Morini F, Zani A, Conforti A, et al. Delphi round 1 The three-­round Delphi process shall be performed using ‘Welphi’, an online data system specifically devel- oped for this use.17 All participants shall be approached simultaneously and asked to rate each of the previously identified outcome parameters on importance as follows: 3 Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 Provenance and peer review  Not commissioned; externally peer reviewed. Author affiliations 1Pediatric Surgery, Erasmus MC Sophia Children's Hospital, Rotterdam, The Netherlands 2Pediatric Surgery, Great Ormond Street Hospital for Children, London, UK 3Pediatric Pulmonology, Ankara University Faculty of Medicine, Ankara, Turkey 4Pediatric Pulmonology, Hospital Universitari Vall d'Hebron, Barcelona, Spain 5Department of Medical and Surgical Neonatology, Ospedale Pediatrico Bambino Gesu, Roma, Italy 6Pediatric Surgery, Karolinska Institutet, Stockholm, Sweden 7Pediatric Surgery, Padua University Hospital, Padova, Italy 8Pediatric Surgery, ZNA-­GZA Paola Children's Hospital, Antwerp, Belgium 9Pediatric Surgery, Medical University of Graz, Graz, Austria 10Pediatric Surgery, University of Helsinki Children's Hospital, Helsinki, Finland 11Pediatric Surgery, Radboud University Medical Centre Amalia Children's Hospital, Nijmegen, The Netherlands 12Pediatric Surgery, Ghent University Faculty of Medicine and Health Sciences, Gent Belgium 13Pediatric Surgery, University of Liverpool, Liverpool, UK 14Pediatric Surgery, Oslo University Hospital, Oslo, Norway 15Pediatric Pulmonology, Erasmus MC Sophia Children's Hospital, Rotterdam, The Netherlands Data collection and confidentiality Participants will complete questionnaires using the ‘Welphi’ survey tool.17 Anonymised data will be stored on a secure online server and will be managed according to the European General Data Protection Regulation.18 Competing interests  None declared. Patient and public involvement  Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. Patient consent for publication  Not required. Patient consent for publication  Not required. Provenance and peer review  Not commissioned; externally peer reviewed. Provenance and peer review  Not commissioned; externally peer reviewed. Author affiliations 1 Open access  This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://​creativecommons.​org/ licenses/​by/​4.​0/. ORCID iD ORCID iD Sergei Hermelijn http://​orcid.​org/​0000-​0002-​7296-​9932 REFERENCES Current Management of Congenital Pulmonary Airway Malformations: A “European Pediatric Surgeons’ Association” Survey. Eur J Pediatr Surg 2018;28:001–5. 6 Wong KKY, Flake AW, Tibboel D, et al. Congenital pulmonary airway malformation: advances and controversies. Lancet Child Adolesc Health 2018;2:290–7. 7 Stanton M. The argument for a non-­operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:183–6. 7 Stanton M. The argument for a non-­operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:183–6. 7 Stanton M. The argument for a non-­operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:183–6. 8 Singh R, Davenport M. The argument for operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:187–95. 7 Stanton M. The argument for a non-­operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:183–6. 8 Singh R, Davenport M. The argument for operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:187–95. y p g g 8 Singh R, Davenport M. The argument for operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:187–95. y p g g 8 Singh R, Davenport M. The argument for operative approach to asymptomatic lung lesions. Semin Pediatr Surg 2015;24:187–95. 9 Hermelijn SM, Wolf JL, Dorine den Toom T, et al. Early KRAS oncogenic driver mutations in nonmucinous tissue of congenital pulmonary airway malformations as an indicator of potential malignant behavior. Hum Pathol 2020;103:95–106. 9 Hermelijn SM, Wolf JL, Dorine den Toom T, et al. Early KRAS oncogenic driver mutations in nonmucinous tissue of congenital pulmonary airway malformations as an indicator of potential malignant behavior. Hum Pathol 2020;103:95–106. g 10 Boers M, Kirwan JR, Gossec L, et al. How to choose core outcome measurement sets for clinical trials: OMERACT 11 approves filter 2.0. J Rheumatol 2014;41:1025–30. Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 4 Open access study protocol for an international Delphi survey. BMJ Open 2019;9:e028861. study protocol for an international Delphi survey. BMJ Open 2019;9:e028861. 11 Williamson PR, Altman DG, Bagley H, et al. The comet Handbook: version 1.0. Trials 2017;18:280. 16 Guyatt GH, Oxman AD, Kunz R, et al. Grade guidelines: 2. framing the question and deciding on important outcomes. J Clin Epidemiol 2011;64:395–400. 12 Kirkham JJ, Davis K, Altman DG, et al. Core outcome Set-­STAndards for development: the COS-­STAD recommendations. PLoS Med 2017;14:e1002447. 17 Welphi. Welphi online survey platform, 2020. Available: www.​welphi.​ com 13 Bruce I, Harman N, Williamson P, et al. 18 (GDPR) Gdpr. Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing directive 95/46/EC (General data protection regulation), OJ 2016 L 119/1, 2018. 17 Welphi. Welphi online survey platform, 2020. Available: www.​welphi.​ com REFERENCES The management of otitis media with effusion in children with cleft palate (mOMEnt): a feasibility study and economic evaluation. Health Technol Assess 2015;19:1–374. 17 Welphi. Welphi online survey platform, 2020. Available: www.​welphi.​ com 18 (GDPR) Gdpr. Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing directive 95/46/EC (General data protection regulation), OJ 2016 L 119/1, 2018. 18 (GDPR) Gdpr. Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing directive 95/46/EC (General data protection regulation), OJ 2016 L 119/1, 2018. 14 Sherratt FC, Eaton S, Walker E, et al. Development of a core outcome set to determine the overall treatment success of acute uncomplicated appendicitis in children: a study protocol. BMJ Paediatr Open 2017;1:e000151. 15 Knaapen M, Hall NJ, van der Lee JH, et al. Establishing a core outcome set for treatment of uncomplicated appendicitis in children: 15 Knaapen M, Hall NJ, van der Lee JH, et al. Establishing a core outcome set for treatment of uncomplicated appendicitis in children: 5 Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544
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Phospholipid analysis in sera of horses with allergic dermatitis and in matched healthy controls
Lipids in health and disease
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* Correspondence: raija.hallamaa@elisanet.fi 1Veterinary Clinic, Nummela, Finland 2University of Helsinki, Faculty of Veterinary Medicine, Helsinki, Finland Full list of author information is available at the end of the article © 2016 Hallamaa and Batchu. 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. Phospholipid analysis in sera of horses with allergic dermatitis and in matched healthy controls Raija Hallamaa1,2* and Krishna Batchu3 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 DOI 10.1186/s12944-016-0209-4 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 DOI 10.1186/s12944-016-0209-4 Open Access Results The main phospholipid classes analysed were phosphat- idylcholine (PC), phosphatidylethanolamine (PE), phos- phatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM). Of these, all except for PS were detected across all the samples. Relative contents of the major phospholipid classes before (stage 0) and after the 4-week therapy (stage 4) of eczema horses are listed in Table 1. In addition to the major phospholipid classes, all the samples also contained PA 36:2 and lysoPC species of 16:0, 18:2 and 18:0. Insect bite hypersensitivity (Fig. 1a) is the most com- mon allergic skin disease of the horse [24] displaying many features similar to atopic dermatitis (AD) of humans [25, 26]. Intense and recurrent pruritus, IgE- mediated responses, perivascular mast cell, eosinophil and T-lymphocyte infiltrations are typical findings of both disorders [25, 27–32]. In addition, horses with in- sect hypersensitivity show IgE-mediated sensitization also to various environmental allergens [27], as do patients with atopic dermatitis [31, 32]. However, disturbed skin barrier function due to inherited defects of filaggrin has not been studied on horses [26]. Treatment of equine IBH is challenging, since total isolation from biting insects is impossible. Antihistamines or allergen-specific immuno- therapy have not been beneficial [33, 34]. Therefore there is a need for comprehensive understanding of this harmful disease. In our previous study [35], we analysed phospho- lipid contents of autoserum preparations that have been recently used in the therapy of insect hypersensitivity in horses. The idea is to collect remnant lipid particles in these preparations after serial washings [35]. Concentra- tions of phosphatidylcholine (PC) and sphingomyelin (SM) were significantly more abundant in the preparations Phosphatidylcholine was the most abundant of all the phospholipid classes detected (Table 1). Its major mo- lecular species in both eczema and control horses at stages 0 and 4 are presented in Figs. 2 and 3, respect- ively. Of the molecular species, PC 36:2 was the most abundant. Additionally, trace amounts of PC 26:0, 28:0, 31:1, 33:1, 33:2, 33:3, 36:2, 36:3, 36:6 and 38:8 were de- tected. In the samples collected later in the summer (Table 2), PC concentrations were found to be more abun- dant than in the samples taken earlier (Figs. 2 and 3). Horses in the eczema group had a significantly lower total concentration of PC than healthy horses before the start of the therapy (Table 3). Abstract Background: Lipids have become an important target for searching new biomarkers typical of different autoimmune and allergic diseases. The most common allergic dermatitis of the horse is related to stings of insects and is known as insect bite hypersensitivity (IBH) or summer eczema, referring to its recurrence during the summer months. This intense pruritus has certain similarities with atopic dermatitis of humans. The treatment of IBH is difficult and therefore new strategies for therapy are needed. Autoserum therapy based on the use of serum phospholipids has recently been introduced for horses. So far, serum lipids relating to these allergic disorders have been poorly determined. The main aim of this study was to analyse phospholipid profiles in the sera of horses with allergic dermatitis and in their healthy controls and to further assess whether these lipid profiles change according to the clinical status after therapy. Methods: Sera were collected from 10 horses with allergic dermatitis and from 10 matched healthy controls both before and 4 weeks after the therapy of the affected horses. Eczema horses were treated with an autogenous preparation made from a horse’s own serum and used for oral medication. Samples were analysed for their phospholipid content by liquid chromatography coupled to a triple-quadrupole mass spectrometer (LC-MS). Data of phospholipid concentrations between the groups and over the time were analysed by using the Friedman test. Correlations between the change of concentrations and the clinical status were assessed by Spearman’s rank correlation test. Results: The major phospholipid classes detected were phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Eczema horses had significantly lower total concentrations of PC (p < 0.0001) and SM (p = 0.0115) than their healthy controls. After a 4-week therapy, no significant differences were found between the groups. Changes in SM concentrations correlated significantly with alterations in clinical signs (p = 0.0047). Conclusions: Horses with allergic dermatitis have an altered phospholipid profile in their sera as compared with healthy horses and these profiles seem to change according to their clinical status. Sphingomyelin seems to have an active role in the course of equine insect bite hypersensitivity. Keywords: Horse, Allergy, Allergic dermatitis, Lipids, Phospholipids, Insect bite hypersensitivity, Summer eczema, Autoserum therapy Page 2 of 9 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 2 of 9 Background made from the sera of the affected than from healthy horses and the amounts of these phospholipids showed significant associations with the severity of prevailing clin- ical status [35]. Various new and important insights into the roles of lipids have been demonstrated over the past few decades. Most of these findings are related to inflammatory and immune responses including studies on pro-resolving lipid media- tors [1, 2], lipid antigens [3, 4] and lipid presenting CD1 molecules [5–7]. Mass spectrometry with specific software applications has made it possible to identify and quantify a multitude of lipid species both in mammalian cells and also of those existing in circulation [8–12]. Although phospholipids are commonly present in mammalian sera [13, 14], changes in their concentrations under various pathological conditions are so far poorly understood [9, 15, 16]. Presently, there is an increasing need to find new biomarkers feasible for diagnosis, treatment and prognosis – not only in allergic diseases, but also in other immune mediated and neurological disorders [2, 16–23]. The purpose of the current study was to analyse by li- quid chromatography-mass spectrometry (LC-MS) phos- pholipids in the sera of horses with insect bite allergic dermatitis and in their matched healthy controls. We hypothesize that phospholipid profiles may differ be- tween the groups. The additional aim was to assess whether these profiles change according to the clinical status after autoserum therapy. Results After the 4-week therapy, no sig- nificant difference could be found between the groups (Table 3, Fig. 3). In the eczema group, the total PC con- centration had increased significantly, while in the control Fig. 1 Typical clinical signs in the tail of a horse with insect bite allergic dermatitis (a) and the same horse after a 4-week therapy (b) Fig. 1 Typical clinical signs in the tail of a horse with insect bite allergic dermatitis (a) and the same horse after a 4-week therapy (b) Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 3 of 9 Page 3 of 9 Table 1 Relative contents of the major phospholipids detected in sera of the horses Phospholipids Relative content (mean ± sd/%) Eczema group Control group Stage 0 PC 76.0 ± 4.8 83.3 ± 9.8 SM 21.9 ± 3.8 14.8 ± 8.5 PI 1.9 ± 1.4 1.7 ± 1.5 PE 0.2 ± 0.2 0.2 ± 0.2 Stage 4 PC 87.4 ± 1.7 85.6 ± 2.7 SM 11.0 ± 1.6 12.4 ± 1.9 PI 1.4 ± 0.7 1.8 ± 1.3 PE 0.2 ± 0.2 0.2 ± 0.2 Samples of horses with allergic dermatitis (n = 10) and their matched healthy controls (n = 10) are collected before treatment (stage 0) and when affected horses have been on autoserum therapy for 4 weeks (stage 4). Phospholipid classes: phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidylethanolamine (PE). Table 1 Relative contents of the major phospholipids detected in sera of the horses the stages in both eczema and healthy horses. Concentra- tions of SM, like PC, were more abundant in the samples collected later in the summer (Table 2, Figs. 4 and 5). Phosphatidylinositol and PE displayed small amounts of the phospholipids detected (Table 1). Of PI, molecular species of 32:0, 34:1, 34:2, 36:1, 36:2, 36:3, 36:4 and 38:4 were found. The pairwise comparisons showed no sig- nificant differences between the two groups at any stages (Table 3). The detected molecular species of PE were that of 32:0, 36:1, 36:2, 36:3, 36:4, 38:4 and 38:5. Unlike PI, the amounts of PE increased significantly from stage 0 to 4 in both groups (Table 3). However, its relative content did not change due to its small total amount (Table 1). No significant differences between the groups were observed at any of the stages. Results Five of the horses had mild and 5 moderate clinical signs at the beginning of the study and the total score of signs was 15, according to the 3-graded scale used. After a 4-week autoserum therapy, the signs had relieved in 6 horses (Fig. 1b), remained unchanged in 3, while one horse suffered from aggravated clinical signs, the total score being 10. Horses with positive clinical outcome showed a milder decrease in SM concentrations than horses with poorer response or controls (Fig. 6). The change in total SM concentrations correlated signifi- cantly with alterations in the clinical signs (P = 0.0047) and of the major molecular species, SM 15:0 exhibited a significant correlation (P = 0.0268). None of the other phospholipid classes showed significant correlations with the change of clinical signs. Samples of horses with allergic dermatitis (n = 10) and their matched healthy controls (n = 10) are collected before treatment (stage 0) and when affected horses have been on autoserum therapy for 4 weeks (stage 4). Phospholipid classes: phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidylethanolamine (PE). Samples of horses with allergic dermatitis (n = 10) and their matched healthy controls (n = 10) are collected before treatment (stage 0) and when affected horses have been on autoserum therapy for 4 weeks (stage 4). Phospholipid classes: phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidylethanolamine (PE). group there was a small decrease (Figs. 2 and 3). Concen- trations of all the major molecular species presented in Figs. 2 and 3 increased significantly (at level p < 0.05) in the affected horses between stages 0 and 4, while in the healthy horses, the only significant change observed was a decrease in the concentration of PC 40:8 (P = 0.0007). Concentrations of SM were found to be significantly lower in eczema horses in comparison with their healthy controls at stage 0 (Table 3), although the relative con- tent of SM was higher in the affected horses (Table 1). The total SM concentrations decreased in both groups during the study period (Table 3, Figs. 4 and 5) and at the second sampling there was no significant difference observed between the two groups (Table 3). The major SM species detected and their changes between the stages 0 and 4 are shown in Figs. 4 and 5. Results The most abundant species was SM 20:0 in both groups and its concentration differed neither between the groups nor the stages. Concentrations of SM 16:0, 18:0, 24:0 and 24:1 decreased significantly (at level p <0.05) between No side effects relating to the therapy were observed. Control horses did not develop signs typical of IBH dur- ing the study period. Discussion our previous study [35], we found that autoserum prepa- rations made from the sera of healthy horses and from horses with allergic eczema showed significantly different phospholipid profiles. Additionally, lipid concentrations displayed significant relationship with the severity of clin- ical signs. Therefore, the present study was focused on serum and changes observed in its lipid profiles in re- sponse to autoserum therapy, but not on the clinical effi- cacy of this therapy per se. Horses with insect hypersensitivity showed signifi- cantly lower total concentrations of PC and SM than their matched healthy controls. After a 4-week therapy, those differences faded. The cause of these originally lower concentrations in eczema horses is unclear. Obser- vations from earlier studies indicated that small children with elimination diets or horses fed with oil supplements show altered lipid compositions in their sera [36, 37]. In addition, fasting influences lipid concentrations [9] which stresses the relevance of matching and equal tim- ing when blood is collected from non-fasting horses and their controls. Horses suffering from severe clinical signs may spend time for scratching and thus eat differently than healthy horses. However, none of the horses were so severely affected. Eczema horses were otherwise in a Phosphatidylcholine, the major phospholipid present in mammalian sera [13, 14], was found to be the most abun- dant also in the present study. In humans, alterations in its concentrations have been linked to various pathological conditions, such as cancer, atopic dermatitis and type-1 diabetes, where in altered levels of both PC and SM in sera have been observed [17, 22, 23, 38]. Fuchs et al. showed that in rheumatoid arthritis, PC/lysoPC ratio acted as an indicator of this autoimmune disease and add- itionally, an increased ratio seemed to be associated with the success of the therapy [39]. Discussion The purpose of this study was to analyse the major phos- pholipids typical of mammals in the sera of healthy horses and horses with allergic dermatitis. The additional aim was to assess whether profiles of the detected phospho- lipids change according to clinical status when horses were treated with an autogenous serum preparation. In Fig. 2 Concentrations of the most abundant phosphatidylcholine (PC) species at the start of therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. All species showed significantly lower concentrations in eczema horses, at level P < 0.0001 i h f h E h ( 0) d h i h d Fig. 2 Concentrations of the most abundant phosphatidylcholine (PC) species at the start of therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. All species showed significantly lower concentrations in eczema horses, at level P < 0.0001 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 4 of 9 Fig. 3 Concentrations of the most abundant phosphatidylcholine (PC) species after a 4-week therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. Concentrations of PC 34:1 and 36:1 were significantly lower in eczema horses, while 40:8 was in the controls, at level P < 0.05 Fig. 3 Concentrations of the most abundant phosphatidylcholine (PC) species after a 4-week therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. Concentrations of PC 34:1 and 36:1 were significantly lower in eczema horses, while 40:8 was in the controls, at level P < 0.05 normal physical condition without signs of emaciation and their feeding regimen and daily activities were similar to their controls, neither had the owners observed any changes in their appetite. Although the feeding regimen was similar between the horses, there could be a possibil- ity that some unknown factors might have influenced the lipid concentrations over time, since sera collected in the late summer showed more abundant concentrations of PC and SM than did sera collected earlier in the summer and this trend was detectable in both affected and healthy horses. This emphasizes the importance of matching ac- companied by simultaneous samplings in these kinds of studies that are prone to environmental impacts. 0 horses with allergic dermatitis and B) their 10 matched healthy controls. The horse and its matched control are numbered similarly Discussion LysoPC is a derivative of Table 2 Matching of the horses A Breed Gender Age (years) Feed B Breed Gender Age (years) Feed Date of 1stsample 1 Finnhorse ♀ 3 same fodder 1 Finnhorse ♀ 9 same fodder 24 April 2 Icelandic horse ♀ 22 same fodder 2 Shetland pony ♀ 22 same fodder 21 May 3 Icelandic horse ♀ 23 same fodder 3 Icelandic horse ♂ 9 same fodder 21 May 4 Finnhorse ♂ 8 same fodder 4 Finnhorse ♂ 7 same fodder 12 June 5 Finnhorse ♂ 8 same fodder 5 Finnhorse ♂ 7 same fodder 12 June 6 Finnhorse ♀ 8 same pasture 6 Finnhorse ♀ 8 same pasture 12 June 7 Finnhorse ♀ 4 same pasture 7 Finnhorse ♀ 7 same pasture 17 July 8 Finnhorse ♀ 4 same pasture 8 Finnhorse ♀ 6 same pasture 11 Aug. 9 Icelandic horse ♀ 10 same fodder 9 Icelandic horse ♀ 29 same fodder 7 Sept. 10 Finnhorse ♀ 11 same pasture 10 Finnhorse ♀ 19 same pasture 12 Sept. A) 10 horses with allergic dermatitis and B) their 10 matched healthy controls. The horse and its matched control are numbered similarly A) 10 horses with allergic dermatitis and B) their 10 matched healthy controls. Discussion The horse and its matched control are numbered similarly Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 5 of 9 Page 5 of 9 Table 3 Pairwise comparisons of serum phospholipid concentrations PC 0 PC 4 PC 0 c PC 4 c PC 0 p = 0.0007 p < 0.0001 p < 0.0001 PC 4 p = 0.0007 ns ns PC 0 c p < 0.0001 ns ns PC 4 c p < 0.0001 ns ns SM 0 SM 4 SM 0 c SM 4 c SM 0 ns p = 0.0115 ns SM 4 ns p = 0.0005 ns SM 0 c p = 0.0115 p = 0.0005 p = 0.0186 SM 4 c ns ns p = 0.0186 PI 0 PI 4 PI 0 c PI 4 c PI 0 ns ns ns PI 4 ns ns ns PI 0 c ns ns ns PI 4 c ns ns ns PE 0 PE 4 PE 0 c PE 4 c PE 0 p = 0.0058 ns p < 0.0001 PE 4 p = 0.0058 ns ns PE 0 c ns ns p = 0.0018 PE 4 c p < 0.0001 ns p = 0.0018 Phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) detected from horses with allergic dermatitis and from their matched healthy controls (c); samples collected before (0) and 4 weeks after (4) the therapy of the affected horses Table 3 Pairwise comparisons of serum phospholipid concentrations clinical signs, neither was there any prognostic association between the first PC concentrations and the response to therapy. In contrast to our horse study, higher levels of PC predicted better outcome in patients with atopic ec- zema after monoclonal antibody therapy [38]. y y Concentrations of SM were significantly lower in the affected than in healthy horses before the treatment, but not afterwards. Therefore, the elevated relative content of SM in eczema horses at the first sampling is in par- ticular due to the lower levels of PC. Surprisingly, the amounts of SM decreased significantly among the con- trol horses during the study period, which demonstrates that concentrations of certain lipids may fluctuate in healthy horses, at least to some extent without largely undermining the relative lipid content. This fluctuation possibly resulted from environmental changes, since SM concentrations decreased also in eczema horses. Discussion However, the decrease was evident only in those eczema horses that showed a poorer outcome in response to therapy. This suggests that affected horses have a specific need for SM during recovery. Sphingomyelins are important lipids in- volved in multiple biochemical reactions, especially in the skin and nervous tissue [15, 41–43]. These phospho- lipids are necessary not only as structural components, but also as bioactive regulators and messengers in cuta- neous homeostasis and immune mediated responses, particularly via the main derivative, sphingosine-1- phosphate [41, 42, 44, 45]. Sphingosine-1-phosphate (S1P) plays a crucial part in mast cell regulation by af- fecting mast cell degranulation and by activating other immune cells involved [44, 45]. The engagement of S1P to its receptors on the plasma membrane seems to be the most critical point in the pathway of mast cell acti- vation [44–46]. In addition to S1P, ceramide is another important metabolite of SM and is one of the main lipid components in skin barrier [41]. In the affected skin, ceramides are continuously utilised for repairing [41]. Although various lipid combinations and their changes Phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) detected from horses with allergic dermatitis and from their matched healthy controls (c); samples collected before (0) and 4 weeks after (4) the therapy of the affected horses PC and it is easily formed from PC during storage at room temperature [40]. In horse sera, the relative content of lysoPC is small [14] and therefore it is not an applicable marker in horses as it is in humans. In the present study, lysoPC was detected in minor concentrations and conse- quently was not evaluated further. Although there was a highly significant difference in PC concentrations between the healthy and the eczema horses at the first sampling, PC did not show any correlation with the change in Fig. 4 Concentrations of the most abundant sphingomyelin (SM) species at the start of therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. Species of SM 15:0 and 24:1 showed significantly lower concentrations in eczema horses, at level P < 0.05 Fig. 4 Concentrations of the most abundant sphingomyelin (SM) species at the start of therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. Discussion Species of SM 15:0 and 24:1 showed significantly lower concentrations in eczema horses, at level P < 0.05 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 6 of 9 Fig. 5 Concentrations of the most abundant sphingomyelin (SM) species after a 4-week therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. Concentrations of SM 18:0 and 21:0 were significantly lower in eczema horses, at level P < 0.05 Fig. 5 Concentrations of the most abundant sphingomyelin (SM) species after a 4-week therapy. Eczema horses (n = 10) and their matched healthy controls (n = 10) are numbered similarly. Concentrations of SM 18:0 and 21:0 were significantly lower in eczema horses, at level P < 0.05 in response to therapy have been linked to atopic dermatitis in humans [38, 47], SM seems to be a poten- tial player in insect bite allergy of the horse, since alter- ations in its concentrations were significantly associated with changes in clinical signs. Similarly, sphingolipids have been shown to act as biomarkers in delayed type hypersensitivity of mice [16]. Horses in the present study were treated with an auto- serum preparation and phospholipids were analysed in response to this treatment. However, we had no control group for the therapy; therefore it is not relevant to con- clude that the observed changes could be specific for the current treatment. In fact, further studies are needed to evaluate a relationship between phospholipid concentra- tions and responses to therapy, comprising placebo or other therapeutic interventions and a greater number of horses than those enrolled in this study. In our study, both PI and PE were detected in minor amounts, which is consistent with the previous studies [14]. These phospholipids are probably not involved in insect bite hypersensitivity, since there was no difference between the groups in the first sampling and the con- centrations of PE changed similarly in both groups. Traces of PA were found in all samples and as an inner leaflet phospholipid, it is not normally detected from the horse serum [14]. Presence of PA is probably related to blood cells that remained when the samples were har- vested without centrifugation [35]. Blood sampling that make lipids less water-soluble and enable them to concentrate into autoserum preparations, although ac- cording to the current study the total concentrations of PC and SM were lower in these horses. This interpret- ation could explain with caution the underlying mechan- ism of this therapy, possibly culminating in the ability of signalling molecules to engage their corresponding re- ceptors which has been recognized to be a critical event for the following mast cell responses [44–46]. The first sample (marked as stage 0 in text, tables and figures) was taken when the horse had shown typical clinical signs of IBH for at least 2 weeks. Blood sample was first collected using 10 ml plain vacuum tubes that were filled in full and kept at room temperature for at least 3 hours before harvesting. The serum was har- vested without using a centrifuge, thus avoiding destruc- tion of serum lipids. With a calibrated pipette, 0.065 ml of the serum was taken from the superficial layer and stored in 4.2 ml of 48 % ethanol. The second sample (marked as stage 4 in text, tables and figures) was col- lected after a 4-week therapy of the affected horse and handled equally. Samples were drawn from the counter- part horse similarly and simultaneously. Therapy of the eczema horses All horses in the eczema group were treated with an oral administration of an autoserum preparation for 4 weeks. Sera used for the preparations were drawn at the time of the first blood sampling. Sera were harvested without using a centrifuge and 0.065 ml of the serum was taken from the superficial, lipid-containing layer of the tube. Lipid particles were washed twice with sterile water (1:100), accordingly 0.065 ml collected from the superfi- cial layer of the previous dilution. After the second washing, 0.065 ml was taken from the superficial layer of the latter solution and added finally in 48 % ethanol (1:100) and absorbed in sugar granules [49]. A dose of granules was given orally once a day for 2 weeks, followed by a 1-week pause, after which horses were medicated for one further week. Horses in the control group received no therapy. Discussion y The lower concentration of PC and SM detected in the sera of affected horses was an unexpected finding, since our earlier study demonstrated that autoserum preparations made from the sera of horses with allergic dermatitis showed significantly higher concentrations of PC and SM than the preparations made from the sera of healthy horses [35]. This contradictory result may be as- sociated with the hydrophobic/hydrophilic interactions of the lipid particles. The serum for autoserum prepara- tions was collected from the superficial layer of the blood sample, washed twice with water and finally added in ethanol; all consecutive transfers were collected from the superficial layer of the previous solution [35]. Thus the most hydrophobic and water-insoluble lipid mole- cules were presumably concentrated in such autoserum preparations. Instead of washings, samples of the present work were mixed directly with alcohol. Therefore, these samples included not only the most hydrophobic but also the less hydrophobic lipid particles, such as PC 26:0 with a short acyl chain or PC 36:6, 38:8 and 40:8 with several double bonds; species that were not detected in autoserum preparations [35]. The discrepancy with the amounts of PC and SM in the serum versus autoserum preparations may be interpreted as so far unknown, hydrophilic/hydrophobic interactions possibly linked to mineral salts that have been found to affect peculiarly the distribution of lipids after successive washings [48]. Sera of affected horses may contain those mineral salts Fig. 6 Difference of sphingomyelin (SM) concentrations (pmol in total of 0.5 ml) after therapy. The change between the pre (stage 0-week) and the post (stage 4-week) concentrations in eczema horses with (A, n = 6) and without (B, n = 4) a positive clinical response and in their matched healthy controls (Ac and Bc, respectively) Fig. 6 Difference of sphingomyelin (SM) concentrations (pmol in total of 0.5 ml) after therapy. The change between the pre (stage 0-week) and the post (stage 4-week) concentrations in eczema horses with (A, n = 6) and without (B, n = 4) a positive clinical response and in their matched healthy controls (Ac and Bc, respectively) Page 7 of 9 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 7 of 9 Methods Horses A total of 20 horses entered in this study in 2014, be- tween the seasons of spring and autumn, the period when horses are exposed to bites of insects. The horses comprised 10 animals with clinical signs of insect bite hypersensitivity (eczema group) and 10 matched healthy controls (control group). The matching is presented in Table 2. Clinical signs of eczema were categorised as mild, moderate or severe at the time of blood sampling and scored 1–3, respectively. Signs were mild, if the horse had only pruritus without skin lesions. Pruritus with mild skin affections in the mane, tail (Fig. 1a) and/ or body was graded as moderate, while pruritus over large skin lesions was regarded as a severe sign. Clinical signs were evaluated after the 4-week therapy and con- sidered as relieved when signs became milder on this scale (Fig. 1a and b). The changes in clinical signs were recorded as relieved, no change or aggravated and re- spectively scored as −1, 0 or +1. Five of the horses had mild and 5 moderate clinical signs in the beginning of the study. The matched healthy controls without a his- tory of IBH lived in the same farms and were fed with the similar fodder as their affected counterparts. Horses were also matched with breed, gender and age as closely as possible (Table 2). The eczema horse and its matched control are numbered similarly in the tables and figures throughout this article. This study has been approved by the Regional State Administrative Agency of Southern Finland (ESAVI/1016/04.10.07/2014) and owners’ signed permission for inclusion was obtained. Conclusions Horses with an allergic dermatitis have an altered lipid profile in their sera as compared with healthy controls and these differences seem to change according to the clinical status of the horses. Sphingomyelin shows a functional role in equine insect bite hypersensitivity. Lipid profiling may become an important target for further studies, since relevant fingerprints delineating different patho- logical disorders are needed – in both human and veter- inary medicine. Acknowledgements We thank Doc Marja Raekallio for her valuable advice, suggestions and supervision and Prof Pentti Somerharju for the cooperation with this study. We are grateful to Mrs Tarja Grundstrom and Mrs Leena Wathen for their excellent laboratory assistance. We also wish to thank the horse owners for their cooperation. 19. Quehenberger O, Armando AM, Brown AH, Milne SB, Myers DS, Merrill AH, et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res. 2010;51:3299–305. doi:10.1194/jlr.M009449. 20. Fischer K, Kettunen J, Würtz P, Haller T, Havulinna AS, Kangas AJ, et al. Biomarker profiling by nuclear magnetic resonance spectroscopy for the prediction of all-cause mortality: an observational study of 17,345 persons. PLoS Med. 2014;11(2):e1001606. doi:10.1371/journal.pmed.1001606. Author details 1V i Cli i 1Veterinary Clinic, Nummela, Finland. 2University of Helsinki, Faculty of Veterinary Medicine, Helsinki, Finland. 3Department of Medical Biochemistry and Developmental Biology, Institute of Biomedicine, University of Helsinki, Helsinki, Finland. References 1. Serhan CN, Petasis NA. Resolvins and protectins in inflammation-resolution. Chem Rev. 2011;111(10):5922–43. doi:10.1021/cr100396c. 1. Serhan CN, Petasis NA. Resolvins and protectins in inflammation-resolution. 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Received: 25 January 2016 Accepted: 23 February 2016 Received: 25 January 2016 Accepted: 23 February 2016 Received: 25 January 2016 Accepted: 23 February 2016 Lipid analysis of the sera p y Phospholipid classes analysed were PC, PE, PS, PI, PA and SM. All the samples stored in ethanol were sub- jected to Folch’s method [48] for lipid extraction, dried under a N2 stream, reconstituted in 500 μl chloroform/ methanol (1:2) and further spiked with the following la- belled standardsa corresponding to each head group: D9(di-44:2) and D9(di-40:2) for PC, D4(di-40:2) and D4(di-20:0) for PE, D3(di-40:2) and D3(di-44:2) for PS, D6(di-36:2) and D6(di-28:0) for PI and finally unlabelled 25:0-SM. Liquid chromatography-mass spectrometry (LC-MS) with selective reaction monitoring (SRM) was used for the analyses. Waters ACQUITY Ultra Perform- ance LC systemb equipped with a Waters ACQUITY BEH C18 column (1.0 × 100 mm) was used to separate the mo- lecular species using gradient elution. Solvent A was aceto- nitrile/H2O (60:40) with 10 mM ammonium formate and 1 % NH4OH, while solvent B was isopropanol/acetonitrile (90:10) containing 10 mM ammonium formate and 1 % NH4OH. The flow rate was 0.13 ml/min and the column Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 8 of 9 Page 8 of 9 Page 8 of 9 temperature 60 °C. Solvent B was set to 40 % at in- jection and increased linearly to 100 % in 14 min, remained at this value for 3 min, decreased back to 40 % in 1 min and then remained there until the end of the gradient at 20 min. The eluent was directed to the electrospray ionization (ESI) source of Waters Quattro Premier triple-quadrupole mass spectrome- terb operated in the positive ion mode. For SRM tran- sitions, proton adducts of the PC, PE, PS and PI species were selected as the precursors, while the product ion was either the head group (PC, SM, PI) or the diacylglycerol fragment (PE, PS). 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Eur J Pharmacol. 2015; doi:10.1016/j.ejphar.2015.02.058. 47. Laitinen K, Sallinen J, Linderborg K, Isolauri E. 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: 40. Breier M, Wahl S, Prehn C, Fugmann M, Ferrari U, Weise M, et al. Targeted metabolomics identifies reliable and stable metabolites in human serum and plasma samples. PLoS One. 2014;9:1–11;e89728. doi:10.1371/journal. pone.0089728. • 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 p y y p 41. Proksch E, Jensen J-M, Elias PM. Skin lipids and epidermal differentiation in atopic dermatitis. 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https://wjbphs.com/sites/default/files/WJBPHS-2024-0235.pdf
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Analysis of biological organic extract of plants in suppressing aphid (Myzus persicae) infestations
World Journal of Biology Pharmacy and Health Sciences
2,024
cc-by
4,852
Abstract Myzus persicae commonly known as the peach-potato aphid, poses a significant threat to agricultural productivity, particularly in potato cultivation, where infestations lead to substantial yield losses and quality degradation. In this study, we investigated the potential of botanical extracts from neem (Azadirachta indica), clove (Syzygium aromaticum), black pepper (Piper nigrum), and bay leaf (Laurus nobilis) in controlling Myzus persicae infestations. Experimental trials were conducted to assess the mortality effects of these botanical extracts on Myzus persicae populations. Results revealed that neem extract exhibited the highest mortality effect, with an impressive 97.7% mortality rate. Clove and black pepper extracts followed closely, demonstrating mortality rates of 92.2% and 77.7%, respectively. In contrast, bay leaf extract exhibited the least mortality effect, with only 48.8% mortality observed. Keywords: Myzus persicae; Natural pesticides; Eco-friendly pest control; Botanical extracts; Toxicity; Mortality. Analysis of biological organic extract of plants in suppressing aphid (Myzus persicae) infestations Rahmah Khan and Newton Paul * Department of Zoology, Isabella Thoburn college, Lucknow, India. World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 ublication history: Received on 15 March 2024; revised on 27 April 2024; accepted on 29 April 2024 Article DOI: https://doi.org/10.30574/wjbphs.2024.18.2.0235 Analysis of biological organic extract of plants in suppressing aphid (Myzus persicae) infestations * Corresponding author: Newton Paul. Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons or(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. 2.1. Rearing of insect Myzus persicae specimens were reared under controlled conditions using non-infested fresh daisy twigs as nutritive substrates to establish and maintain a standardized insect culture for experimental purposes. Plastic rearing containers, equipped with perforated cloth to ensure adequate ventilation are utilized. Uninfected fresh daisy twigs sourced from pesticide-free environments serve as nutritive substrates maintaining experimental integrity. Myzus persicae specimens are introduced onto these twigs with precision using a brush ensuring controlled inoculation. Environmental parameters were maintained. This includes maintaining a lower temperature range of 18-20°C and humidity levels sustained at 50-60% to simulate more temperate conditions conducive to experimental requirements. Additionally, a photoperiod of 16 hours simulates daylight conditions. Continuous twig replenishment ensures consistent aphid nutrition. After a period of 25-30 days, new instances of M. persicae were observed on the twigs indicating the completion of their developmental cycle. These aphids were carefully separated from the twigs to ensure accurate and controlled experimental conditions (Plate-1: Figure-5). 2.3. Preparation of extracts: The prepared extracts were inspired by the research paper cited (Erdogan et. al., 2020), (Khalid et. al., 2020), (Madanat et. al., 2016) though few changes were made. The process involved mixing 10 g of fine powder of spices or leaves with 100 ml of water. Boiling the mixture for 30 minutes on medium-high flame with constant stirring, followed by cooling and filtration using Whatman filter paper no. 1 yielded an aqueous solution. To this solution 100 ml of ethanol was added. The resulting solution was kept in the dark for 48-72 hours before being used in spray bottles for it to ferment (Plate-1: Figure-4). The Neem extract was combined with the other spice extracts in a 1:1 w/w ratio to create three distinct mixtures for demonstrating their joint efficacy against Myzus persicae thereby facilitating the assessment of potential synergistic effects. World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Botanical pesticides, derived from natural sources like plants, animals, microbes, and specific minerals, are known for their low toxicity towards non-target organisms such as humans, animals, beneficial insects, and the environment (Adusei & Azupio, 2022) (Adusei & Azupio, 2022) 2. Materials and methods The experiment was conducted at 15°±5°C and 94 ± 5% humidity. The experimental design comprised 4 treatments, each replicated 5 times. The experimental setup involved the deployment of plastic containers as controlled environments, with oasis serving as the substrate for experimental conditions, and nylon synthetic fabric employed for precise filtration purposes. 2.4. Experimental setup 2.4.1. BIOASSAY 1: Analysis of toxic effect of plants extracts on Myzus persicae 2.2. Preparation of powder and extracts Four extract treatments were prepared from two leaf specimens, Azadirachta indica (Neem) and Laurus nobilis (Bay Leaf), as well as two spice specimens, Syzygium aromaticum (Clove) and Piper nigrum (Black Pepper). Neem leaves (Azadirachta indica) were collected from the campus of Isabella Thoburn College, Lucknow, while fruits of black pepper (Piper nigrum), flower buds of clove (Syzygium aromaticum), and leaves of bay laurel (Laurus nobilis) were obtained from domestic sources. After air-drying for 5 days, Neem leaves were desiccated until crisp, while all specimens were then ground to a fine powder using an electric grinder. The resulting powders, each weighing 10 grams, underwent sieving for uniformity, yielding final samples weighing 8-10 grams for precise experimental conditions. 1. Introduction Potato holds significant agricultural importance both in India and across Asia (Scott & Saurez, 2011). In Central India, the cultivation of potatoes occurs during the short winter season, typically from October to March. Within this agricultural context, diverse species of aphids assume a significant role as vectors for viral diseases affecting potato crops, consequently contributing to seed degeneration (Bhatnagar et. al., 2012). In India, numerous researchers have previously conducted extensive studies on the appearance and population dynamics of aphids on potatoes, as well as their correlation with virus transmission. This exploration stands as a crucial prerequisite in enhancing our comprehension of aphids and their ecosystem (Bhatnagar et. al., 2012). Aphids, belonging to the family Aphididae within the order Hemiptera, are notable agricultural nuisances present worldwide. They cause considerable financial harm through their direct consumption of plant tissues, transmission of plant viruses, and secretion of honeydew (Kareem Ullah et al., 2023). A wealth of research has consistently emphasized the economic importance of aphids as agricultural pests (Emden & Harrington, 2017). The swift reproductive rate of aphids coupled with their capacity to transmit plant pathogens presents a formidable menace to crop yields on a global scale, thus demanding the implementation of efficient control strategies (Emden & Harrington, 2017). The transmission of over 110 plant viruses is attributed solely to Myzus persicae (Sulzer) (Singh & Agarwal, 2022). Wingless parthenogenetic adult females are oval-shaped, 1.2 to 2.1 mm long, displaying colours such as whitish green, pale yellow-green, grey- green, mid-green, dark green, pink, or red (CABI, 2021). The tobacco variant (nicotianae) ranges from bright yellow to nearly black. Genetic colour diversity exists, with some intensifying green or magenta pigmentation in colder climates. Immature stages have a noticeable sheen, while adults are less shiny. Winged variants feature a black dorsal patch on the abdomen (CABI, 2021). M. persicae has the potential to diminish the photosynthetic capacity of plants through the secretion of sugary honeydew, consequently impacting both the quality and quantity of crops (Frantz, et. al., 2004). 2.5. Statistical analysis The collected data were analysed in a completely randomized design to find significant difference between Neem, Black pepper, Clove and Bay leaf extract treatment. The analysis of variance (ANOVA) was applied. The test significant value is 𝑃< 0 ∙05, the calculation was performed by using IMB SPSS statistical ver. 27 software and graphs are plotted using the GraphPad prism 9 software for Microsoft. 2.4.3. BIOASSAY 3: Analysis of repellent action of different plants extract on Myzus persicae The repellency assay involved using a petri dish with filter paper divided into treated and untreated halves. Each half was subjected to extract sprayed. Ten adults Myzus persicae were introduced onto each filter paper, covered to prevent escape. After approximately 12 hours, the number of insects in treated and untreated areas was recorded. Three replications were conducted for each spice. Repellence percentage was calculated using the following formula (Plate-1: Figure-3). 𝑟𝑒𝑝𝑒𝑙𝑙𝑒𝑛𝑐𝑦% = (𝑁𝐶−𝑁𝑇) (𝑁𝐶+ 𝑁𝑇) × 100 𝑟𝑒𝑝𝑒𝑙𝑙𝑒𝑛𝑐𝑦% = ( 𝐶 ) (𝑁𝐶+ 𝑁𝑇) × 1 Where, NC number of insects in control (untreated) area, And NT number of insects in treated area Where, NC number of insects in control (untreated) area, And NT number of insects in treated area 2.4.1. BIOASSAY 1: Analysis of toxic effect of plants extracts on Myzus persicae Plastic containers 3mm in diameter and 56mm in height, underwent thorough cleaning and drying procedures to ensure sterility. Oasis, a hydrophilic foam was hydrated in water for 20-25 minutes to facilitate water absorption. Employing a smaller container with a 4mm diameter, Oasis was precisely cut to fit the container with a 1cm height to support the twigs. Uninfected twigs measuring 5cm in length were meticulously positioned within the Oasis substrate. 30 Myzus persicae aphids were introduced onto the twigs under controlled conditions. To maintain a pest and contaminant free environment, synthetic nylon fiber was used to cover the container opening secured with rubber bands. This methodology ensures a controlled and sterile experimental environment conducive to scientific investigation. Each 25 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 container was diligently labelled with the corresponding extract identifier to ensure precise investigation and treatment allocation, adhering to rigorous scientific protocols. The experiment involves counting the mortality rate by counting the number of dead Myzus persicae after 6hr, 12hr, 18hr, 24hr, 30hr, 36hr, 42hr, 48hr, 54hr, 60, 66hr, 72hr. In order to enhance result accuracy and facilitate comprehensive data analysis, each aphid-extract container was duplicated across five distinct sets. Additionally, to establish a comparative baseline a control group consisting of 30 untreated Myzus persicae within 5cm stems was also replicated across five separate sets, conducted concurrently (Plate-1: Figure-1). container was diligently labelled with the corresponding extract identifier to ensure precise investigation and treatment allocation, adhering to rigorous scientific protocols. The experiment involves counting the mortality rate by counting the number of dead Myzus persicae after 6hr, 12hr, 18hr, 24hr, 30hr, 36hr, 42hr, 48hr, 54hr, 60, 66hr, 72hr. In order to enhance result accuracy and facilitate comprehensive data analysis, each aphid-extract container was duplicated across five distinct sets. Additionally, to establish a comparative baseline a control group consisting of 30 untreated Myzus persicae within 5cm stems was also replicated across five separate sets, conducted concurrently (Plate-1: Figure-1). Neem + black pepper; Neem + clove and Neem + bay leaf Plastic containers were individually prepared, each containing a 5cm twig submerged in oasis substratum. Subsequently, 20 Myzus persicae were introduced into each container. A specific combination of extracts in a 1:1 v/v ratio was applied via spraying. Containers were carefully labelled to facilitate proper identification of the extract combinations. After 12 and 24 hours, the containers were examined, and the number of surviving and deceased Myzus persicae was recorded. The mortality percentage was computed using a designated formula. An analysis of the results was conducted to ascertain the efficacy of various spice combinations in repelling the insects (Plate-1: Figure-2). 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦% = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑖𝑛𝑠𝑒𝑐𝑡𝑠 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑠𝑒𝑐𝑡𝑠 × 100 2.4.3. BIOASSAY 3: Analysis of repellent action of different plants extract on Myzus persica mortality% = number of dead insects total number of insects × 100 mortality% = number of dead insects total number of insects × 100 2.4.2. BIOASSAY 2: Analysis of mixed plants extract toxicity effect on Myzus persicae Another aspect of the experiment involved investigating synergistic and antagonistic interactions by combining different extracts to assess their combined effects. Synergism, also known as potentiation, occurs when the combined effect of two or more extracts exceeds the sum of their individual effects. Antagonism, on the other hand occurs when the combined effect is less than the cumulative sum of their individual effects. The experiment was done by using a mixture of neem extract with other extracts. Prepared different combinations of extracts in equal parts. The mixtures were: Neem + black pepper; Neem + clove and Neem + bay leaf 3. Result During analysis it was found that the efficacy of specific extracts against Myzus persicae, a common agricultural pest, was evaluated. The study focused on assessing the mortality rates, combined effects and repellent actions of four distinct extracts. Through rigorous observation and analysis, it was determined that Azadirachta indica (neem) and Syzygium aromaticum (clove) exhibited the most notable insecticidal properties, demonstrating significant effectiveness against Myzus persicae in Piper nigrum (black pepper) displayed comparatively lower potency than clove. Conversely, Laurus nobilis was identified as the least efficacious among the extracts tested. These findings contribute to the understanding 26 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 of natural insecticidal agents and their potential applications in pest management strategies (Table1 and Figure6, in Table2 and Figure7 and in Table3 and Figure8). The highest mortality rates observed within 72 hours post-application. Neem powder treatments exhibited the most substantial impact, achieving a mortality rate of 97.7%. Following closely behind, clove and black pepper treatments recorded mortality rates of 92.2% and 77.76% respectively. Conversely, other treatments displayed comparatively lower levels of mortality, with bay leaf treatment resulting in a 48.8% mortality rate (Neem>Clove>Black pepper>Bay leaves) in Table1 and Figure6. In a combined action study, mortality counts were assessed 24 hours post-treatment. Neem extract served as the standard, mixed in a 1:1 v/v ratio with various prepared extracts in Table2 and Figure7. Depict the outcomes, with the Azadirachta indica and Syzygium aromaticum mixture demonstrating the highest toxicity, resulting in a mortality rate of 64.43%. Following this, the combination of Azadirachta indica and Piper nigrum exhibited a mortality rate of 35.53%. Conversely, the combination of Azadirachta indica and Laurus nobilis displayed the lowest toxicity, with a mortality rate of 24.4%. (Neem + Clove > Neem + Black pepper> Neem + Bay leaves) (Table 2 and Figure 7). In the analysis of variance (ANOVA), it was observed that neem extract demonstrated the highest repellency efficacy, registering a significant percentage of 83.3%. Following neem, clove and black pepper exhibited repellency percentages of 66.6% and 56.6% respectively. Bay leaf demonstrated a repellency effect of 23.3% against Myzus persicae, as illustrated in Table3 and Figure8. Figure 1 Experimental set up for mortality effect of prepared extract. Figure 2 Experimental set up for joint effect of prepared extracts Figure 3 Experimental setup for repellency Figure 4 Prepared extracts Figure 2 Experimental set up for joint effect of prepared extracts Figure 1 Experimental set up for mortality effect of prepared extract. Figure 2 Experimental set up for joint effect of prepared extracts Figure 1 Experimental set up for mortality effect of prepared extract. Figure 3 Experimental setup for repellency Figure 4 Prepared extracts 27 27 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Figure 5 Rearing of aphids Figure 5 Rearing of aphids Table 1 Mean mortality effect of different extracts on Myzus persicae S. No. Extract No. Three of the five replicas were considered; Test significance is 𝒑> 𝟎. 𝟎𝟓 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 of Myzus persicae (N) Mortality (Mean±S.E) 6hr 12hr 18hr 24hr 30hr 36hr 42hr 48hr 54hr 60hr 66hr 72hr 1. Control 30 0.33± 0.33 0.33± 0.33 1.00± 0.00 1.33± 0.33 1.33± 0.33 1.33± 0.33 1.66± 0.33 1.66± 0.33 1.66± 0.33 2.00± 0.33 2.33± 0.33 2.33± 0.33 2. Neem 30 2.33± 0.33 4.33± 0.33 6.33± 0.33 8.33± 0.33 12.66± 0.33 14.66± 0.66 17.33± 0.33 19.33± 0.33 21.66± 0.88 25.00± 0.57 26.00± 0.57 29.33± 0.33 3. Black pepper 30 1.66± 0.33 2.33± 0.66 5.33± 0.33 7.66± 0.33 8.66± 0.33 10.00± 0.57 11.33± 0.88 12.66± 0.66 14.66± 0.66 16.33± 0.88 18.33± 1.20 23.33± 0.57 4. Clove 30 2.00± 0.57 4.33± 0.88 6.00± 0.57 7.00± 0.57 8.33± 0.33 12.66± 0.33 14.33± 0.33 16.66± 0.33 18.33± 0.33 21.33± 0.88 24.33± 0.33 27.66± 0.33 5. Bay leaf 30 0.33± 0.33 1.33± 0.33 2.66± 0.33 4.33± 0.33 6.00± 0.57 7.33± 0.33 8.66± 0.33 10.00± 0.57 11.33± 0.33 12.66± 0.88 13.33± 0.33 14.66± 0.88 Data represented in the table are the mean of three out of five replications; Test significance is 𝑷˂𝟎. 𝟓 Table 1 Mean mortality effect of different extracts on Myzus persicae Table 2 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae S.No Extract Number of aphids Mortality (Mean ±S.E) Type of action 12hr 24hr 1 Control 30 1.33±0.33 1.66±0.33 2 Neem + black pepper 30 7.33± 0.66 10.66± 0.33 Synergistic 3 Neem +clove 30 10.66± 0.66 19.33± 0.88 Synergistic 4 Neem+ bay leaf 30 4.66± 0.33 7.33± 0.33 Synergistic Three of the five replicas were considered; Test significance is 𝒑> 𝟎. 𝟎𝟓 Table 2 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae 28 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Table 3 Repellency effect of different extracts on Myzus persicae S. No Plants used for Extract Number of aphids Number of aphids on untreated area Number of aphids on treated area Mean±S.E A B C a b c 1 Neem 10 9 8 8 1 2 2 8.33±0.33 2 Black pepper 10 5 6 6 5 4 4 5.66±0.33 3 Clove 10 7 7 6 3 3 4 6.66±0.33 4 Bay leaf 10 2 2 3 8 8 7 2.33±0.33 Three out of five replications are considered; Test significance is𝑷< 𝟎. 4. Discussion Parallel work found out that, during the nymphal period of Aphid, mortality rates reached 100.0% at the highest concentration, indicating significant and potentially lethal effects when treated with neem bio-pesticide (Santos et. al., 2004), which stands true for this experiment as the mortality rate of replica treated with Neem extract reaches 97.76% (Table1 and Figure6). Another study showed, Neem seed oil (NSO) at a 1.0% concentration induced mortality ranging from 94% to 100% in second instar nymphs of the green peach aphid, Myzus persicae (Sulzer) (Murray, 1994). Another showed that the Hydroethanolic extract derived from neem leaves exhibited mortality rates exceeding 95% on Myzus persicae, indicating significant efficacy against this species (Déla, et. al., 2014). Neem leaf extract contains saponins, meliantriol, and azadirachtin, acting as vegetable insecticides against aphids. Saponins are stomach and contact poisons, meliantriol works as a repellent, while azadirachtin inhibits aphids' hormone function, crucial for their development. (Tobing, et. al., 2023), which stands true with this experiment (Table1 and Figure6). The estimated lethal concentration (LC95) of clove essential oil, designed to terminate 95% of aphids, in the work of (Pedro, et al., 2020), which stands true for this experiment as the mortality rate of replica treated with Neem extract reaches 97.7% (Table1 and Figure6). Neem oil exhibited the highest nymph mortality rates at 96.67%, 100%, and 100% in concentirations of 1%, 3%, and 5% respectively, after 72 hours. Clove oil followed with 80.00% mortality of Myzus persicae seen in (Sonowal, et al., 2023). The methanol extracts from Azadirachta indica leaves, at concentrations of both 5% and 10%, provided complete protection (100%) to lentil and chickpea seeds from aphids by (Bhuiyah, et al., 2003). Therefore, this study indicates that neem exhibits the highest mortality rates, followed by clove, attributed to the presence of Azadirachta and eugenol respectively. Our findings align closely with (Haddi, et al., 2015) studies indicating that clove oil possesses insecticidal toxicity against Sitophilus zeamais Motschulsky, attributed to eugenol and the sesquiterpene β-caryophyllene. (Table1 and Figure6) Another study of (Ahmed, et. al., 2021) states that, Black pepper essential oil has been identified as a potent insecticide, demonstrating significant efficacy with an impressive 80% mortality rate upon contact application. The bay leaf extract demonstrated minimal efficacy, with less than 50 percent mortality recorded during the shortest exposure time by (Kaur, et. al., 2018) (Table1 and Figure6). World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 𝟎𝟓 Figure 6 Mean mortality effect of different extracts on Myzus persicae Table 3 Repellency effect of different extracts on Myzus persicae S. No Plants used for Extract Number of aphids Number of aphids on untreated area Number of aphids on treated area Mean±S.E A B C a b c 1 Neem 10 9 8 8 1 2 2 8.33±0.33 2 Black pepper 10 5 6 6 5 4 4 5.66±0.33 3 Clove 10 7 7 6 3 3 4 6.66±0.33 4 Bay leaf 10 2 2 3 8 8 7 2.33±0.33 Three out of five replications are considered; Test significance is𝑷< 𝟎. 𝟎𝟓 Table 3 Repellency effect of different extracts on Myzus persicae Figure 6 Mean mortality effect of different extracts on Myzus persicae Figure 6 Mean mortality effect of different extracts on Myzus persicae Figure 7 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae Figure 7 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae Figure 7 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae 29 29 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Figure 8 Mean of repellency effect of different extracts on Myzus persicae Figure 8 Mean of repellency effect of different extracts on Myzus persicae References [1] Adhikari, K., Bhandari, S., Niraula, D. & Shrestha, J., (2020). Use of neem (Azadirachta indica A. Juss) as a biopesticide in agriculture. Journal of Agriculture and Applied Biology, 1(10), pp. 100-117. [1] Adhikari, K., Bhandari, S., Niraula, D. & Shrestha, J., (2020). Use of neem (Azadirachta indica A. Juss) as a biopesticide in agriculture. Journal of Agriculture and Applied Biology, 1(10), pp. 100-117. [2] Adusei, S. & Azupio, S., (2022). Neem: A Novel Biocide for Pest and Disease Control of Plants. Journal of Chemistry, p. 12. [3] Qasim Ahmed, Manjree Agarwal, Ruaa Al-Obaidi, Penghao Wang, Yonglin Ren (2021). Evaluation of Aphicidal Effect of Essential Oils and Their Synergistic Effect against Myzus persicae (Sulzer) (Hemiptera: Aphididae). molecules, 26(10). [4] Ali, H., Ameer, S., Qasim, M., Fiaz, S., Ali, S., Noor shah, A., Zaheer, S., ALI, B., Nawaz, M., Ali, Y., & Ahmad, N. (2022). Efficacy of Botanical Plant Extracts on the Population Dynamics of Cotton Aphid, Aphis gossypii Glover (Hemiptera; Aphididae), Journal of Bioresource Management, 9 (2). ISSN: 2309-3854 online [5] Baig, B. & Yousaf, S., (2021). Phytochemical Screening of Neem and Black Pepper for Bio efficacy against Insect Pests of Okra and Potato. Sarhad Journal of Agriculture, 37(2), pp. 697-705. [6] Bhatnagar, A., Somi, A. K. & Chander, S., (2012). Buildup of Myzus persicae and development of mosaics on potato in central India. Potato Journal 39(2):208-210, p. 3. [7] Bhuiyah, M., Karim, A., Islam, B. & Alam, M., (2003). Control of pulse beetle in stored chickpea and lentil by treating sacks with methanol extract of some selected botanicals. Bangl J Entomol., Volume 13, pp. 59-69. [8] CABI, (2021). Myzus persicae (green peach aphid). CABI Campendium. [8] CABI, (2021). Myzus persicae (green peach aphid). CABI Campendium. [9] Déla, M. A., Ketoh, K. G., Amevoin, K. & Ameline, A., (2014). Evaluation of neem leaves-based preparations as insecticidal agents against the green peach aphid, Myzus persicae. African Journal of Agricultural Research, 9(2), pp. 1344-1352. [10] Emden, H. v. & Harrington, R., (2017). Aphids as crop pest. CABI. [10] Emden, H. v. & Harrington, R., (2017). Aphids as crop pest. CABI. [11] Erdogan P, Aksu P, Esin Kilinc G, Kahyaoglu Murat, Numan E Babaroglu (2020). Formulation of Allium sativum L. and Melia azedarach L. plant extracts and their effects on Myzus persicae Sulzer, 1776 (Hemiptera: Aphididae). J Bacteriol Mycol Open Access. 2020;8(3):72‒76. DOI: 10.15406/jbmoa.2020.08.00277. [12] Frantz, J. D., Gardner, J., Hoffmann, M. P. 5. Conclusion In conclusion, the conducted experiment confirmed the high effectiveness of neem against Myzus persicae, with a repellency rate exceeding that of clove and black pepper extracts. However, while clove and black pepper demonstrated notable repellent properties, bay leaf extract emerged as the least effective bio-pesticide in combating aphids. These findings underscore the potential of neem as a natural and potent solution for aphid control, highlighting the importance of continued research into botanical alternatives for pest management in agriculture. 4. Discussion In a recent study, the efficacy of neem against aphids was examined, revealing a repellency rate of 68.9%. This finding underscores the potential of neem as a natural and effective means of aphid control in agricultural settings. In another recent study (Hartati, 2020), the synergistic effect of a blend containing clove and neem extracts was assessed, resulting in an observed repellency rate of more than 30.33% against pest. The combined utilization of biopesticides, specifically A. indica and E. globulus, at a concentration of 50%, has proven highly efficacious in combating wheat aphids (Baig & Yousaf, 2021). Furthermore, the synergistic potency of these botanicals surpasses their individual effectiveness when applied alone (Table2 and Figure7). In the recent scientific investigation, (Adhikari, et. al., 2020) empirical data indicated that neem exhibits an 83% repellent efficacy, thereby providing further validation for the results obtained in the conducted experiment. In (Lacotte, et. al., 2023) it was demonstrated that the repellent efficacy of clove, black pepper and bay leaves is measured at 40%, 35% and 23.3% respectively, thus providing empirical validation for the outcomes of the conducted experiment (Table3 and Figure8). 30 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Statement of informed consent Informed consent was obtained from all individual participants included in the study. Disclosure of conflict of interest Disclosure of conflict of interest Disclosure of conflict of interest The authors have no any conflict of interest for publishing this article. The authors have no any conflict of interest for publishing this article. Acknowledgments The author is thankful to the in-charge Prof. (Mrs.) Chitra Singh, Department of Zoology and Principal Prof. (Mrs.) Panzy Singh of Isabella Thoburn College, Lucknow all the necessary laboratory facilities and support. World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 References Jahn, M. M., Frantz JD, Gardner J, Hoffmann MP, Jahn M.M (2004). Greenhouse screening of Capsicum accessions for resistance to green peach Aphid (Myzus persicae). hortscience, 39(6), pp. 1332-1335. 31 31 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 [13] Khalid Haldi, Eugenio E Oliveria, Leda R A Faroni, Daniela C Guedes, Natalie N S Miranda (2015). Sublethal exposure to clove and cinnamon essential oils induces hormetic-like responses and disturbs behavioral and respiratory responses in Sitophilus zeamais (Coleoptera: Curculionidae) Journal of eco entomology, 108(6). [14] Hartati, S., (2020). Effectiveness of citronella, clove, and neem essential oil mix formulas against budok disease of patchouli plant. IOP Conference Series: Earth and Environmental Science, Volume 458, pp. 012-013. [15] Kareem Ullah, R. M., Gao, F., Sikandara, A. & Haiyan, W., (2023). Insights into the Effects of Insecticides on Aphids (Hemiptera:Aphididae):Resistance Mechanisms and Molecular Basis. international journal of molecular science, p. 18. [16] Kaur, M. KK Chahal, Amit Kumar, Ramandeep Kaur and Urvashi. (2018). Nematicidal activity of bay leaf (Laurus nobilis L.) essential oil and its components against Meloidogyne incognita. Journal of Entomology and Zoology Studies, 6(2), pp. 1057-1064. [17] Khalid, M. R., Aqueel, M. A., Majeed, M. Z. & Raza, A. B. M., (2020). In-vitro Response of Cereal Aphid Sitobion avenae (Homoptera: Aphididae) against some Selected Botanical Extract. Journal of Agriculture and Food, 1(2), pp. 1-12. [18] LacotteV., Marjolaine Rey, Sergio Peignier, PierreEdouard Mercier, Isabelle Rahioui, Catherine Sivignon, Lionel Razy, Sylvain Benhamou, Sébastien Livi, Pedro da Silva. (2023). Bioactivity and chemical composition of forty plant essential oils against the pea aphid Acyrthosiphon pisum revealed peppermint oil as a promising biorepellent. Industrial Crops and Products, Volume 197. [19] Madanat, H. M., Al Antary, T. M. & Abu Zarqa, M. H., (2016). Toxicity of six Ethanol plant extracts against the green peach aphid Myzus persicae Sulzer (Homoptera: Aphididae). Fresenius Environmental Bulletin, 25(3), pp. 706- 718. [20] Murray, B. I., (1994). Insect growth regulating effects of neem extract and azadirachtin on aphids. Entomologia Experimentalis et Applicata, Volume 72, pp. 77-84. [21] Santos, T. M. d., Costa, N. P., Torres, A. L. & Arlindo Júnior, A. L., (2004). Effect of neem extract on the cotton aphid. Core, Volume 39, pp. 1071-1076. [22] Scott, G. & Saurez, V., (2011). Growth rates for potato in India 1961-2009 and their implications for industry. potato J. [23] Singh, R. & Agarwal, R., (2022). References Association of the aphids (Insecta: Homoptera: Aphididae) with monocots flowering plants in India. International Journal of Biological Innovations, 4(2), pp. 252-281. [24] Sonowal, S., Rajkumari, P., Borah, N., Bora, B., Neog, P. P., & Borah, B. K. (2023). Efficacy of Different Essential Oils Against Nymphal Stage of Aphid (Aphis craccivora Koch) in Green Gram (L.). International Journal of Environment and Climate Change, 13(12), 473–478. https://doi.org/10.9734/ijecc/2023/v13i123704 [25] Tobing, O. L., Mulyaningsih, Y. & Safitri, A. D., (2023). The effect of concentration and frequency of neem leaf extract on aphid attacks on chili plants. Indonesian Journal of applied research, 4(2), pp. 146-158. [26] Pedro F.S. Toledo, Luis O. Viteri Jumbo, Sarah M. Rezende, Khalid Haddi, Bruno A. Silva, Tarcísio S. Mello, Terezinha M.C. Della Lucia, Raimundo W.S. Aguiar, Guy Smagghe, Eugenio E. Oliveira. (2020). Disentangling the ecotoxicological selectivity of clove essential oil against aphids and non-target ladybeetles, Science of The Total Environment, Volume 718,137328, ISSN 0048-9697, https://doi.org/10.1016/j.scitotenv.2020.137328. 32 32
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И.М. Микайлов, П.В. Григорьев, Д.А. Пташников, С.В. Майков ФГБУ «Российский научно-исследовательский институт травматологии и ортопедии им. Р.Р. Вредена» Минздрава России Санкт-Петербург, Россия Цель исследования – оценить результаты эндопротезирования плечевого сустава после радикальной резекции опу- холи проксимального отдела плечевой кости, а также сравнить полученные функциональные результаты с исходами эндопротезирования плечевого сустава у пациентов с обширным повреждением проксимального отдела плечевой кости неопухолевого генеза. у Материал и методы. Основную группу составили 38 пациентов, прооперированных в РНИИТО им. Р.Р. Вредена в период с 2001 по 2013 г., из них замещение пострезекционного дефекта однополюсными эндопротезами проводилось у 26 (68%) человек, ревизионными и модульными системами с реверсивными головками – у 12 (32%). Контрольную группу составили 46 пациентов с обширным поражением проксимального отдела плечевой кости не- опухолевого генеза. Сроки наблюдения составили от 7 месяцев до 9 лет. Оценка функциональных результатов прово- дилась в сроки от 6 месяцев до года. Результаты. Анализ семилетней выживаемости по методу Каплана – Мейера, проведённый для пациентов с первич- но злокачественными опухолями проксимального отдела плеча (25 больных), составил 77%. В основной группе среднее значение функциональных результатов по шкале MSTS составило 77,7%. Однополюсное эндопротезирование показало плохие результаты как в контрольной (61,3% по шкале MSTS, 60,7 по шкале Neer), так и в основной (67,7% – MSTS, 61,1 – Neer) группах. ) ру Заключение. На сегодняшний день методом выбора, не ухудшающим онкологическую составляющую лечения паци- ентов с опухолевым поражением плечевого сустава, является его эндопротезирование модульными реверсивными систе- мами в сочетании с дополнительными средствами фиксации мягких тканей. Ключевые слова: опухоли плечевого сустава, эндопротезирование плечевого сустава, модульные эндопротезы. оригинальные статьи оригинальные статьи т р а в м а т о л о г и я и о р т о п е д и я р о с с и и оригинальные статьи ограничения амплитуды движения (особенно отведения и наружной ротации) встречалось в 95% случаев и было связано с болевым син- дромом либо механическим препятствием. Патологические переломы и литическое разру- шение кости произошли у 14 (36,9%) пациен- тов: 9 (23,7%) – с патологическим переломом хирургической шейки плеча, 5 (13,2%) – с тотальной литической деструкцией прокси- мального отдела плечевой кости. ограничения амплитуды движения (особенно отведения и наружной ротации) встречалось в 95% случаев и было связано с болевым син- дромом либо механическим препятствием. Патологические переломы и литическое разру- шение кости произошли у 14 (36,9%) пациен- тов: 9 (23,7%) – с патологическим переломом хирургической шейки плеча, 5 (13,2%) – с тотальной литической деструкцией прокси- мального отдела плечевой кости. Так, по данным литературы, использование аллотрасплантата с целью рефиксации ротатор- ной манжеты обеспечивает лучшую функцию оперированной конечности, однако сопряжено с такими осложнениями, как перелом аллотран- сплантата и инфекции [5, 10, 17]. Использование аутотрансплантата из мало- берцовой кости – технически сложная опера- ция, требующая соответствующего уровня от оперирующего хирурга и дающая далеко не лучшую функцию. Самым частым осложнени- ем при этом виде оперативного лечения явля- ется перелом трансплантата [10]. Перед операцией проводились следующие виды обследования: –  онкологическое, когда при отсутствии на предоперационном этапе результатов гистоло- гии и данных о распространении опухоли вы- полняли детальный онкологический поиск, в том числе и пункционную биопсию; –  онкологическое, когда при отсутствии на предоперационном этапе результатов гистоло- гии и данных о распространении опухоли вы- полняли детальный онкологический поиск, в том числе и пункционную биопсию; Мы в своей практике для замещения постре- зекционного дефекта у данной категории паци- ентов используем ревизионные и модульные онкологические системы со стандартными и ре- версивными головками. у у –  общесоматическое – традиционное пред­ операционное исследование соматического ста- туса пациента, выявление сопутствующей пато- логии и подготовка его к операции; Цель исследования – оценить результаты эндопротезирования плечевого сустава после радикальной резекции опухоли проксимального отдела плечевой кости, а также сравнить полу- ченные функциональные результаты с исходами эндопротезирования плечевого сустава у пациен- тов с обширным повреждением проксимального отдела плечевой кости неопухолевого генеза. р –  местное, целью которого являлось опреде- ление локализации процесса, степени поражения кости, распространенности на окружающие ткани для определения объема операции, ее прогноза и подбора эндопротеза. Для этого выполняли рент- генологическое обследование пораженного плече- вого сустава в двух стандартных проекциях с за- хватом плеча до локтевого сустава. оригинальные статьи При обширных распространенных процессах выполняли КТ по- раженной области, дополняя это исследование ан- гиографией для выяснения характера кровоснаб- жения опухолевого очага и его взаимоотношения с магистральными сосудами. С целью уточнения степени распространенности процесса, взаимо- отношения опухолевых масс с мягкими тканями, окружающими сустав, и протяженности распро- странения опухоли по костному каналу выполня- ли МРТ данной области [1, 3, 4, 13, 16, 17]. –  местное, целью которого являлось опреде- ление локализации процесса, степени поражения кости, распространенности на окружающие ткани для определения объема операции, ее прогноза и подбора эндопротеза. Для этого выполняли рент- генологическое обследование пораженного плече- вого сустава в двух стандартных проекциях с за- хватом плеча до локтевого сустава. При обширных распространенных процессах выполняли КТ по- раженной области, дополняя это исследование ан- гиографией для выяснения характера кровоснаб- жения опухолевого очага и его взаимоотношения с магистральными сосудами. С целью уточнения степени распространенности процесса, взаимо- отношения опухолевых масс с мягкими тканями, окружающими сустав, и протяженности распро- странения опухоли по костному каналу выполня- ли МРТ данной области [1, 3, 4, 13, 16, 17]. Введение ния, за последний период значительно увеличи- лись [9, 11, 17, 18]. Это привело к росту неудачных исходов малоинвазивных операций, таких как внутрикостный остеосинтез. Повысилось коли- чество ревизий на фоне прогрессирования ло- кального литического опухолевого процесса и, как следствие, нестабильности конструкции. В связи с этим выросла актуальность более ради- кальных первичных операций [16–18]. ния, за последний период значительно увеличи- лись [9, 11, 17, 18]. Это привело к росту неудачных исходов малоинвазивных операций, таких как внутрикостный остеосинтез. Повысилось коли- чество ревизий на фоне прогрессирования ло- кального литического опухолевого процесса и, как следствие, нестабильности конструкции. В связи с этим выросла актуальность более ради- кальных первичных операций [16–18]. Проксимальный отдел плечевой кости явля- ется достаточно частой локализацией первич- ных опухолей и метастатических поражений костей [1, 3, 9, 11, 17, 18]. Органосохраняющие операции при опухоле- вом поражении проксимального отдела плечевой кости у взрослых пациентов всегда сопряжены со значительными хирургическими трудностя- ми. Чаще всего это связано с распространением опухолевого процесса за пределы кости, что при- водит к необратимому поражению ротаторной манжеты (надостная, подостная, малая круглая и подлопаточная мышцы) и капсулы плечевого сустава, в результате чего при выполнении резек- ции опухоли теряются основные стабилизаторы плечевого сустава. В то же время современные исследования показывают, что при использова- нии реверсивного эндопротеза существует воз- можность восстановления функции плечевого сустава в тех случаях, когда удается сохранить дельтовидную мышцу и ее иннервацию без ущер- ба для онкологического результата (сохраняя от- рицательный хирургический край) [1, 4, 15, 16]. р р [ ] На сегодняшний день существует целый ряд органосохраняющих операций, используемых при опухолевом поражении проксимального отдела плечевой кости: аллопластика прокси- мального отдела плеча в сочетании с накостным остеосинтезом пластиной; использование алло- трансплантата в сочетании с эндопротезом пле- чевой кости; изолированное использование эндо- протезов плечевого сустава, как однополюсных, так и реверсивных; аутопластика дефекта васку- ляризированным трансплантатом малоберцовой кости в сочетании с накостным остеосинтезом. Все эти методики широко используются онко- ортопедами, и каждая имеет ряд преимуществ и недостатков [5, 10, 17]. б р рур р ) [ ] Средние сроки выживаемости пациентов с вторичным поражением костей, вызванным раз- витием комплексного противоопухолевого лече- Выбор того или иного вида операции зависит от предпочтений хирурга, его опыта, наличия банка донорской кости. т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) 27 2014 – 4 (74) оригинальные статьи Материал и методы В клинике РНИИТО им. Р.Р. Вредена в пе- риод с 2001 по 2013 г. было выполнено 38 ор- ганосохраняющих операций пациентам с опу- холевым поражением проксимального отдела плечевой кости. Возраст пациентов составил от 15 лет до 71 года (38,5±3,34). Мужчин было 10 (26,3%), женщин – 28 (73,7%). Распределение по нозоло- гии: хондросаркома – 5 (13,2%) случаев; ГКО – 10 (26,3%); остеосаркома – 3 (7,9%); саркома Юинга – 1 (2,6%); плазмацитома – 1 (2,6%); лимфо- ма кос­ти – 1 (2,6%); гемангиоэндотелиома – 1 (2,6%); метастатическое поражение кости – 10 (26,3%), из них рак молочной железы (РМЖ) – 5 (13,2); рак почки – 3 (2,6%), рак легких – 2 (5,2%); аневризмальная костная киста – 3 (7,9%), хондробластома – 3 (7,9%). [ ] Все пациенты были условно разделены на три группы: 1-я группа. Пациенты с опухолями прокси- мального отдела плечевой кости, не выходящи- ми за пределы кости и не распространяющимися по костному каналу ниже уровня прикрепления дельтовидной мышцы (рис. 1). Все пациенты получали соответствующую неоадъювантную, химио- и/или лучевую тера- пию согласно стандартным протоколам лече- ния. Хондросаркомы и доброкачественные но- вообразования плечевой кости лечили только хирургическим методом. 2-я группа. Пациенты с опухолями, поражаю- щими проксимальный отдел плеча, с мягкоткан- ным компонентом, выходящим за пределы кости, с вовлечением в опухолевый процесс ротаторной манжеты, но не распространяющимися ниже при- крепления дельтовидной мышцы (рис. 2). Сроки наблюдения за пациентами составили от 7 месяцев до 9 лет, средний срок – 52 месяца. 3-я группа. Пациенты с опухолями, поража- ющими проксимальный отдел плеча, с мягко­ тканным компонентом, выходящим за пределы кости, с вовлечением в опухолевый процесс ро- таторной манжеты, распространяющимися ниже прикрепления дельтовидной мышцы (рис. 3). Клинически опухолевое поражение прок- симального отдела плечевой кости у всех пациентов характеризовалось постоянным болевым синдромом, усиливающимся при дви- жении. Нарушение функции сустава в виде 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 28 оригинальные статьи Рис. 1. Рентгенограммы пациента Л., 62 лет, с метастатическим поражением головки плечевой кости, в анамнезе рак почки: а, б – до операции; в, г – после операции а б в г в г б г в а Рис. 1. Рентгенограммы пациента Л., 62 лет, с метастатическим поражением головки плечевой кости, в анамнезе рак почки: а, б – до операции; в, г – после операции а б в Рис. 2. т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) Рис. 3. Пациентка Т., 48 лет, с гемангиоэндотелиомой проксимального отдела левой плечевой кости: а – ренгенограмма до операции; б – КТ с ангиографией до операции; в – рентгенограмма после операции т р а в м а т о л о г и я и о р т о п е д и я р о с с и и Материал и методы Рентгенограммы пациентки Ф., 56 лет, в анамнезе рак молочной железы, метастатическое поражение проксимального отдела левой плечевой кости: а, б – до операции; в – через 1,5 месяца после операции а б в Рис. 2. Рентгенограммы пациентки Ф., 56 лет, в анамнезе рак молочной железы, метастатическое поражение проксимального отдела левой плечевой кости: а, б – до операции; в – через 1,5 месяца после операции б а в б а в б в а Рис. 3. Пациентка Т., 48 лет, с гемангиоэндотелиомой проксимального отдела левой плечевой кости: а – ренгенограмма до операции; б – КТ с ангиографией до операции; в – рентгенограмма после операции а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) 2014 – 4 (74) 29 оригинальные статьи Пациентам 1-й группы выполнялась резек- ция пораженного проксимального отдела пле- чевой кости с сохранением вращательной ман- жеты (16 человек). Пациетам 2-й и 3-й групп выполнялась объемная резекция очага ново- образования с соблюдением онкологических принципов (22 человека). Величина резекции составила от 5 до 17 см. женное дегенеративное поражение ротаторной манжеты плеча. 30 пациентам был установлен однополюсный протез, 16 пациентам – ревер- сивный эндопротез. Особенности техники операции. Имплантацию эндопротеза осуществляли в положении больного на спине в положении «пляжного кресла» с полностью свободной опе- рируемой конечностью. Для замещения пострезекционных дефек- тов проксимального отдела плечевой кости у 26 (68%) пациентов использовались ревизионные ножки цементной фиксации с однополюсными головками, у 12 (32%) – ревизионные и стан- дартные ножки цементной фиксации с ревер- сивными головками. р ру Мы использовали два варианта доступа, применяемых при стандартном эндопротези- ровании: трансдельтовидный или дельтовидно- пекторальный [2, 11, 14, 15, 18]. При наличии большого мягкотканного компонента опухоли для увеличения обзора и облегчения выделе- ния опухоли мы выполняли акромионтомию. Резекцию проксимального отдела плечевой кос­ ти производили в пределах здоровых тканей. В случаях, когда операция проводилась по поводу злокачественного новообразования, остеотомию плеча выполняли из расчета захвата 2–3 см ви- зуально здорового участка кости [2, 9, 14, 17]. Выполнив остеотомию, отправляли содержимое костномозгового канала с края опила на срочное цитологическое исследование. Удостоверившись в достаточности уровня резекции, абластично, в пределах здоровых тканей, проводили выделе- ние проксимального отдела плеча с полным его удалением единым блоком вместе с головкой и капсулой сустава. Образовавшийся пострезек- ционный дефект кости замещали длинной реви- зионной ножкой цементной фиксации или мо- дульным эндопротезом. Материал и методы Глубина посадки ножки эндопротеза составляла от 7 до 12 см (в среднем 9 см). При использовании ревизионной ножки эндопротеза выполняли пластику ее не погру- женной в кость части костным цементом с анти- биотиком. С целью рефиксации отсечённых со- храненных мышц в цемент внедряли лавсановые нити. Использование этой методики способство- вало уменьшению полости, формирующейся пос­ле удаления опухоли, и стабилизации плече- вого сустава (рис. 4). Во всех случаях суставной отросток ло- патки не был вовлечен в опухолевый процесс. Пациентам выполнялся первый тип резекции по М.М. Малауэру [11, 13, 17]. Край опила плечевой кости не содержал опу- холевых клеток (R-0), подмышечный нерв не был вовлечен в опухолевый процесс либо по- врежден в ходе операции. Оценка результатов лечения больных про- водилась с учетом как ортопедических (ана- лиз динамики клинико-рентгенологической картины и функции сустава), так и онко- логических критериев (число местных ре- цидивов новообразований и выживаемость пациентов). Функциональный результат лечения оцени- вали по шкалам Musculoskeletal Tumour Society (MSTS) и Neer [7, 8, 12]. В клиническом раз- деле шкалы MSTS учитываются 6 параметров: боль, функция конечности, позиционирование руки, ловкость, подъем тяжести и эмоциональ- ное восприятие результата лечения. Каждый параметр оценивается по 5-балльной шкале, после чего по сумме набранных баллов вычис- ляется процент от максимально возможного количества баллов 30. Шкала Neer учитывает как восприятие пациентом функции опериро- ванной конечности, так и оценку амплитуды всех возможных движений. Максимальный результат – 100 баллов, хороший – более 89 баллов, удовлетворительный 80–89 баллов, неудовлетворительный 70–79 баллов, плохой – менее 70 баллов. Рис. 4. Использование цементной пластики и лавсановых нитей с целью рефиксации отсеченных мышц Контрольную группу составили 46 пациен- тов с обширным поражением проксимального отдела плечевой кости неопухолевого гене- за (асептический некроз, последствия много­ оскольчатых застарелых переломов головки плечевой кости), которым было выполнено эн- допротезирование в РНИИТО им. Р.Р. Вредена в период с 2006 по 2012 г. Дефект кости составил от 3 до 5 см, во всех случаях наблюдалось выра- Рис. 4. Материал и методы Использование цементной пластики и лавсановых нитей с целью рефиксации отсеченных мышц 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 30 оригинальные статьи У шести пациентов для рефиксации отсечён- ных мышц мы использовали тубы из полимер- ного волокна. После операции пациенты получали профи- лактическую антибактериальную и антикоагу- лянтную терапию. Дренажи удаляли на 2–5-е сутки после операции. Во всех случаях удалось достигнуть хороше- го натяжения мягких тканей, восстановить дли- ну конечности. Активизация больного начиналась на 2-е сутки после операции с участием специалистов по лечебной физкультуре. Рентгенологический контроль осуществлялся ежеквартально. В первой группе пациентов мы пытались максимально сохранить ротаторную манжету, что способствовало лучшей стабилизации сус­ тава и раннему восстановлению функции ко- нечности. После удаления опухолевого очага выполняли установку эндопротеза с ножкой цементной фиксации в положении головки 20–25° ретроверсии. Результаты Онкологический результат. Cроки наблюде- ния составили от 7 месяцев до 9 лет, в среднем 3,4 ± 0,7 года. Местный рецидив опухоли после резекции проксимального отдела плечевой кос­ ти и замещения дефекта эндопротезом отмечен в одном случае, что составляет 2,6% от обще- го числа больных. У данного пациента через 8 месяцев выявлено распространение новообра- зования (хондросаркомы) по мягким тканям в области оперативного вмешательства. Больной направлен в онкологический стационар для выполнения ампутации. В течение 18 месяцев пациент умер от прогрессирования онкологи- ческого заболевания. На момент контрольного осмотра в разные сроки (3 и 7 лет) умерли еще 2 пациента из-за прогрессирования основного заболевания (метастатического поражения лег- ких), из них один случай остеосаркомы и один случай саркомы Юинга. Анализ семилетней вы- живаемости по методу Каплана – Мейера, про- ведённый для пациентов с первично злокаче- ственными опухолями проксимального отдела плеча (25 больных), составил 77% (рис. 5). Во второй группе больных ввиду вовлечения в опухолевый процесс ротаторной манжеты в ряде случаев мы использовали эндопротезы с реверсивными головками. В третьей группе пациентов принципиаль- ным моментом было восстановление места прикрепления дельтовидной мышцы, по воз- можности большой грудной мышцы, что спо- собствовало стабилизации эндопротезапроте- за, а также в значительной степени влияло на функциональный результат. Имплантацию метаглена и гленосферы осу- ществляли по стандартной технологии с допол- нительной фиксацией 3–4 винтами. Послеоперационное ведение больных. Конечность фиксировали на отводящей шине, что облегчало уход за больным в раннем после- операционном периоде, а также способствовало формированию первичных рубцов в правиль- ном положении конечности. Рис. 5. Выживаемость пациентов со злокачественными опухолями проксимального отдела плечевой кости Рис. 5. Выживаемость пациентов со злокачественными опухолями проксимального отдела плечевой кости т р а в м а т о л о г и я и о р т о п е д и я р о с с и и т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) 31 31 2014 – 4 (74) оригинальные статьи Оценка функциональных результатов по MSTS проводилась в сроки от 6 месяцев до года после операции у 38 пациентов. Результаты сравнительной оценки функци- ональных результатов, полученные в основной (онкологической) и контрольной (ортопедиче- ской) группах, представлены в таблице 3. Распределение функциональных результа- тов по группам отражено в таблице 1. ) ру , р Осложнения при эндопротезировании прок- симального отдела плечевой кости. Обсуждение Вывих (подвывих) эндопротеза. Про­филак­ тика вывиха обеспечивается за счет фиксации сохраненных мышц (ротаторный манжеты) непо- средственно к протезу, что дает сбалансированное натяжение мягких тканей, центрацию головки эн- допротеза, объем движений, а также способствует стабилизации сустава. Для этого желательно ис- пользовать толстые нерезорбируемые нити. Мы в ряде случаев использовали полимерные тубы, что значительно упрощало восстановление точек прикрепления мышечного аппарата, ускоряло восстановление функции конечности. Еще одним фактором стабилизации является восстановление длины конечности, что дает хорошее натяжение как мышц, так и фасции и способствует равно- мерному распределению нагрузки. С целью про- филактики вывиха эндопротеза головка должна быть установлена в ретроверсии от 15 до 30° [2, 11, 13, 14, 16]. Функциональные результаты. Как показал анализ литературы, оценка функциональных ре- зультатов у данной категории пациентов прово- дится исключительно по шкале MSTS. Для более детального изучения данного вопроса мы реши- ли ввести в исследование контрольную группу пациентов с поражением плечевого сустава не- опухолевого генеза и оценить функциональный результат при помощи шкал MSTS и Neer. р у р По данным различных авторов, при использо- вании модульных онкологических эндопротезов плечевого сустава среднее значение функцио- нальных результатов по MSTS колеблется от 65% до 88% [1, 2, 6, 17, 18, 19]. По результатам нашего исследования, этот показатель составил 87,6% при использовании реверсивных эндопротезов и 67,7% при использовании однополюсных эн- допротезов, средний показатель – 77,7%. Однако стоит отметить, что на функцию реверсивного эндопротеза значительно влияет сохранность естественного места прикрепления дельтовид- ной мышцы и ее целостность. Как показало наше исследование, суммарный результат в 1-й и 2-й группах (пациенты с сохраненной дельтовидной мышцей) составил 93,1% по MSTS. В нашем исследовании вывих протеза отме- чен у 1 (2,6%) пациента через 6 месяцев после операции. Пациенту было выполнено ревизи- онное оперативное вмешательство с заменой эндопротеза на реверсивный. Контрольный ос- мотр через 6 месяцев показал хорошую функ- цию – 83% по MSTS. ) В контрольной группе функциональные по- казатели для реверсивных эндопротезов соста- вили 95% по MSTS и 89,25 по шкале Neer. Для пациентов основной группы при использовании реверсивных эндопротезов функциональный ре- зультат составил 87,6% по MSTS и 83,5 по Neer. Разницу в показателях можно объяснить резким снижением функции в 3-й группе «онкологи- ческих» пациентов, которым выполнялась ре- фиксация дельтовидной мышцы к эндопротезу. Суммарный результат в 1-й и 2-й группах сопо- ставим с результатами в контрольной группе. Инфекционные осложнения. По данным раз- личных авторов, инфекционные осложнения являются достаточно распространенной при- чиной, приводящей к ревизионным операциям и удалению эндопротеза. Результаты Общее ко- личество осложнений, приведших к реэндо- протезированию, составило 3 (7,9%) случаев. Инфекционные осложнения развились у од- ного пациента на фоне нарушения двигатель- ной активности на 14-е сутки после операции. Пациенту было выполнено ревизионное опе- ративное вмешательство, протез сохранен. Мы наблюдали один случай вывиха однополюс- ного эндопротеза, развившегося через 6 ме- сяцев после первичного эндопротезирования. Также был выявлен один случай асептической нестабильности однополюсного эндопротеза, развившейся через 3,5 года после первичного оперативного вмешательства, потребовавший ревизионного эндопротезирования с заменой протеза на реверсивный. Разрушения конструк- ции мы не наблюдали. Таблица 1 Средние функциональные результаты онкологического эндопротезирования по шкале MSTS Группа Однополюсный эндопротез Реверсивный эндопротез n оценка, % n оценка, % 1 13 70,4 3 95,6 2 8 68,5 6 90,6 3 5 64,2 3 76,6 В среднем по группам – 67,7 – 87,6 n – количество пациентов. Таблица 1 Средние функциональные результаты онкологического эндопротезирования по шкале MSTS n – количество пациентов. По шкале Neer результаты однополюсного эндопротезирования у 23 пациентов были оце- нены в разные сроки наблюдения (в среднем 3 года), а результаты применения реверсивных эндопротезов оценивались у 12 пациентов в сроки от 6 месяцев до года (табл. 2). При использовании однополюсных эндо- протезов в отдаленном периоде (через 2–3 года с момента операции) мы наблюдали подвывих головки эндопротеза у 8 (35%) больных, что не повлияло на результат. Таблица 2 NEER Таблица 2 Средняя оценка функциональных результатов онкологического эндопротезирования по шкале NEER Группа Однополюсный эндопротез Реверсивный эндопротез количество пациентов средний результат, баллы количество пациентов средний результат, баллы 1 13 68,1 (плохой) 3 90,6 (хороший) 2 7 63 (плохой) 6 87,5 (удовлетворительный) 3 3 52,2 (плохой) 3 72,6 (неудовлетворительный) В среднем по группам – 61,1 (плохой) 12 83,5 (удовлетворительный) Таблица 3 Средняя оценка функциональных результатов эндопротезирования по шкалам Neer и MSTS в основной и контрольной группах Группа Шкала MSTS, % Шкала Neer, баллы Однополюсное эндопротезирование Реверсивное эндопротезирование Однополюсное эндопротезирование Реверсивное эндопротезирование Контрольная 61,3 95 60,7 плохой результат 89,25 хороший результат Основная 67,7 87,6 61,1 плохой результат 83,5 удовлетворительный результат т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 32 оригинальные статьи Обсуждение Потери онкологиче- ских эндопротезов плечевого сустава вслед- ствие развития местного инфекционного вос- палительного процесса составляют от 5 до 15% [9, 11, 13, 14, 17, 18]. В нашем исследовании парапротезная ин- фекция наблюдалось у 1 (2,6%) пациента. Срок развития осложнения составил 14 дней. Нам удалось купировать инфекционный процесс путем хирургической обработки раны без уда- ления эндопротеза. Однополюсное эндопротезирование показа- ло плохой результат как в контрольной (61,3% MSTS, 60,7 Neer), так и в основной (67,7% MSTS, 61,1 Neer) группах. Асептическая нестабильность эндопротеза. Асептическая нестабильность эндопротеза. Нестабильность эндопротеза отмечена у 1 (2,6%) пациента через 3,5 года после операции. Данному пациенту первоначально был уста- новлен однополюсный эндопротез на ножке цементной фиксации. Нами было выполнено ревизионное эндопротезирование с установкой реверсивного эндопротеза на длинной ревизи- онной цементной ножке. По данным различных авторов, асептическая нестабильность у этой категории пациентов встречается в пределах 1,5–5,5% [11, 13, 14, 16–18]. О Это дает возможность с уверенностью говорить о том, что использование реверсивных систем у пациентов с опухолевым поражением прокси- мального отдела плеча – перспективная методи- ка, которая не только сохраняет конечность па- циенту, но и восстанавливает хорошую функцию, соизмеримую с функцией у пациентов, подверг- шихся эндопротезированию плечевого сустава с патологией неопухолевой природы. Однако из-за малого количества и небольших сроков наблюде- ний не представляется возможным получить ста- тистически достоверные результаты, поэтому мы считаем необходимым продолжать работу в этом направление с целью накопления большего коли- чества материала и клинических наблюдений. Основными ортопедическими критериями, определяющими удачный исход оперативного лечения пациентов с объемными резекциями проксимального отдела плечевой кости, явля- р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 33 2014 – 4 (74) оригинальные статьи 3. Тепляков В.В., Карпенко В.Ю., Франк Г.А., Буланов А.А., Державин В.А., Шаталов А.М. Эндопротезирование при опухолевом поражении длинных костей. Российский он- кологический журнал: 2009; (5):11–14. ются: восстановление длины конечности, пра- вильная установка и надежная фиксация со- ответствующего эндопротеза, восстановление точек естественного прикрепления мышц. Все это позволяет сохранить конечность и достаточ- ную для нормальной жизни функцию плечевого сустава. Стоит отметить, что при использовании ревизионных (длинных) ножек эндопротеза хи- рург может столкнуться с определенными тех- ническими трудностями во время их установки. Заключение g 9. Fuhrmann R.A., Roth A., Venbrocks R.A. Salvage of the upper extremity in cases of tumorous destruction of the proximal humerus. J Cancer Res Clin Oncol. 2000; 126:337–344. Применяемая нами методика эндопротези- рования плечевого сустава с использованием реверсивных систем показала в основном от- личные и хорошие функциональные результа- ты, вполне сопоставимые с результатами эндо- протезирования, выполненного у пациентов с поражениями плечевого сустава неопухолевого генеза. В то же время однополюсное эндопроте- зирование показало плохие результаты в обеих исследуемых группах. Из этого следует, что на сегодняшний день методом выбора, не ухудша- ющим онкологическую составляющую лечения этой категории пациентов, является эндопроте- зирование плечевого сустава модульными ре- версивными системами в сочетании с дополни- тельными средствами фиксации мягких тканей. 10. Manfrini M., Vanel D., De Paolis M., Malaguti C., Innocenti M., Ceruso M., Capanna R., Mercuri M. Imaging of vascularized fibula autograft placed inside a massive allograft in reconstruction of lower limb bone tumors. Am J Roentgenol. 2004;15(4):963–970. 11. Mayilvahanan N., Paraskumar M., Sivaseelam A., Natarajan S. Custom mega-prosthetic replacement for proximal humeral tumours. Int Orthop. 2006;30:158–162. p 12. Neer C.S. Displaced proximal humeral fractures. I. Classification and evaluation. J Bone Joint Surg Am. 1970;52:1077–1089. 13. Potter B.K., Adams S.C., Pitcher J.D. Jr, Malinin T.I., Temple H.T. Proximal humerus reconstructions for tumors. Clin Orthop. 2009;467:1035–1041. 14. Raiss P., Kinkel S., Sauter U., Bruckner T., Lehner B. Replacement of the proximal humerus with MUTARS tumor endoprostheses. Eur J Surg Oncol. 2010;36:371–377. p g 15. Rispoli D.M., Athwal G.S., Sperling J.W., Cofield R.H. The anatomy of the deltoid insertion. J Shoulder Elbow Surg. 2009;18:386–390. 16. Rödl R.W., Gosheger G., Gebert C., Lindner N., Ozaki T., Winkelmann W. Reconstruction of the proximal humerus after wide resection of tumours. J Bone Joint Surg Br. 2002;84:1004–1008. Обсуждение При погружении ножки в костный канал необ- ходимо точно выставить ротацию компонента и глубину посадки, восстановить дефект кости. Ошибка на любом из этих этапов может при- вести к нестабильности сустава и повлиять на функцию конечности. В то же время использо- вание модульных систем в значительной степе- ни упрощает восстановление длины конечности и пространственное расположение компонентов эндопротеза [11, 13, 14, 17]. у 4. Тепляков В.В., Карпенко В.Ю., Шаталов А.М., Бухаров В.А., Державин В.А., Мысливцев И.В. Эндопротезирование длинных трубчатых костей и су- ставов при дефиците мягких тканей. Вестник москов- ского онкологического общества. 2011 (4). Режим до- ступа netoncology.ru/.uploads/press/1191/V-11-0495.file. doc (дата посещения 22.09.2014) 5. Aponte-Tinao L.A., Ayerza M.A., Muscolo D.L., Farfalli G.L. Allograft reconstruction for the treatment of musculoskeletal tumors of the upper extremity. Sarcoma. 2013;2013:925413. 6. Cannon C.P., Paraliticci G.U., Lin P.P., Lewis V.O., Yasko A.W. Functional outcome following endoprosthetic reconstruction of the proximal humerus. J Shoulder Elbow Surg. 2009;18:705–710. 7. Enneking W.F., Dunham W., Gebhardt M.C., Malawer M., Pritchard D.J. A system for the functional evaluation of reconstructive procedures after surgical treatment of tumors of the musculoskeletal system. Clin Orthop. 1993;(286):241–246. ( ) 8. Enneking W.F. Modification of the system for functional evaluation of surgical management of musculoskeletal tumors. In: Bristol–Myers/Zimmer Orthopaedic Symposium. Limb salvage in musculoskeletal oncology. New York: Churchill Livingstone; 1987. P. 626–639. Литература 1. Алиев М.Д., Соколовский В.А., Дзампаев А.З., Нисиченко Д.В., Сергеев П.С., Хестанов Д.Б. Отдаленные результаты применения мегапротезов в хирургии опухолей костей и суставов. Вестник московского онкологического обще- ства. 2011; (4). Режим доступа netoncology.ru/.uploads/ press/1191/V-11-0495.file.doc (дата посещения 22.09.2014) 1. Алиев М.Д., Соколовский В.А., Дзампаев А.З., Нисиченко Д.В., Сергеев П.С., Хестанов Д.Б. Отдаленные результаты применения мегапротезов в хирургии опухолей костей и суставов. Вестник московского онкологического обще- ства. 2011; (4). Режим доступа netoncology.ru/.uploads/ press/1191/V-11-0495.file.doc (дата посещения 22.09.2014) 17. Van de Sande M.A., Dijkstra P.D., Taminiau A.H. Proximal humerus reconstruction after tumour resection: biological versus endoprosthetic reconstruction. Int Orthop. 2011;15(9):1375–1380. ( ) 18. Yang Q., Li J., Yang Z., Li X., Li Z. Limb sparing surgery for bone tumours of the shoulder girdle: the oncological and functional results. Int. Orthop. 2010;15(6):869–875. p (д щ ) 2. Майков С.В. Пути повышения эффективности эндопротези- рования плечевого сустава. Дис. канд. мед. наук. СПб.; 2012. p (д щ ) 2. Майков С.В. Пути повышения эффективности эндопротези- рования плечевого сустава. Дис. канд. мед. наук. СПб.; 2012. Сведения об авторах: Микайлов Илкин Мугадасович – лаборант-­исследователь научного отделения нейроортопедии с костной онкологией; Григорьев Петр Владимирович – лаборант-­исследователь научного отделения нейроортопедии с костной онкологией; Пташников Дмитрий Александрович – д.м.н. профессор заведующий отделением № 18; Майков Сергей Валерьевич – младший научный сотрудник отделения спортивной травматологии и реабилитации СВЯЗЬ С АВТОРАМИ: СВЯЗЬ С АВТОРАМИ: СВЯЗЬ С АВТОРАМИ: e-mail: mim17@mail.ru (Микайлов И.М.) СВЯЗЬ С АВТОРАМИ: e-mail: mim17@mail.ru (Микайлов И.М.) Рукопись поступила 03.10.2014 т р а в м а т о л о г и я и о р т о п е д и я р о с с и и Рукопись поступила 03.10.2014 т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 34 оригинальные статьи Vreden Russian Research Institute of Traumotology and Orthopedics, St. Petersburg, Russia Vreden Russian Research Institute of Traumotology and Orthopedics, St. Petersburg, Russia The purpose of the study was to evaluate the results of surgical reconstructions of the proximal humerus after transarticular tumour resection, сcompare the functional results with the results of shoulder arthroplasty in patients with extensive damage of the proximal humerus of non-neoplastic origin. Material and methods. Between 2001 and 2013 38 proximal humeral reconstructions were performed in our clinic: using monopolar endoprostheses – 26 (68%) and modular systems with reversible head -12 (32%). The control group included 46 patients with extensive lesions of the proximal humerus of non-neoplastic origin, operated in our clinic in the period from 2006 to 2012. Term follow-up of patients ranged from 7 months to 9 years. Assessment of functional results was carried out in a period from six months to one year. Results. The seven-year survival according the Kaplan – Meier method for patients with primary malignant tumors of the proximal humerus (25 patients) was 77%. In the study group the average value of functional outcome MSTS score was 77.7%. Unipolar prosthesis showed a bad result, both in the control (61,3% MSTS, 60,7 Neer) and in the main (67,7% MSTS, 61,1 Neer) study groups. C l i C tl th th d f h i hi h d ’t i i th l i t f t t t ti t ith Results. The seven-year survival according the Kaplan – Meier method for patients with primary malignant tumors of the proximal humerus (25 patients) was 77%. In the study group the average value of functional outcome MSTS score was 77.7%. Unipolar prosthesis showed a bad result, both in the control (61,3% MSTS, 60,7 Neer) and in the main (67,7% MSTS, 61,1 Neer) study groups. Conclusion. Currently the method of choice which doesn’t impair the oncologic component of treatment patients with shoulder neoplastic lesions is its replacement with modular reversible systems in combination with additional soft tissue fixation. Conclusion. Currently the method of choice which doesn’t impair the oncologic component shoulder neoplastic lesions is its replacement with modular reversible systems in combination with ad Key words: tumors of the shoulder joint, shoulder prosthesis, modular endoprosthesis. 8. Enneking WF. Modification of the system for functional evaluation of surgical management of musculoskeletal tumors. In: Bristol–Myers/Zimmer Orthopaedic Symposium. Limb salvage in musculoskeletal oncology. New York: Churchill Livingstone; 1987. 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Dis. kand. med. nauk [Ways to improve the efficiency of replacement of the shoulder joint. Dr. med. nauk sci. diss.]. SPb.; 2012. 11. Mayilvahanan N, Paraskumar M, Sivaseelam A, Natarajan S. Custom mega-prosthetic replacement for proximal humeral tumours. Int Orthop. 2006;30:158–162. shoulder joint. Dr. med. nauk sci. diss.]. SPb.; 2012. 3. Teplyakov VV, Karpenko VYu, Frank GA, Bulanov AA, Derzhavin VA, Shatalov AM. Endoprotezirovaniye pri opukholevom porazhenii dlinnykh kostey [Endoprosthesis with neoplastic lesions of the long bones] Rossiyskiy pediatricheskiy zhurnal [Russian Journal of Pediatrics]. 2009; (5):11–14. p 12. Neer CS. Displaced proximal humeral fractures. I. Classification and evaluation. J Bone Joint Surg Am. 1970;52:1077–1089. g 13. Potter BK, Adams SC, Pitcher JD Jr, Malinin TI, Temple HT. Proximal humerus reconstructions for tumors. Clin Orthop. 2009;467:1035–1041. 4. Teplyakov VV, Karpenko VYu, Shatalov AM, Bukharov VA, Derzhavin VA, Myslivtsev IV. Endoprotezirovaniye dlinnykh trubchatykh kostey i sustavov pri defitsite myagkikh tkaney [Endoprosthesis of the long bones and joints with a deficit of soft tissues] Vestnik moskovskogo onkologicheskogo obshchestva [Internet]. 2011 (4). Available at: netoncology.ru/. uploads/press/1191/V-11-0495.file.doc (accessed 22.09.2014) 14. Raiss P, Kinkel S, Sauter U, Bruckner T, Lehner B. Replacement of the proximal humerus with MUTARS tumor endoprostheses. Eur J Surg Oncol. 2010;36:371–377. 15. Rispoli DM, Athwal GS, Sperling JW, Cofield RH. The anatomy of the deltoid insertion. J Shoulder Elbow Surg. 2009;18:386–390. 5. Aponte-Tinao LA, Ayerza MA, Muscolo DL, Farfalli GL. Allograft reconstruction for the treatment of musculoskeletal tumors of the upper extremity. Sarcoma. 2013;2013:925413 16. Rödl RW, Gosheger G, Gebert C, Lindner N, Ozaki T, Winkelmann W. Reconstruction of the proximal humerus after wide resection of tumours. J Bone Joint Surg Br. 2002;84:1004–1008. 6. т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) Author’s information: Mikailov Ilkin M. – research assistant of the scientific department of neuroorthopedics and bone tumors; Grigoriev Petr V. – research assistant of the scientific department of neuroorthopedics and bone tumors; h k A f h h d f d Mikailov Ilkin M. – research assistant of the scientific department of neuroorthopedics and bone tumors; Grigoriev Petr V. – research assistant of the scientific department of neuroorthopedics and bone tumors; Ptashnikov Dmitry A. – professor, the head of department N 18; Grigoriev Petr V. – research assistant of the scientific department of neuroorthopedics and bone tumors; Ptashnikov Dmitry A. – professor, the head of department N 18; g p p Ptashnikov Dmitry A. – professor, the head of department N 18; Ptashnikov Dmitry A. – professor, the head of department N 18; k h f d h b l d Maykov Sergei V. – researcher of sport trauma and rehabilitation department. Corresponding author: e-mail: mim17@mail.ru (Mikailov IM) I.M. Mikailov, P.V. Grigoriev, D.A. Ptashnikov, S.V. Maykov I.M. Mikailov, P.V. Grigoriev, D.A. Ptashnikov, S.V. Maykov т р а в м а т о л о г и я и о р т о п е д и я р о с с и и Corresponding author: e-mail: mim17@mail.ru (Mikailov IM) 35 р а в м а т о л о г и я и о р т о п е д и я р о с с и и 2014 – 4 (74) 2014 – 4 (74)
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Paradigma indiciário: contribuições para a investigação da construção das identidades de futuros professores de línguas
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10.5216/sig.v27i2.35551 10.5216/sig.v27i2.35551 10.5216/sig.v27i2.35551 * Professora Associada do Instituto de Letras da Universidade Federal da Bahia (UFBA). E-mail: liviarad@yahoo.com. Resumo Este trabalho trata das contribuições do paradigma indiciário para o estudo das identidades docentes e, particularmente, as dos futuros professores de línguas, com ênfase para os do espanhol. Com essa finalidade, explicitaremos em que consiste esse modelo e como pode contribuir para gerar maior inteligibilidade a respeito de como futuros professores se envolvem nos processos de construção identitária ao longo de seus percursos formativos. Sendo assim, neste artigo indicaremos algumas contribuições desse paradigma para o exame da problemática em foco, refletindo a respeito do seu potencial como construto metodológico de cunho qualitativo e interpretativista. Este trabalho trata das contribuições do paradigma indiciário para o estudo das identidades docentes e, particularmente, as dos futuros professores de línguas, com ênfase para os do espanhol. Com essa finalidade, explicitaremos em que consiste esse modelo e como pode contribuir para gerar maior inteligibilidade a respeito de como futuros professores se envolvem nos processos de construção identitária ao longo de seus percursos formativos. Sendo assim, neste artigo indicaremos algumas contribuições desse paradigma para o exame da problemática em foco, refletindo a respeito do seu potencial como construto metodológico de cunho qualitativo e interpretativista. Palavras-chave: paradigma indiciário, formação de professores de espanhol, identidades, Linguística Aplicada. Paradigma Indiciário: contribuições para a investigação da construção das identidades de futuros professores de línguas Lívia Márcia Tiba Rádis BAPTISTA* Lívia Márcia Tiba Rádis BAPTISTA* 1 Sobre o paradigma indiciário: detalhes e singularidades O historiador italiano Carlo Ginzburg (2001, p. 143-179) examina como, por volta do final do século XIX, emergiu, no campo das Ciências Humanas, um modelo epistemológico conhecido como paradigma indiciário, fundado na análise minuciosa do residual, do episódio, ou, ainda, da singularidade. Apresenta, entre outras questões, os princípios metodológicos desse modelo amplamente operante apesar de não ter sido teorizado de forma explícita, o que contribuiria conforme Ginzburg (2001, p. 143) para “sair dos incômodos da contraposição entre racionalismo e irracionalismo”. Nesta seção, portanto, revisitaremos em que consiste o paradigma indiciário e, com esse propósito, refletiremos sobre como ele pode orientar a compreensão de determinados aspectos relacionados à construção das identidades docentes, no âmbito da formação inicial de futuros professores de línguas, em especial, a espanhola. Remontando à história desse paradigma, Ginzburg (2001, p.143- 144) menciona que entre 1874 e 1876 surgiu uma série de artigos sobre pintura italiana na Zeitschrift für bildende Kunst assinada por um estudioso russo, Ivan Lermolieff e traduzida para o alemão pelo desconhecido Johannes Schwarze. Esses artigos propunham um método novo para a atribuição da autoria dos quadros antigos em que se recomendava examinar os pormenores considerados desprezíveis e, consequentemente, não observados pelos imitadores no original, fato que gerou polêmicas na época. Descobriu-se depois que o responsável por essa proposta tinha sido o historiador de arte Giovanni Morelli; razão pela qual passou a ser conhecido como método morelliano ou método indiciário de Morelli. De acordo com Ginzburg (2001, p. 145-146), além de Morelli, esse método foi empregado por Sherlock Holmes, personagem de Arthur Conan Doyle e por Freud. Ginzburg (2001, p.150-151) esclarece que o emprego do paradigma indiciário pelos três deu-se no final do século XIX, no período de 1870 a 1880, fato que coincide com a afirmação desse método no âmbito das Ciências Humanas. Ora, esse método proporcionou aos que dele se valeram captar uma realidade “mais profunda” e “de forma inatingível” através de uma análise pormenorizada de pistas: os sintomas (no caso de Freud), os indícios (no caso de Sherlock Holmes) e os signos pictóricos (no caso de Morelli). Contudo, as raízes desse paradigma se remontam aos primórdios de nossa história, uma vez que como ressalta Ginzburg (2001, p.151) por milênios o homem foi caçador. 1 Sobre o paradigma indiciário: detalhes e singularidades Durante inúmeras perseguições, ele aprendeu a reconstruir as formas e movimentos das presas invi- síveis pelas pegadas na lama, ramos quebrados, bolotas de esterco, tufos de pelos, plumas emaranhadas, odores estagnados. Aprendeu a farejar, registrar, interpretar e classificar pistas infinitesimais como fios de barba. Aprendeu a fazer operações mentais complexas com por milênios o homem foi caçador. Durante inúmeras perseguições, ele aprendeu a reconstruir as formas e movimentos das presas invi- síveis pelas pegadas na lama, ramos quebrados, bolotas de esterco, tufos de pelos, plumas emaranhadas, odores estagnados. Aprendeu a farejar, registrar, interpretar e classificar pistas infinitesimais como fios de barba. Aprendeu a fazer operações mentais complexas com 566 Baptista, L. M. T. R. Paradigma Indiciário... rapidez fulminante, no interior de um denso bosque ou numa clarei- ra cheia de ciladas. Ainda de acordo com Ginzburg (2001, p.152-155), “o caçador teria sido o primeiro a ‘narrar uma história’ porque era o único capaz de ler, nas pistas mudas (se não imperceptíveis) deixadas pela presa, uma série coerente de eventos”. Assim, foi nessa sociedade de caçadores que a narração e a leitura dos indícios se destacaram, mas foram igualmente empregadas nas antigas civilizações mesopotâmica e grega, o que sugere que esse saber indiciário não foi abandonado. Dando um salto temporal e situando-nos mais a frente, Ginzburg (2001, p. 143-179) observa que o paradigma indiciário, ao emergir no final do século XIX se defrontou com o galileano e, consequentemente, o cartesiano. Quanto ao modelo proposto por Descartes, sabemos que seguiu a tradição de Galileu e despontou nas sociedades modernas do Renascimento no século XVI e passou a ser amplamente incorporado à ciência. Consistiu em um conjunto institucionalmente articulado de práticas e saberes, norteado por uma concepção de indivíduo livre e racional, consciente de seus atos, sujeito de si e do conhecimento bem como capaz de descobrir as leis da natureza. Segundo Japiassú e Marcondes (2001, p. 194), essa concepção foi inaugurada por Francis Bacon, Galileu e Descartes e amadureceu, por exemplo, com as contribuições de Isaac Newton (1642-1727) no campo da Física, com a formalização da Mecânica de Galileu e, ainda, com as investigações em óptica e sobre a natureza da luz. 1 Sobre o paradigma indiciário: detalhes e singularidades Nietzsche questionou a noção de sujeito racional de Descartes, embora com os filósofos iluministas do século XVIII, a razão, a ciência e a lei natural tenham passado a ser o alicerce de uma verdade universal que levaria ao progresso da humanidade, libertando o homem da religião e do obscurantismo. Em suma, estabeleceu-se o primado da razão e da vontade. Ginzburg (2001, p. 143) contrapõe o modelo galileano e, por conseguinte, o cartesiano ao indiciário e salienta que, ao longo do tempo, coexistiram, a despeito da ascensão do primeiro. Dada essa coexistência tais paradigmas produziram seus efeitos e impactos epistemológicos e, ao contrário do que se possa supor, o paradigma indiciário não é propriamente uma perspectiva contemporânea nem tampouco foi gestado no cenário atual. Portanto, interessa acentuar que esse modelo Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 567 não é uma invenção ou uma moda a ser adotada sem nenhuma reflexão a priori, mas faz parte de certa tradição interpretativista, ou de forma mais ampla, de certa filosofia da ciência. Ora, em face desse panorama é que podemos dizer que certos movimentos da ciência, em especial, aqueles situados no campo epistemológico são reveladores das não linearidades, incongruências, embates e conflitos acerca da definição da natureza do conhecimento, da relação intricada entre os objetos e os sujeitos. l Avançando em nossa reflexão, a seguir abordaremos a relação entre paradigma, epistemologia, subjetividade e objetividade, o que nos levará a apreciar como o paradigma indiciário pode contribuir para a compreensão de questões concernentes à problemática das identidades docentes, alvo de nossas considerações neste texto. Baptista, L. M. T. R. Paradigma Indiciário... 2 Paradigmas, epistemologias e subjetivismo/objetivismo: algumas breves considerações Ao longo dessa seção, trataremos do que entendemos por paradigma e, por extensão da relação entre epistemologias, subjetivismo e objetivismo. Principiamos essa exposição com a definição platônica de paradigma, com base em Japiassú e Marcondes (2001, p. 206), segundo a qual esse está relacionado às formas ou ideias, ou seja, aos arquétipos, modelos perfeitos ou “eternos imutáveis” dos objetos existentes no mundo natural do qual seriam cópias e dos que, em certa medida, participariam. Já o filósofo da ciência Thomas Samuel Kuhn (2003, p. 67-76), em seu livro A Estrutura das Revoluções Científicas, considera o paradigma uma visão de mundo, a Wetanschaun, que envolve não somente a teoria científica dominante como também determinados princípios filosóficos, leis, procedimentos técnicos padronizados empregados na resolução dos problemas e concepção metodológica. De acordo com essa perspectiva, os modelos seguidos e dos quais surgem as tradições da pesquisa científica são paradigmas, como, por exemplo, a Mecânica de Galileu, a Mecânica Quântica. Conforme esse autor, um paradigma é aquilo que os membros de uma determinada comunidade científica partilham em termos de saber. Baptista, L. M. T. R. Paradigma Indiciário... 568 Contudo, ainda que se suponha certa homogeneidade ou unidade constitutiva, o paradigma não deve ser visto como imutável e estático, ou ainda, com uma natureza centrada e hermética. Desse modo, se ocorre uma mudança de paradigma há mudanças de ordem epistemológica, ou seja, transformações que afetam a própria teoria do conhecimento em diferentes níveis, relacionados à forma da crítica, à filosofia e à história das ciências. Quanto a esses níveis, conforme Japiassú e Marcondes (2001, p. 84-85), o da forma da crítica do conhecimento científico engloba o exame dos princípios, das hipóteses e das conclusões das distintas ciências, considerando-se seu alcance e o seu valor objetivo, enquanto os da filosofia e história das ciências referem-se às modalidades como o Empirismo, o Racionalismo etc. Portanto, um exemplo de mudança de paradigma e, por extensão, mudança epistemológica foi o da passagem da teoria astronômica geocêntrica, de inspiração aristotélica-ptolomaica, para a heliocêntrica, de inspiração copernicana e galileana e, além dessa, a da passagem do modelo escolástico ao cartesianismo. Com estes modelos assumiu-se a existência de uma verdade no mundo da natureza, distante da visão teocêntrica. 2 Paradigmas, epistemologias e subjetivismo/objetivismo: algumas breves considerações Devido a esse deslocamento, defende-se o pressuposto de que se pode aceder à verdade, na medida em que essa é passível de ser descoberta e transformada em leis capazes de expressar as regularidades que dominam o movimento da natureza. Consoante com esse ponto de vista, o método correto permitiria ao observador distinguir o verdadeiro e o falso e, assim, o cientista descobriria algo pré-existente e exterior a ele, ou seja, o objeto seria independente de sua subjetividade. Com essa mudança de paradigma impôs-se o princípio de neutralidade científica, que afirmou a isenção e imunidade do cientista em nome de sua racionalidade objetiva. Mencionamos que no final do século XIX, o paradigma indiciário veio à tona novamente, e no século XX observamos uma tendência à redefinição da ideia de verdade/objetividade e falsidade/subjetividade. Dessa perspectiva, o objeto – quer fosse a sociedade ou quer fosse a natureza – não existiria de modo objetivo a priori, mas seria construído pelos sujeitos. Recolocou-se, assim, a problemática do ponto de vista, da relação entre o sujeito e sua percepção e compreensão do real. 569 Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 Esse questionamento, no seio dos mais diversos campos, a respeito da natureza da verdade e da objetividade, esteve presente na filosofia da ciência contraposta àquela que constitui o modelo cartesiano. Neste sentido, problematizou-se a forma de representação da relação estabelecida entre sujeito e objeto, entre indivíduo, natureza e sociedade. Além disso, mostrou-se mais cautelosa quanto à afirmação das verdades, ou, de modo mais radical, renunciou a propor qualquer forma de verdade, fosse essa provisória ou não. Ainda quanto aos questionamentos a respeito da relação entre sujeito e objeto, entre indivíduo, natureza e sociedade destacamos a noção de regime de verdade proposta por Foucault (1985, p. 12), conforme a qual cada sociedade tem seu regime de verdade, sua “política geral” de verdade: isto é, os tipos de discurso que aceita e faz funcionar como verdadeiros; os mecanismos e instâncias que permitem distinguir entre sentenças verdadeiras e falsas, os meios pelos quais cada um deles é sancionado; as técnicas e procedimentos valorizados na aquisição da verdade; o status daqueles que estão encarregados de dizer o que conta como verdadeiro. 2 Paradigmas, epistemologias e subjetivismo/objetivismo: algumas breves considerações Ora, os regimes de verdade (FOUCAULT, 1985) instaurados na e para a ciência evidenciam a tensa relação existente entre produção do conhecimento e o poder na e para as sociedades humanas ao longo dos séculos. Ainda segundo esse raciocínio, há tipos de discursos recebidos e acolhidos que passam a funcionar como verdadeiros, embora condicionados pelas mais diversas instâncias sociais, culturais e históricas. E, além desses discursos, há mecanismos que diferenciam os enunciados falsos dos verdadeiros, modos através dos quais uns e outros enunciados são sancionados bem como procedimentos e formas valorizados para a obtenção da verdade. Assim, a determinação da noção de verdade está associada a processos históricos e linguísticos, a estruturas de poder, segundo Foucault. Como observa Vaitsman (1995, p. 4), no interior dessa filosofia da ciência sobre a qual estamos tratando, o conhecimento científico e, particularmente o das Ciências Humanas, não se reduz a uma Baptista, L. M. T. R. Paradigma Indiciário... 570 “representação exata da realidade”, a uma espécie de “espelho da natureza”. Ao contrário, é uma forma de representação, entre outras, da realidade e as descrições científicas não são transparentes e objetivas, são “construções da realidade” e “discursos sobre o mundo”; portanto, são provisórias e reformuláveis, pois para essa autora (1995, p. 4), incerteza, contextualidade, contingência, singularidade, particulari- dade, passaram a conviver ou mesmo a substituir, nesta visão de ciência, a antiga concepção de conhecimento que pudesse abarcar a totalidade, descobrir a verdade e estabelecer leis gerais sobre o desenvolvimento histórico ou fenômenos da natureza. Diante do exposto, dada à natureza heterogênea, dinâmica, negociável das identidades, assim como a dos processos identitários, torna-se preciso orientar-se por uma abordagem que considere a contingência do conhecimento, a complexa inter-relação entre a subjetividade e os objetos, ou seja, as distintas vias de interpretação e ressignificação do real/realidade. Quanto ao paradigma indiciário, se filia a essa filosofia da ciência, enfatiza a idiossincrasia e a singularidade dos dados, e, igualmente, propõe interpretações geradas a partir de uma análise minuciosa das pistas para a compreensão do observado. i Por fim, gostaríamos de concluir anunciando que a seguir trataremos de algumas das contribuições do paradigma indiciário para a compreensão e análise das identidades no âmbito formativo. 2 Paradigmas, epistemologias e subjetivismo/objetivismo: algumas breves considerações Com tal finalidade, discorreremos sobre alguns pontos em particular relacionados com aspectos metodológicos, a saber, a natureza de dados e o conceito de rigor e suas implicações para os estudos voltados à problemática da constituição e construção das identidades, o que nos permitirá avançar em nossa reflexão sobre a temática proposta. 3 Paradigma indiciário e estudo das identidades docentes: algumas questões sobre os dados e o rigor Científico A opção pelo paradigma indiciário, assim como qualquer outra, tem implicações que devem ser examinadas, em especial, pelas repercussões na compreensão e na análise do objeto. Dessa maneira, eleger esse modelo requer deslocar o foco da repetibilidade Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 571 ou da replicabilidade para o da singularidade e da idiossincrassia dos dados. Pressupõe, conforme Abaurre et al. (2001, p. 14-15), definir critérios quanto à identificação dos dados a serem tomados como representativos, ou seja, quanto à definição da singularidade que revela e, igualmente, quanto ao conceito de rigor metodológico. Portanto, aqui nos ocuparemos fundamentalmente desses dois aspectos, relacionando-os ao estudo das identidades docentes. Consistentes com o paradigma indiciário, consideramos que os fenômenos sociais e, mais particularmente, os educativos, conforme propõe Pérez Gómez (2007, p. 100) se definem por seu “caráter radicalmente inacabado”, por sua “dimensão criativa, autoformadora, aberta à mudança intencional” e, igualmente, por sua dimensão semiótica, dada a “relação indeterminada” e “polissêmica” instituída entre “o significante observável e o significado latente de todo fenômeno social ou educativo”. Para a compreensão de como se configura a dinâmica da construção identitária, em concreto no que se refere à relação entre os sujeitos e suas identidades, acreditamos que as contribuições do paradigma indiciário podem orientar a geração e análise de dados. Assim sendo, a análise minuciosa de certos indícios, reveladores da singularidade da ação dos sujeitos, presentes nos diversos posicionamentos assumidos para ressignificar e interpretar os saberes mobilizados, possibilita entrever a complexa tessitura de significados que afetam as distintas maneiras de pensar, sentir, expressar e atuar dos sujeitos nos percursos formativos. Essa análise, por sua vez, precisa seguir o que seria o “rigor flexível”, como veremos. Neste sentido, nos interessa focalizar a dinâmica dos processos identitários, para cuja compreensão é relevante considerar as sucessivas interações estabelecidas entre os sujeitos e os vários meios sociais, culturais e profissionais nos quais se inserem nas distintas ocasiões. Consideramos, assim, que essas interações promovem a construção de identidades docentes diferenciadas, de acordo com as distintas maneiras através das quais os sujeitos estabelecem relações entre os saberes, experiências e contextos. Dessa forma, as identidades são permanentemente renegociadas, reformuladas e reformuláveis, visto que os sujeitos instauram processos de ressignificação e Baptista, L. M. T. R. Paradigma Indiciário... 572 interpretação, caracterizados, segundo Hoffnagel (2010, p. 64) como uma “realização interacional e alcançada em eventos comuns, como traços constitutivos dos encontros sociais”. 3 Paradigma indiciário e estudo das identidades docentes: algumas questões sobre os dados e o rigor Científico Portanto, o processo identitário se produz ao longo das constantes negociações dos sujeitos nos diversos espaços em que esse circula e nos encontros em que participa. Destarte, é, simultaneamente, uma construção dos sujeitos, situada histórica, social, cultural e politicamente, na qual se imbricam diversos processos de interação e socialização, diversas práticas discursivas e processos de autorreconhecimento e heterorreconhecimento. No caso da formação inicial de professores e, em especial, a de espanhol a qual nos dedicamos, esperamos contribuir para a proposição de itinerários formativos críticos, nos quais se questionem modelos profissionais que limitem a ação e a prática docentes à dimensão técnica e/ou instrucionista e repercutam na construção de suas identidades docentes. Por isso, acreditamos que problematizar como os sujeitos negociam suas identidades e como se percebem no processo de construção de suas identidades docentes constitui uma alternativa mais condizente com uma formação reflexiva. Reiteramos, portanto, que esse olhar pressupõe admitir que a identidade não é algo permanente e que sua construção implica os sujeitos, já que como postula Pimenta (1997, p. 6) a identidade não é um dado imutável. Nem externo, que possa ser adquirido. Mas, é um processo de construção do sujeito historica- mente situado. A profissão de professor, como as demais, emerge em dado contexto e momento históricos, como resposta a neces- sidades que estão postas pelas sociedades, adquirindo estatuto de legalidade. Diante das dimensões históricas e contextuais, no tocante aos processos identitários docentes e à formação de professores, Silva (2009, p. 48) refere-se aos impactos provocados pelas mudanças sociais e educacionais vividas nos últimos vinte anos sobre a identidade docente. Neste sentido, a crise de identidade docente se traduz em diferentes manifestações que abrangem a insatisfação com os problemas reais da prática e a identidade ideal, solicitações de transferências, inibição com respeito ao trabalho realizado em sala Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 573 de aula, desejo expresso de abandonar a docência, ausência laboral, esgotamento, sentimentos como stress, ansiedade e depreciação do eu e depressões. Além dessa crise, Silva (2009, p. 49) chama a atenção para o fato de que [...] a formação prática da formação inicial deve permitir ao futuro professor identificar-se a si próprio como professor e identificar-se com os estilos de ensino que é capaz de utilizar, levando em con- sideração os efeitos que os referidos estilos produzem nos alunos. 3 Paradigma indiciário e estudo das identidades docentes: algumas questões sobre os dados e o rigor Científico [...] a formação prática da formação inicial deve permitir ao futuro professor identificar-se a si próprio como professor e identificar-se com os estilos de ensino que é capaz de utilizar, levando em con- sideração os efeitos que os referidos estilos produzem nos alunos. Ora, em virtude do exposto é necessário estudar como, ao longo desse processo formativo, se dá a construção identitária e, particularmente, como os futuros professores de línguas percebem-se e interpretam os diversos saberes docentes. Importa contribuir para que os sujeitos reflitam sobre como certas percepções repercutem na sua futura ação e prática e como se posicionam enquanto profissionais. li No entanto, não basta a reflexão com fim em si mesma, uma vez que essa precisa alcançar a práxis pedagógica, considerando as distintas possibilidades contextuais e de atuação e, igualmente, a plausibilidade do ensino de línguas no contexto histórico e social em que nos situamos. Contudo, essa opção não implica assumir uma visão ingênua e idílica do papel da escola e dos professores, como observa acertadamente Nóvoa (2014, p. 40-41) ao dizer que “os professores não são certamente os ‘salvadores do mundo’, mas também não são ‘meros agentes’ de uma ordem superior que os ultrapassa”. Segundo esse autor, somente por meio de uma reelaboração permanente de uma identidade profissional “os professores poderão definir estratégias de ação que não podem mudar tudo, mas que podem mudar alguma coisa. E esta alguma coisa não é coisa pouca”. Condizente com nossas considerações anteriores é preciso rever e problematizar concepções, tais como a do ensino como transmissão cultural ou treinamento de habilidades (PÉREZ GÓMEZ, 2007, p.67-97), contrastadas com a do ensino como mudanças conceituais, afinado com uma perspectiva ecológica, que considera os contextos de aprendizagem. Lembramos que o ensino centrado na transmissão cultural, apoiado nas teorias comportamentalistas, destaca a primazia dos ajustes ambientais, em especial, os materiais, conteúdos, Baptista, L. M. T. R. Paradigma Indiciário... 574 avaliação e demais recursos multissensoriais como fundamentais para a aprendizagem. De acordo com essa concepção, se atingem os objetivos ao empregarem-se corretamente as estratégias de ensino e aprendizagem, daí o protagonismo do método. Já para o ensino como treinamento de habilidades, importa vincular a formação de capacidades aos conteúdos e ao contexto cultural, no qual habilidades e tarefas tenham sentido. 3 Paradigma indiciário e estudo das identidades docentes: algumas questões sobre os dados e o rigor Científico Ambas as visões valorizam os procedimentos didáticos e podem ser relacionadas à primazia do método, uma vez que propõem identificar e comparar métodos, abordagens e técnicas com o intuito de determinar os que melhores, e, portanto, mais eficientes. Ainda que reconheçamos a dimensão técnica e procedimental, é relevante a compreensão das diversas influências dos diferentes contextos nos processos de aprendizagem, uma vez que como propõem Williams e Burden (2008, p. 197) os enfoques sistêmicos ou ecológicos enfatizam o em torno social do aluno, para que se explique como e por que aprendem bem, como destacam o caráter dinâmico e interativo das variáveis presentes e refutam a adoção de um ponto de vista do tipo linear de causa e efeito. Em face de nossas observações anteriores, promover um processo formativo crítico se fundamenta por uma relação dialógica, por um constante movimento questionador e, no caso, um fio condutor e desencadeador desse processo se relaciona às experiências vividas pelos futuros docentes com respeito à conformação de suas identidades. Neste sentido, vemos nos estágios supervisionados, em suas diversas modalidades (participação, regência ou observação), um dos primeiros e importantes espaços de socialização docente, ainda que não o único e definitivo. Historiar as vivências coletivas nele materializadas nos proporciona elementos para uma compreensão de como são ressignificados os saberes docentes, relacionados à(s) língua(s) e cultura(s), às relações entre sua autopercepção e a realidade mais imediata, entre o ideal e o real bem como os constantes reajustamentos e renegociações dos sujeitos com os sentidos que vão estabelecendo para a sua ação. Dessa ótica, conforme Santos (2014, p. 19), os estudos sobre a formação assumem novas perspectivas e, sendo assim, importa entender como o professor constrói sua identidade profissional. Ainda para essa autora (2014, p.19), na Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 575 análise predominam os procedimentos interpretativos bem como se enfatizam aspectos específicos da realidade e valorizam-se os aspectos microssociais, em lugar das questões estruturais, além de interessar-se pelo papel do agente-sujeito e pelas identidades sociais. Consideramos, por conseguinte, que os diversos contextos nos quais os sujeitos participam, interagem e se relacionam são o eixo dos processos de construção das identidades docentes. Os sujeitos se implicam nessa construção, atribuindo sentidos ao ser professor e, no caso, ao ser professor de línguas e da língua espanhola no e para o contexto no qual estão inseridos ou se inserindo. 3 Paradigma indiciário e estudo das identidades docentes: algumas questões sobre os dados e o rigor Científico As identidades, dada à sua heterogeneidade, se embaralham, se confundem, se mesclam e nessa dinâmica vão se delineando os processos identitários, com a tentativa sempre fugaz de fixar determinados papéis e identidades. Assim, concordamos com Silva (2009, p. 54) que a identidade docente é uma construção social, na qual convergem múltiplos fatores que interagem entre si de forma contínua, como podem ser suas histórias de vida, condições de trabalho, os imaginários sobre a profissão e discursos que circulam nas diversas práticas sociais nas quais os docentes se inserem e participam. Dessa forma, trazer à tona questionamentos, confrontar visões, desnaturalizar sentidos e práticas, implica ouvir a voz aos sujeitos, tendo em vista uma contextura mais ampla que transcende, muitas vezes, modismos metodológicos, vocações idealizadas ou pressupostos equivocados. No caso da formação inicial de professores de espanhol, os relatórios produzidos nos estágios supervisionados contêm dados reveladores sobre como esses sujeitos percebem-se e são percebidos: se como estagiários, se como alunos, ou ainda, se como futuros professores; sobre que significados esses sujeitos atribuem à ação docente e à língua espanhola no contexto das práticas e das ações; sobre quais saberes docentes e visões de ensino emergem nessas falas e como são mobilizados e interpretados por eles. Esses indícios, daí nossa aproximação do paradigma indiciário, nos revelam como os sujeitos ressignificam a experiência vivenciada ao longo dos estágios e, obviamente, ao longo do próprio percurso formativo. Dessa ótica, os relatórios, registros de aulas, diários e narrativas elaboradas por esses sujeitos são fontes de dados privilegiados ou cruciais, pois Baptista, L. M. T. R. Paradigma Indiciário... 576 geram um grande número de observações a respeito do observado e nos permitem estabelecer interpretações e hipóteses sobre como se processa a construção dos processos identitários em dados contextos e sob dadas condições, evidenciando mais uma vez a não neutralidade da relação entre conhecimento, saber e poder. Acrescentamos ainda que o rigor que orienta essas análises, em consonância com o paradigma indiciário, diferencia-se daquele exigido pelas metodologias experimentais, conforme observa A.A. Quartarolla (apud Abaurre et al., 2001, p.14-15). Neste sentido, o olhar do pesquisador volta-se para a singularidade dos dados, para o que eles nos dizem sobre essa relação entre sujeitos, identidades e práxis pedagógica. Além disso, o modelo indiciário apoia-se na ideia de que a realidade não é transparente e, igualmente, que a análise detalhada de alguns sinais é central para a compreensão do observado. Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 3 Paradigma indiciário e estudo das identidades docentes: algumas questões sobre os dados e o rigor Científico Passaremos a seguir a tratar de duas questões relevantes para aprofundarmos o exame acerca do paradigma indiciário e suas contribuições para a compreensão dos processos identitários, no âmbito da formação inicial de professores. A primeira delas se refere à atuação do analista e à relação entre o sujeito e o objeto no paradigma indiciário, já que de modo similar ao historiador e ao médico, o nosso conhecimento acerca das identidades docentes em construção é indireto, conjetural, indiciário e, portanto, jamais absoluto. A segunda questão relaciona-se à forma de tratamento reservada aos dados e sua análise. Como visto, no paradigma indiciário, prevalece o tratamento qualitativo, a abordagem interpretativista e se defende a singularidade do observado. Ora, essa opção metodológica implica lidar com o contingente, com o idiossincrásico, com a provisoriedade e com a contextualidade na proposição das interpretações e condução das análises. Sendo assim, ao optar pelo paradigma indiciário valorizamos os indícios que permitam traçar a história do sujeito ao vivenciar as experiências docentes e os diversos conflitos e questionamentos emergentes, materializados nos seus relatos, nas suas narrativas. Cabe-nos examinar como os sujeitos, de seu lugar de fala, percebem- se por meio da análise de como se designam, como se categorizam ou/e se denominam; por meio das representações que se revelam; do Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 577 modo como enunciam, reforçando determinadas posições discursivas ou apagando outras. Como analista, por conseguinte, é preciso explorar as ocorrências que evidenciam um trabalho do sujeito com a linguagem em dois sentidos: quando tomam posição e enunciam e, ainda, quanto aos efeitos de sentido materializados/produzidos nessa tomada de posição e suas implicações para e na construção de suas identidades. Ainda no tocante à atuação do analista, optar pelo paradigma indiciário para investigar a construção da identidade e, em especial, os processos identitários pressupõe rever o conceito de rigor. Trata- se de um “rigor flexível”, que orientará tanto a seleção dos dados singulares quanto a sua interpretação. Lembramos, igualmente, que a subjetividade será um elemento constitutivo do percurso investigativo e, dessa maneira, inegável na condução e elaboração das propostas de interpretações a respeito dessa história particular dos sujeitos. 4 Considerações finais Gostaríamos de finalizar dizendo que eleger o paradigma indiciário pressupõe privilegiar a singularidade reveladora como dado crucial e seguir procedimentos abdutivos, diferenciados dos indutivos e dedutivos. Além disso, supõe assumir a existência, conforme postula Foucault (1996, p. 26-27), da vinculação entre os sistemas de verdade e as práticas sociais e políticas de onde são provenientes e aplicadas, o que nos leva a falar do “caráter perspectivo” do conhecimento. De acordo com essa ótica, não há uma essência do conhecimento, visto que esse é “histórico e pontual”, contextual, contingente e provisório. Sendo assim, como propõe Foucault (1996, p. 25), o conhecimento é (e será) “sempre uma certa relação estratégica em que o homem se encontra situado”; será essa relação estratégica responsável pela definição do efeito de conhecimento e, por isso, esse será sempre “parcial, oblíquo, perspectivo”. Em outras palavras, segundo esse autor (1996, p. 25) “o conhecimento esquematiza, ignora as diferenças, assimila as coisas entre si, e isto sem nenhum fundamento em verdade. Devido a isso, o conhecimento é sempre um desconhecimento”. Baptista, L. M. T. R. Paradigma Indiciário... 578 Consoante com essa visão de conhecimento, concordamos com a afirmação de Garcia et al. (2005, p. 54) de que, ao estudar as identidades docentes, estamos diante de um saber que não é permanente e, assim, torna-se necessário perder a ilusão de um conhecimento definitivo e de totalidade acer- ca dessa questão, é uma precaução epistemológica importante se considerarmos a heterogeneidade da categoria docente e a própria instabilidade das identidades no mundo contemporâneo. perder a ilusão de um conhecimento definitivo e de totalidade acer- ca dessa questão, é uma precaução epistemológica importante se considerarmos a heterogeneidade da categoria docente e a própria instabilidade das identidades no mundo contemporâneo. Em face de nossas considerações anteriores, o estudo das identidades em construção, e, em especial, dos processos identitários precisa considerar o complexo das relações entre sujeitos, práticas, ações e contextos, consoante com uma visão perspectiva do conhecimento. Acreditamos, assim, que as identidades sociais são móveis, reformuláveis e plásticas e que a construção identitária pressupõe a instauração de um fluxo ininterrupto de (re)ssignificação e (re)interpretação, por parte dos sujeitos, dos sentidos atribuídos à docência e à sua ação. Ainda conforme Garcia et al. (2005, p. Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 Abstract This paper focuses on the contributions of the “indiciary paradigm” to the study of teacher identity and specially the identity of prospective language teachers with emphasis on the ones who teach Spanish. To this aim, we present the model as well as its contributions to create more intelligibility about the way that prospective teachers experience the processes of identity construction through their formative paths. Thus, in this article, we indicate some possibilities of this paradigm for the study of our central problem, reflecting on its potentiality as a methodological construct for a qualitative, interpretive research. Keywords: indiciary paradigm, Spanish teacher education, identities, applied linguistics. 4 Considerações finais 54) as investigações recentes que problematizaram a relação entre a identidade e a profissionalização docente parecem ter se fixado em “aspectos unificadores e fundantes da identidade” como, por exemplo, a classe social, a natureza do processo de trabalho, o gênero, a história de vida, a formação profissional, etc”. Porém, diferentemente dessa via, os autores sugerem que se busquem diferenças, descontinuidades e, ainda, que se privilegiem as narrativas dos docentes sobre si mesmos bem como seus contextos de trabalho. Ora, isso sugere ser preciso nos aproximarmos do que seria a “dinâmica contraditória e fragmentada” dessa construção identitária e neste sentido o paradigma indiciário e, em especial, o foco no residual, nos indícios reveladores da singularidade e idiossincrasia, representa um ganho em termos de possibilidades de análise. Nessa direção, poderá trazer maior inteligibilidade sobre como os sujeitos se assumem, se percebem e se reconhecem como docentes de língua espanhola em seu processo inicial de formação. Consideramos, ainda, que essa perspectiva de análise pode nos levar à problematização das diferentes negociações que os sujeitos realizam quando refletem sobre esse Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 579 processo, em consonância com uma (trans)formação crítico-reflexiva situada histórica, espacial e temporalmente, mais próxima do cotidiano das práticas. processo, em consonância com uma (trans)formação crítico-reflexiva situada histórica, espacial e temporalmente, mais próxima do cotidiano das práticas. Paradigma indiciario: sus aportes a la investigación de la construcción de las identidades de futuros profesores de lenguas Resumen Resumen Este trabajo trata de los aportes del paradigma indiciario para el estudio de las identidades docentes y, particularmente, las de los futuros profesores de lenguas, con énfasis en los del español. Con esa finalidad, explicitaremos en qué consiste ese modelo y cómo puede aportar para generar mayor inteligibilidad a respecto de cómo futuros profesores se involucran en sus procesos de construcción identitaria vividos a lo largo de sus itinerarios formativos. Así, en este artículo indicamos algunas contribuciones de ese paradigma para el examen de la problemática enfocada, reflexionando acerca de su potencial como constructo metodológico de naturaleza cualitativa e interpretativista. Palabras clave: Paradigma Indiciario, Formación de profesores de español, Identidades, Lingüística Aplicada. 580 Baptista, L. M. T. R. Paradigma Indiciário... Referências ABAURRE, Maria Bernadete Marques; FIAD, Raquel Salek; MAYRINK- ABAURRE, Maria Bernadete Marques; FIAD, Raquel Salek; MAYRINK- SABINSON, Maria Laura Trindade. Cena I- Indícios. In: ______. Cenas de aquisição da escrita. O sujeito e o trabalho com o texto. 2. ed. Campinas: ABAURRE, Maria Bernadete Marques; FIAD, Raquel Salek; MAYRINK- SABINSON, Maria Laura Trindade. Cena I- Indícios. In: ______. Cenas de SABINSON, Maria Laura Trindade. Cena I- Indícios. In: ______. Cenas de aquisição da escrita. O sujeito e o trabalho com o texto. 2. ed. Campinas: Mercado das Letras, 2001. p. 13-36. FOUCAULT, Michel. Conferência I. In: ______. A verdade e as formas jurídicas. Rio de Janeiro: Nau, 1996. p. 7-28. FOUCAULT, Michel. Microfísica do poder. Organização e Tradução Roberto Machado. 5. ed. Rio de Janeiro: Edições Graal, 1985. GARCIA, Maria Manuela Alves; HYPOLITO, Álvaro Moreira; VIEIRA, Jarbas Santos. As identidades docentes como fabricação da docência. In: Educação e Pesquisa, São Paulo, v. 31, n. 1, jan./abr. ,2005. p. 45-56. GINZBURG, Carlo. Sinais: raízes de um paradigma indiciário. In: ______. Mitos, Emblemas, Sinais. Morfologia e história. 2. ed. São Paulo: Companhia das Letras, 2001. p. 143-179. HOFFNAGEL, Judith Chambliss. Temas em Antropologia e Linguística. Recife: Bagaço, 2010. JAPIASSÚ, Hilton; MARCONDES, Danilo. Dicionário básico de filosofia. 3. ed. Rio de Janeiro: Zahar, 2001. KUHN, Thomas Samuel. “A prioridade dos paradigmas”. In: ______. A Estrutura das Revoluções Científicas. 8. ed. Perspectiva, São Paulo, 2003, p. 67-76. NÓVOA, Antonio. Diz-me como ensinas, dir-te-ei quem és e vice-versa. In: FAZENDA, Ivani (Org.). A pesquisa em educação e as transformações do conhecimento. 12. ed. Campinas: Papirus, 2014. p. 29-41. PÉREZ GÓMEZ, Ángel Ignacio. Ensino para a compreensão. In: GIMENO SACRISTÁN, José; PÉREZ GÓMEZ, Ángel Ignacio. Compreender e transformar o ensino. 4. ed. Artmed. Porto Alegre: 2007. p. 67-97. PÉREZ GÓMEZ, Ángel Ignacio. Compreender o ensino na escola: modelos metodológicos de investigação educativa. In: GIMENO SACRISTÁN, José; PÉREZ GÓMEZ, Ángel Ignacio. Compreender e transformar o ensino. 4. ed. Porto Alegre: Artmed. 2007. p. 99-117. PIMENTA, Selma Garrido. Formação de professores - saberes da docência e identidade do professor. In: Nuances, v. III, setembro de 1997, p. 5-14. 581 Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 Disponível em: <http://revista.fct.unesp.br/index.php/Nuances/article/ view/50>. Acesso em: 24 abr. 2015. SANTOS, Lucíola Licinio de Castro Paixão. Formação do professor e pedagogia crítica. In: FAZENDA, Ivani (Org.). A pesquisa em educação e as transformações do conhecimento. 12. ed. Campinas: Papirus, 2014. Baptista, L. M. T. R. Paradigma Indiciário... Referências p. 17-28. SILVA, Maria de Lourdes Ramos. A complexidade Inerente aos Processos Identitários Docentes. In: Notandum Libro 12 2009 CEMOrOC-Feusp / IJI-Universidade do Porto, p. 45-58. Disponível em: <www.hottopos.com/ notand_lib_12/malu.pdf>. Acesso em: 24 abr. 2015. VAITSMAN, Jeni. Subjetividade e paradigma de conhecimento. In: Boletim Técnico do Senac, v. 21, n. 2, maio/ago., 1995, p. 1-9. Disponível em: <http:// www.senac.br/bts/212/2102003009.pdf>. Acesso em: 28 abr. 2015. Williams, Marion & BURDEN, Robert L. El contexto de aprendizaje. In: ______. Psicología para profesores de idiomas. Enfoque del constuctivismo social. Madrid: Edinumem, 2008. p. 195-209. Submetido em 13 de maio de 2015 Aceito em 12 de junho de 2015 Publicado em 21 de dezembro de 2015 582
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Psychoanalytic Reading of Decadence and Crisis in Ted Hughes' "Relic" and T.S. Eliot's "The Hollow Men"
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INTRODUCTION Literature often serves as a mirror reflecting the complexities of human experience, unraveling the intricate layers of emotion, thought, and societal context. In twentieth century poetry, T.S. Eliot and Ted Hughes emerge as witnesses to the two consecutive turbulent eras. Hughes’ “Relic” and Eliot’s “The Hollow Men” capture the essence of the two eras marked by profound uncertainty and disquiet. Beyond the mere words on the page, these poems invite readers to delve into the depths of the human psyche, exploring the interplay between decadence and crisis. The concept of decadence, often associated with moral and cultural decline, finds its resonance in these two poems. However, a psychoanalytic approach divulges deeper dimensions of the texts, showing the underlying psychological landscapes that shaped their creations. Drawing from the theories of Sigmund Freud and Carl Gustav Jung, this research undertakes a journey to illuminate the subconscious motivations, anxieties, and desires that find expression in “Relic” and “The Hollow Men”. Moreover, crises, whether individual or societal, act as a testing ground for both individuals and cultures. In the times of Eliot and Hughes respectively, there were abundant upheavals including wars, economic changes, and shifts in cultural norms. “Relic” and “The Hollow Men” express the pervasive sense of disillusionment and fragmentation that characterized their times. By using a psychoanalytic framework to explore the underlying psychological crisis, this study aims to unravel how individual minds connect with the broader socio-cultural environment. This research undertakes a two-fold investigation: a psychoanalytic exploration of subconscious landscapes and an analysis of how these landscapes intersect with the major crises of twentieth century. Through an examination of the two works of Hughes and Eliot, this study strives to illuminate the intricate interplay between the human psyche, societal decadence, and existential crisis. While exploring their intricate poetry, this paper invites readers on a journey that goes beyond time and provides deep insights into the human experience International Journal of Social Science And Human Research ISSN (print): 2644-0679, ISSN (online): 2644-0695 Volume 06 Issue 09 September 2023 DOI: 10.47191/ijsshr/v6-i9-14, Impact factor- 6.686 Page No: 5446-5450 International Journal of Social Science And Human Research ISSN (print): 2644-0679, ISSN (online): 2644-0695 Volume 06 Issue 09 September 2023 DOI: 10.47191/ijsshr/v6-i9-14, Impact factor- 6.686 Page No: 5446-5450 International Journal of Social Science And ISSN (print): 2644-0679, ISSN (online): 2644-0695 Volume 06 Issue 09 September 2023 DOI: 10.47191/ijsshr/v6-i9-14, Impact factor- 6.686 Page No: 5446-5450 Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” Md Mehedi Hasan artment of English Language and Literature, North Western University, Khulna-9000, Bangladesh ABSTRACT: This research employs psychoanalytic methods to explore themes of decadence and crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men”. Drawing from Freudian and Jungian theories, it delves into the psychological aspects to reveal expressions of the despair of modernity. The paper illustrates underlying psychological decay within the poems, examining subconscious motives, suppressed desires, and conflicts. Through this exploration, intricate psychological states contributing to perceptions of crisis come to light. Highlighting these literary pieces as reflections of contemporary turmoil, the poems expose the erosion of both individual and collective identities. By using the lens of psychoanalysis, a deeper understanding of the interplay between the human psyche and society emerges, offering insights into the profound implications of modern decadence and crisis. Ultimately, the application of psychoanalysis enriches comprehension of the connections between psychological intricacies and societal disintegration, shedding light on the significance of the poets’ selected works. KEYWORDS: Crisis, decadence, fragmentation, hollow men, “Relic”, Psychoanalysis, Ted Hughes, T.S. Eliot, and twentieth century TEXTUAL ANALYSIS AND INTERPRETATION Symbolism and Imagery: Freud’s view that symbols are the “fulfillments of wishes” (Freud, 1900) aligns with the notion that symbols in literature often reflect subconscious desires and conflicts. By analyzing the symbols and imagery in “Relic” and “The Hollow Men” with psychoanalysis, it is possible to discover deeper meanings beyond the obvious story. Character Analysis: Jung’s concept of individuation, a process of integrating conscious and unconscious elements, is pertinent to character analysis. Jung proposed that “the conscious mind is thrown into the highest relief and revealed in all its peculiarity” during the process of individuation (Jung, 1968). This idea resonates with analyzing the selected poems, revealing the narrator’s inner struggles and psychological transformations. This approach shows how literature carries deep meanings, and creates links between personal psychology and the wider social and cultural context. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” DECADENCE AND CRISIS DECADENCE AND CRISIS Decadence: Both Freud’s “id” representing basic desires and Jung’s “shadow” symbolizing suppressed elements offer insights into understanding decadence. According to Freud, “the id knows no judgments of value” (Freud, 1933), which implies that the portrayal of decadence in the poems might be rooted in the uninhibited expression of suppressed urges. Jung’s idea that the shadow contains both “positive and negative aspects” (Jung, 1968) relates to the complex interplay between societal norms and the darker, hidden facets of the human psyche. Crisis: Freud’s theory of the unconscious mind as a reservoir of repressed traumas aligns with the crises depicted in the selected poems. Freud’s concept of repression highlights the role of crisis in shaping the unconscious. Likewise, Jung’s study of archetypes and the collective unconscious (Shelburne, 1976) offers a way to comprehend how characters in the poems struggle with existential crises tied to universal themes. DISCUSSION Though Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” differ in style and subject, they come together in depicting mental decline, hidden wants, and twentieth century’s existential challenges. The study reveals common themes and profound psychological insights found in the selected poems. LITERATURE REVIEW AND POINT OF DEPARTURE Lok Raj Sharma’s study of T.S. Eliot’s “The Hollow Men” reveals the poem’s profound exploration of men’s spiritual void and inefficacy. In Sharma’s opinion, through vivid sections depicting communication barriers, existential uncertainties, and societal detachment, the poem paints a bleak picture of modern human condition. Sharma’s analysis offers valuable insights into Eliot’s portrayal of isolation, materialism, and spiritual hollowness in the poem: This poem is perhaps the most negative and pessimistic of all Eliot’s poems. It does not only reflect the paralyzed psyche of Post- World War I , but it also reveals Eliot’s mental agony, the painful experience of collapsing marriage with Vivien Eliot, his skepticism towards religious hope and love. (Sharma, 2021) Mariwan M Hasan’s analysis of T.S. Eliot’s “The Hollow Men” employs textual and reader-response approaches to reveal its themes of religion, spirituality, and modernity. Hasan highlights Eliot’s depiction of hollow modern individuals and their quest for meaning in a post-World War I world. The study provides insights into how Eliot’s poem serves as a reflection of societal conditions and the search for faith and morality in a fragmented era: “His concerning about the outcomes of the modern time caused him to be known as the spokesperson of the modern age. He has expressed the noticeable feelings and anxieties exposed to modern man through the complexity of the modern period.” (Hasan, 2019) Dr. Maitali Khanna and Rajeshwari C. Patel analyze T.S. Eliot’s portrayal of moral dilemmas in “The Hollow Men” and The Waste Land, revealing his insights into the societal breakdown during the wartime era. The research highlights Eliot’s keen understanding of modern moral challenges, demonstrated in his poems: “Keeping in mind the disillusionment of the generation, T.S. Eliot initially wrote “The Hollow Men”, to serve as an epilogue to The Waste Land.” (Khanna & Patel, 2022) While numerous research articles have extensively examined T. S. Eliot’s poem “The Hollow Men”, contemporary scholarly investigations have yet to delve into a comparative analysis between Eliot’s “The Hollow Men” and Hughes’ “Relic” from the vantage point of psychoanalysis. This void in existing literature serves as the research gap of this study. THEORETICAL FRAMEWORK Psychoanalytic literary criticism is a way of understanding literature analyzing the hidden thoughts and feelings in people’s minds. Sigmund Freud and Carl Jung came up with this idea. It helps readers see deeper meanings in stories by exploring what people might not even realize they are thinking or feeling. Freud’s assertion “dreams are the royal road to the unconscious” (1900) highlights the significance of exploring the hidden realms of the mind. Psychoanalysis is a process that aligns with the exploration of the underlying psychological dimensions within the poems “Relic” by Ted Hughes and “The Hollow Men” by T.S. Eliot. IJSSHR, Volume 06 Issue 09 September 2023 www.ijsshr.in IJSSHR, Volume 06 Issue 09 September 2023 Page 5446 www.ijsshr.in Page 5446 Psychoanalytic Reading of Decadence and Crisis in T.S. Eliot’s “The Hollow Men” Psychoanalytic Reading of Decadence and Crisis in T.S. Eliot’s “The Hollow Men” T.S. Eliot’s poem “The Hollow Men” serves as a compelling canvas for a psychoanalytic exploration of decadence and crisis. Using Sigmund Freud and Carl Jung's theories as guides, this perspective reveals deep symbolisms and psychological struggles. It sheds light on how identity breaks down and the search for meaning in a world filled with emptiness. The two epigraphs, “Mistah Kurtz—he dead / A penny for the Old Guy,” (Eliot, 1925, p. 2383) echo the sense of loss and the fleetingness of life. This fragmented opening reflects Freud’s notion of the unconscious mind as a repository of suppressed desires and unresolved conflicts. The reference to Mistah Kurtz, an allusion to Conrad’s Heart of Darkness (1899), serves as a reminder of the darkness within the human soul, resonating with Jung’s exploration of the collective unconscious. The line “We are the hollow men” (1925, p. 2383) reflects the fragmented and empty nature of the human psyche. The term “hollow” can be seen as a metaphor for the suppression of authentic emotions and desires. Through a psychoanalytic lens, this can be interpreted as the ego’s attempt to mask the unconscious id, resulting in a sense of emptiness and disconnection. The following lines “We are the stuffed men / Leaning together / Headpiece filled with straw. Alas!” (1925, pp. 2383-2384) express a profound disconnection from authentic emotions and genuine existence. This hollow state reflects the void-like nature of the unconscious, mirroring Freud’s concept of the id—the reservoir of primal instincts that often remain unexamined and untamed. The imagery of “dried voices” (1925, p. 2384) that “whisper together / Are quiet and meaningless” (1925, p. 2384) evokes the hollowness of communication, drawing parallels to the suppressed emotions and concealed desires within the human psyche. This notion aligns with Freud’s belief in the unconscious as a realm where repressed thoughts and emotions linger, shaping one’s behavior without conscious awareness. Eliot’s portrayal of “Shape without form, shade without color” (1925, p. 2384) and “gesture without motion” (1925, p. 2384) captures the disintegration of self and identity, reflecting the fractured psyche. These descriptions mirror the fragmented nature of modern identity, as individuals grapple with reconciling personal desires and societal expectations. This fragmentation parallels Jung’s exploration of individuation—a journey towards integrating the conscious and unconscious aspects of the self. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” In Ted Hughes’ poem “Relic”, the depths of human experience and the shadows of the unconscious come to the forefront, inviting a psychoanalytic exploration of decadence and crisis. Based on the theories of Sigmund Freud and Carl Jung, this interpretation examines intricate layers of symbolism and meaning, focusing on the profound interplay between the human psyche and the dissolution of societal structures. IJSSHR, Volume 06 Issue 09 September 2023 IJSSHR, Volume 06 Issue 09 September 2023 www.ijsshr.in Page 5447 www.ijsshr.in Psychoanalytic Reading of Decadence and Crisis in T.S. Eliot’s “The Hollow Men” The lines “This is the dead land / This is cactus land” (1925, p. 2385) paint a picture of desolation, underscoring the barren psychological landscape that the hollow men inhabit. The supplication of “a dead man’s hand / Under the twinkle of a fading star” (1925, p. 2385) evokes a sense of yearning for salvation amidst spiritual decay, echoing Freud’s concept of unfulfilled desires that continue to influence human behavior. Eliot’s deliberate structural choices—fragmented sections, repetition of phrases—mirrors the repeating cycles of the unconscious mind. The refrain “This is the way the world ends / Not with a bang but a whimper” (1925, p. 2386) encapsulates the sense of disillusionment and crisis, suggesting that the end is not marked by a grand event but rather by a quiet fade into obscurity. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” The core of the poem holds a jawbone, a physical object that serves as a gateway to the mysterious realms of the subconscious. The poem’s vivid imagery of “crabs, dogfish, broken by the breakers” (Hughes, 1960, p. 2588, line 2) creates a powerful scene reflecting the wild sea and the chaotic unconscious. The lack of companionship in these depths shows a separation from awareness, echoing Freud’s idea of the id, a place of basic instincts and desires. The sea’s unending devouring gains metaphorical meaning, reflecting the never-ending desires of the unconscious. The action of being “gnawn bare” (p. 2588, line 8) reflects the ceaseless demands of the id, forever craving satisfaction. The transformation of the jawbone into a “cenotaph” (p. 2588, line 16) evokes a poignant metaphor for the loss of identity and purpose, reflecting the ego’s struggle to reconcile personal desires with societal expectations. Furthermore, the eternal presence of the sea resembles Freud’s idea of the timeless unconscious. As the sea brings ashore “spars of purposes” (p. 2588, line 13), it holds unresolved psychological conflicts that were blocked, echoing Jung’s collective unconscious, a reservoir of universal symbols and shared experiences. Within this complex web, Hughes’ “Relic” goes beyond just a poem—it becomes a reflection of intricate individual psychology and societal collapse, echoing the chaos and disintegration of the modern world. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” on social media or in professional situations, can result in a fragmented self-perception. Many individuals find themselves wearing different deliberate disguises online and offline, struggling to reconcile these various facets into a cohesive whole. Communication and Connection: The poem’s depiction of “dried voices” (1925, p. 2384) and “quiet and meaningless whispers” (1925, p. 2384) reflects the challenges of communication in the digital era. While we are more connected than ever, truly meaningful communication can be rare, often leading to superficial exchanges. The depiction of voices without depth illustrates the reality of virtual interactions that lack emotional richness. Spiritual Vacuum: The references to “dead land” (1925, p. 2385) and “cactus land” (1925, p. 2385) evoke images of spiritual desolation and a world devoid of higher values. In an age marked by consumerism, materialism, and a focus on immediate gratification, many individuals may feel a void where deeper spiritual and ethical foundations should reside. The poem’s portrayal of stone images and supplications to broken stone reflects the emptiness that can arise when material pursuits replace meaningful connections and values. Fear of Irrelevance: The fear of being forgotten or becoming insignificant, as expressed in the refrain “Not with a bang but a whimper,” (1925, p. 2386) echoes the anxieties of modern individuals. In a world saturated with information and fleeting trends, the fear of being rendered irrelevant or forgotten is a genuine concern for many. The poem’s depiction of a quiet and unremarkable end reflects the unease that arises from the potential obscurity that modern life can entail. Yearning for Meaning: The poem’s exploration of the “Shadow” and the yearning for something beyond the mundane can be seen as a reflection of the human need for deeper meaning and purpose. In a world that often emphasizes material success and external achievements, many individuals long for a sense of fulfillment that transcends superficial pursuits. The poem’s themes resonate with the search for significance in an era of rapid change and uncertainty. “The Hollow Men” reflects modern society’s challenges with identity, communication, and meaning. It explores inner struggles and emptiness, remaining relevant in a changing world searching for authenticity and purpose. A Comparative Study of Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” from Psychoanalytic Perspective “The Hollow Men” and “Relic” when analyzed through psychoanalytic perspective, reveal similar insights into the complexities of the human mind. Relevance of “The Hollow Men” to Modern Society T.S. Eliot’s poem “The Hollow Men” resonates with modern society through its profound exploration of existential crisis, disconnection, and the erosion of identity. This haunting portrayal of inner turmoil and societal decay finds uncanny parallels in the complexities of contemporary existence, reflecting the challenges, anxieties, and uncertainties of the modern world. Existential Despair: The “hollow men” embody a sense of emptiness and existential despair that many individuals in modern society can relate to. In a world marked by rapid changes, technological advancements, and shifting cultural norms, individuals may grapple with a loss of meaning and struggle to find their place in the grand scheme of things. The poem’s portrayal of individuals trapped in a state of spiritual desolation echoes the sense of disorientation and detachment experienced by many in the face of a rapidly evolving world. Fragmentation of Identity: The fragmented nature of the hollow men symbolizes the fractured identities that can emerge in the modern age. The widespread use of digital communication and constant exposure to various facets of one’s identity, whether IJSSHR, Volume 06 Issue 09 September 2023 Page 5448 Page 5448 www.ijsshr.in CONCLUSION Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” converge in a psychoanalytic exploration. Themes of suppressed desires, existential turmoil, and fragmented identity intertwine. Through Freudian and Jungian lenses, the selected poems resonate with modern complexities, portraying the clash of conscious control and hidden impulses. The fading of self and the search for purpose highlight twentieth century’s crisis, revealing the outcomes of ignoring hidden desires. These poems offer insights into our own challenges, inviting us to explore the intricacies of the human mind, where hidden wishes and existential longings endure. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” Despite their differences, the poems intersect in portraying psychological decline, repressed wants, and the existential challenges of the poets’ contemporary times respectively. Suppressed Desires and Fragmented Identity: In “Relic”, the sea’s consumption symbolizes insatiable desires, echoing Freud’s concept of the id—the repository of repressed instincts. Similarly, “The Hollow Men” portrays individuals as “stuffed men” (Eliot, 1925, p. 2384) with “dried voices” (1925, p. 2384), suggesting suppression of desires and fractured identity. This fragmentation reflects the struggle to reconcile conscious identity with suppressed unconscious desires. Existential Emptiness and Crisis: Both poems explore existential emptiness. “Relic” presents the jawbone’s transformation into a cenotaph, representing the loss of identity. In “The Hollow Men”, the titular figures embody spiritual emptiness. These portrayals resonate with Freud’s concept of existential crises, wherein suppressed desires can lead to feelings of meaninglessness and despair. Disconnection from Society: “Relic” alludes to the sea’s isolation, mirroring contemporary disconnection amidst desires. “The Hollow Men” speaks of individuals forgotten in death’s “twilight kingdom” (1925, p. 2385), reflecting detachment from societal narratives. These portrayals align with Jung’s concept of individuation—the quest for self-integration amidst societal disintegration. Search for Meaning and Salvation: In “Relic”, the jawbone’s transformation signifies the search for meaning amidst decay, echoing Jung’s individuation journey. “The Hollow Men” portrays yearning for salvation, reflecting both Freud’s idea of unfulfilled desires and Jung’s concept of transcendent aspirations. Fragmented Language and Repetition: Both poems use fragmented language and repetition to mirror the unconscious’s fragmented nature. In “Relic”, phrase like “This is the sea’s achievement” (Hughes, 1960, p. 2588, line 10) demonstrates repetition’s role in reflecting unconscious conflicts. In “The Hollow Men”, repeated lines emphasize how unfulfilled desires and a sense of emptiness continue in an unending cycle. Modern Decay and Crisis: Both poems reflect modern decay and crisis. “Relic” portrays decay within the sea’s depths, paralleling psychological decay. “The Hollow Men” evokes spiritual desolation and societal dissolution. Both poems offer poignant reflections of contemporary anxieties and the psychological challenges posed by modernity. IJSSHR, Volume 06 Issue 09 September 2023 Page 5449 Page 5449 www.ijsshr.in REFERENCES 1) Eliot, T. S. (1925). The hollow men. In M. H. Abrams (Ed.), The Norton Anthology of English Literature (7th ed., Vol. 2, pp. 2383-2386). W. W. Norton. 1) Eliot, T. S. (1925). The hollow men. In M. H. Abrams (Ed.), The Norton Anthology of English Literature (7th ed., Vol. 2, pp. 2383-2386). W. W. Norton. d, S. (1900). The interpretation of dreams. York University. https://psychclassics.yorku.ca/Freud/Dreams/d 2) Freud, S. (1900). The interpretation of dreams. York University. https://psychclassics.yorku.ca/Freud/Dreams/dreams.pdf 3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychology. pp. 116-150. 2) Freud, S. (1900). The interpretation of dreams. York University. https://psychclassics.yorku.ca/Freud/Dreams/dreams.pdf 3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychology. pp. 116-150. C lli B k h // d / i h/ df lib /F d U i df 3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychology. pp. 116-150. Collier Books.https://www.sas.upenn.edu/~cavitch/pdf-library/Freud_Unconscious.pdf 3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychol Collier Books.https://www.sas.upenn.edu/~cavitch/pdf-library/Freud_Unconscious.pdf 4) Freud, S. (1933). New introductory lectures on psycho-analysis. Internet Archive. Pdf. 5) Hasan, M. (2019). A thematic investigation into T. S. Eliot’s “The hollow men:” with approach. Critical Literary Studies, 1(2), pp.159-184. doi: 10.34785/J014.2019.680 6) Hughes, T. (1960). Relic. In M. H. Abrams (Ed.), The Norton Anthology of English Literature (7th e 2588). W. W. Norton. 7) Jung, C. G. (1968). Analytical psychology: its theory and practice. Academia. https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Car 7) Jung, C. G. (1968). Analytical psychology: its theory and practice. Academia. https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Carl_Jung 7) Jung, C. G. (1968). Analytical psychology: its theory and practice. Academia. https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Carl_Jung https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Carl_Jung 8) Khanna, M. & Patel, R. (2022). Moral predicament in T. S. Eliot’s “The hollow men” and “The waste land”. Research Journal of English, 7(1). pp. 7-15. https://www.researchgate.net/publication/357621278_Moral_Predicament_in_T_S_Eliot's_The_Hollow_Men_and_The_ Waste_Land https://www.researchgate.net/publication/357621278_Moral_Predicament_in_T_S_Eliot's_The_Hollow_Men_and_The_ Waste Land 9) Sharma, L. R. (2021). Men’s spiritual vacuity and inefficacy in Eliot’s poem “The hollow men”. Shanlax I t ti l J l f E li h 10(1) 1 5 htt //d i /10 34293/ li h 10i1 4252 9) Sharma, L. R. (2021). Men’s spiritual vacuity and inefficacy in Eliot’s poem “The hollow men”. Shanlax International Journal of English, 10(1), pp. 1-5. https://doi.org/10.34293/english.v10i1.4252 10) 10. Shelburne, W. A. (1976). C. G. Jung’s theory of the collective unconscious: a rational reconstruction. University of Florida. https://ufdcimages.uflib.ufl.edu/UF/00/09/81/33/00001/cgjungstheoryofc00shel.pdf 10) 10. Shelburne, W. A. (1976). C. G. Jung’s theory of the collective unconscious: a rational reconstruction. University of Florida. REFERENCES https://ufdcimages.uflib.ufl.edu/UF/00/09/81/33/00001/cgjungstheoryofc00shel.pdf SSHR, Volume 06 Issue 09 September 2023 www.ijsshr.in Page 5450 There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) IJSSHR, Volume 06 Issue 09 September 2023 IJSSHR, Volume 06 Issue 09 September 2023 IJSSHR, Volume 06 Issue 09 September 2023 Page 5450 www.ijsshr.in Page 5450
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DAMPAK WABAH COVID-19 TERHADAP PENJUALAN JAHE MERAH DI SURAKARTA
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DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 22, No. 2, Desember 2020 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 22, No. 2, Desember 2020 DAMPAK WABAH COVID-19 TERHADAP PENJUALAN JAHE MERAH Hendi Yudhanto Adinugroho1, Liana Mangifera2, Rizcho Louistama Setyana Putra3, Fida Nur Rahma4, Calista Amelia Salsabila5, Diah Ayu Martiningsih6 Manajemen Fakultas Ekonomi dan Bisnis Universitas Muhammadiyah Surakarta Email: Hendi.yudhanto@gmail.com, Abstrak Penelitian ini bertujuan menganalisis dampak wabah Covid-19 terhadap penjualan jahe merah di Kawasan kota Surakarta. Variabel yang digunakan adalah Pemahaman & Pengetahuan tentang Covid-19 dan Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19. Populasi penelitian ini adalah penjual jahe merah yang pernah melakukan transaksi jual beli jahe merah di saat situasi Covid-19 yang berlokasi di kawasan Kota Surakarta dan Sekitarnya . Pemilihan sampel menggunakan metode purposive sampling. Jumlah sampel yang diambil dalam penelitian ini sebanyak 120 orang responden yang tersebar di Surakarta, Sukoharjo, Karanganyar, Boyolali, Sragen, Klaten dan Wonogiri. Penelitian ini menggunakan data primer yang diambil menggunakan wawancara terstruktur dengan bantuan kuesioner. Analisis data yang digunakan adalah analisis regresi berganda. Simpulan yang dihasilkan adalah adalah Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 berpengaruh positif dan signifikan terhadap tingkat penjualan jahe merah, sedangkan Pemahaman dan Pengetahuan tentang Covid-19 berpengaruh positif tetapi tidak signifikan terhadap tingkat penjualan jahe merah. Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 paling berpengaruh dalam penjualan jahe merah di kawasan Kota Surakarta. Kata Kunci: Covid-19, penjualan, jahe merah Abstract This study aims to analyze the impact of the Covid-19 outbreak on red ginger sales in the Surakarta city area. The variables used are Understanding & Knowledge about Covid-19 and Perceptions & Attitudes of the Community to anticipate Covid-19. The population of this study were red ginger sellers who had bought and sold red ginger during the Covid-19 situation, located in the city of Surakarta and its surroundings. The sample selection used purposive sampling method. The number of samples taken in this study were 120 respondents spread across Surakarta, Sukoharjo, Karanganyar, Boyolali, Sragen, Klaten and Wonogiri. This study uses primary data which is taken using a structured interview with the help of a questionnaire. The data analysis used is multiple regression analysis. The resulting conclusion is that the perception & attitude of the community to anticipate Covid-19 has a positive and significant effect on the level of red ginger sales, while the understanding and knowledge of Covid-19 has a positive but not significant effect on the level of red ginger sales Public perceptions & attitudes to anticipate Covid-19 are the most influential in selling red ginger in the Surakarta City area. Keywords: Covid-19, sale, Red Ginger Pendahuluan (Buana, 2020). Penyakit pernapasan virus akut yang dinamai penyakit Corona virus 2019 (COVID-19) berawal mula mewabah di Wuhan Cina disebabkan oleh Coronavirus Maraknya wabah COVID-19 di berbagai negara meyebabkan banyak masyarakat berupaya meningkatkan daya tahan tubuhnya. 93 Dampak Wabah Covid-19... (93-100) novel, merupakan Sindrom Pernafasan Akut Parah Coronavirus 2 (SARS-CoV-2). Menurut penelitian yang dilakukan oleh Li, dkk (2020 ) Coronavirus (CoVs) sejauh ini merupakan kelompok terbesar dari RNA indra-positif yang diketahui virus memiliki berbagai inang alami. Dalam beberapa dekade terakhir, baru Coronavirus yang berkembang telah menjadi ancaman global bagi kesehatan masyarakat. Organisasi Kesehatan Dunia (WHO) telah mendaftarkan epidemi coronavirus pneumonia baru sebagai keadaan darurat kesehatan masyarakat yang menjadi perhatian internasional.(Darmawan et al., 2020) paparan virus tersebut dengan penerapan protokol kesehatan yaitu pencegahan dengan melalukan social distancing, belajar dirumah yang dilakukan oleh instansi pendidikan, physical distance, pemakaian masker hingga lockdown di beberapa wilayah di dunia dan menjaga kebersihan dengan rajin cuci tangan dengan sabun, serta meningkatkan imunitas dalam tubuh. Hal tersebut mempengaruhi perilaku dan sikap masyarakat dalam mengantisipasi penularan virus covid 19 melalui konsumsi minuman herbal jahe. (Redi Aryanta, 2019) dan masyarakat percaya akan manfaat jahe yang dipercaya mampu untuk mengobati sesak nafas serta Di dukung dengan aneka kuliner yang terdapat di daerah Surakarta, masyarakat cenderung lebih mudah mendapatkan minuman-minuman herbal jahe tersebut seperti di warung hik pinggir jalan yang menyediakan berbagai minuman khas tradisional berbahan rempah seperti wedang jahe, susu jahe, the jahe, wedang uwuh yang berkhasiat untuk meningkatkan stamina sekaligus menghangatkan badan (Mangifera, Isa and Wajdi, 2018). Perilaku masyarakat ini merupakan tindakan- tindakan individu yang secara langsung terlibat dalam mendapatkan produk, megkonsumsi dan menghabiskan produk yang diinginkan.(Medika, 2020). Di Indonesia sendiri Pemerintah telah mengeluarkan status darurat bencana terhitung mulai tanggal 29 Februari 2020. Menurut data situs covid 19, per 1 november 2020 data jumlah korban di Indonesia yang positif terinfeksi covid 19 adalah 415.402, sembuh 345.566 dan meninggal 14.044 orang. Hal ini berdampak terhadap seluruh sistem kehidupan dan aktivitas sosial di seluruh sektor terutama bagi masyarakat di wilayah Surakarta dan sekitarnya. Kebijkana yang dilakukan oleh pemerintah untuk dapat menyelesaikan kasus luar biasa ini, antara lain dengan mensosialisasikan gerakan Social Distancing. Konsep ini menjelaskan bahwa untuk dapat mengurangi bahkan memutus mata rantai infeksi Covid-19 seseorang harus menjaga jarak aman dengan manusia lainnya minimal 2 meter, dan tidak melakukan kontak langsung dengan orang lain, menghindari pertemuan massal. Pendahuluan Pemerintah juga meliburkan aktivitas sekolah dan perkuliahan diganti dengan pembelajaran secara online dan memberlakukan bekerja didalam rumah, namun kondisi ini justru malahan dimanfaatkan oleh banyak masyarakat untuk berlibur dan belum bisa mengatasi permasalahan bencana covid 19.(Yanti et al., 2020). Pilihan atau keputusan pembelian jahe dipengaruhi oleh empat faktor psikologis utama yaitu motivasi, persepsi, pembelajaran, serta kepercayaan dan sikap.(Isa, Mardalis and Mangifera, 2018) Pengertian Persepsi Konsumen disini bahwa Persepsi adalah suatu proses memilih, mengatur dan menginterpretasikan informasi mengenai suatu produk barang atau jasa oleh konsumen. (Mauliansyah, 2015). Persepsi tidak hanya terjadi dalam bentuk rangsangan fisik tapi juga dipengaruhi oleh kondisi pemasaran yang ada. Hal ini selaras dengan yang disampaikan beberapa ahli. Menurut Pride & Ferrel dalam Fadila (2013:45), Persepsi adalah proses pemilihan, pengorganisasian dan penginterprestasian masukan informasi, sensasi yang diterima melalui penglihatan, perasaan, pendengaran, penciuman dan Hingga saat ini belum ditemukan obat antivirus covid-19, status kekebalan tubuh sendiri menjadi salah satu faktor paling penting yang mempengaruhi perkembangan penyakit dan prognosis. Himbauan kepada masyarakat untuk melindungi diri dari 94 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 22, No. 2, Desember 2020 sentuhan, untuk menghasilkan makna. Persepsi tersebut muncul karena daya tahan tubuh merupakan kunci yang sangat penting dalam situasi pandemi global COVID-19. Oleh karena itu banyak masyarakat memilih meningkatkan imun daya tahan tubuh melalui alternatif lain dengan membuat dan mengkonsumsi jahe merah sebagai upaya untuk meningkatkan daya tahan tubuh dalam upaya melawan COVID19. mengantisipasi Covid-19 , variable dependent adalah tingkat penjualan Jahe di kawasan kota Surakarta dan sekitarnya. Analisis data yang digunakan adalah analisis regresi berganda, yang terdiri dari koefisien diterminan, uji simultan (uji F), dan uji hipotesis (uji T) dan content analysis. Hasil Penelitian dan Pembahasan Penelitian ini dilakukan didaerah Surakarta yang terdiri dari 6 kabupaten yaitu Kota Surakarta, Kabupaten Sukoharjo, Wonogiri, Klaten, Karanganyar, Sragen, dan Boyolali. Dimana hamper semua wilayah tersebut menjadi zona merah persebaran COVID-19 yang sampai saat ini belum ditemukan anti virusnya atau vaksinnya. Sehingga masyarakat dan sekitarnya mencoba mensiasati hal tersebut dengan mengkonsumsi obat-obatan herbal seperti jahe merah yang di olah menjadi minuman/ wedang. Dengan permintaan jahe merah yang tinggi di duga adanya dampak COVID-19 terhadap tingkat penjualan jahe. Dengan adanya persepsi masyarakat terhadap wabah Covid 19 tersebut di duga memberi dampak terhadap pola perilaku masyarakat dalam mengkonsumsi minuman herbal khususnya jahe merah. Dengan adanya COVID-19 menyebabkan minat masyarakat akan jahe merah tinggi sehingga diduga mengakibatkan tingkat penjualan jahe merah di wilayah solo raya akan mengalami kenaikan. Hal ini merupakan suatu permasalahan yang menarik untuk di teliti. Untuk mendukung hal tersebut, maka diadakan penelitian ini dengan tujuan untuk mengetahui dampak COVID-19 terhadap tingkat penjualan jahe merah di kota Surakarta dan sekitarnya. COVID-19 membawa dampak yang begitu kompleks di semua sector industry dan kehidupan masyarakat Surakarta dan sekitarnya, salah satu sector yang terdampak sangat parah adalah perekonomian, banyak tenaga kerja yang terkena PHK bahkan kondisi UMKM sangat memprihatinkan sehingga banyak UMKM yang gulung tikar. Hampir Mayoritas masyarakat terkena dampak akan situasi dan Kondisi tersebut tanpa terkecuali merah di wilayah kota Surakarta dan sekitarnya.i Metode Penelitian Penelitian ini menggunakan metode diskriptif kuantitatif. Populasi penelitian ini adalah Penjual Jahe merah yang berada di wilayah Surakarta, Sukoharjo, Klaten, Wonogiri, Sragen Karanganyar, Boyolali. Pemilihan sampel menggunakan metode purposive sampling. Kriteria tersebut adalah Penjual jahe Merah yang berdomisili di Kota Surakarta dan Sekitarnya. Jumlah sampel yang diambil dalam penelitian ini sebanyak 120 pedagang jahe merah yang tersebar di Surakarta dann kabupaten Sukoharjo, Wonogiri, Sragen, Klaten, Karanganyar dan Boyolali. Penelitian ini menggunakan data primer yang diambil menggunakan wawancara terstruktur dengan bantuan kuesioner. Variabel independent dalam penelitian ini adalah persepsi responden tentang Pemahaman dan Pengetahuan tentang Covid-19 dan Persepsi dan Sikap Masyarakat untuk Profil responden dalam penelitian ini adalah Sebanyak 120 pedagang yang menjadi responden, yang terdiri dari 15,8 % Laki-laki dan 84,2 % perempuan. Sedangkan untuk aspek Pendidikan sebanyak 1,67% tidak bersekolah, 21,7% Tamatan SD, 37,5% tamatan SMP. 37,5% tamatan SMA, 0,83% tamatan D3 dan 0,83% tamatan S1. Terkait penjualan terdiri dari sistem penjualan Offline dan untuk Distribusi lokasi penjualan berasal dari Surakarta, Boyolali, Wonogiri, Karanganyar, Sukoharjo, Sragen dan Klaten. 95 Dampak Wabah Covid-19... (93-100) Tabel 1. Distribusi Lokasi Penjualan Tabel 1. Distribusi Lokasi Penjualan Rata-rata harga jahe dari 120 pedagang yang menjadi responden diperoleh informasi harga/Kg pada saat Covid-19 yang dihimpun dari berbagai daerah di Surakarta dan sekitarnya yang hasilnya yaitu, karanganyar berkisar Rp. 50.000, Sragen berkisar Rp. 49.100, Surakarta Rp.46.700, Klaten berkisar Rp. 46.500, wonogiri berkisar Rp. 46.250 dan Sukoharjo berkisar Rp. 46.100. Sehingga dapat disimpulkan bahwa disaat Covid-19 wilayah karanganyar menjadi daerah dengan harga komoditas jahe merah paling tinggi yaitu sebesar Rp. 50.000/Kg dan Sukoharjo menjadi daerah dengan harga jahe merah yang paling rendah yaitu sebesar Rp. 46.100/Kg. Boyolali berkisar Rp.47.200. Tabel 1. Distribusi Lokasi Penjualan Tabel 1. Distribusi Lokasi Penjualan Dari Sebanyak 120 pedagang yang menjadi responden diperoleh informasi mengenai rata-rata harga jahe merah/Kg pada saat sebelum Covid-19 yang dihimpun dari berbagai daerah di Surakarta dan sekitarnya yang hasilnya yaitu, Wonogiri berkisar Rp.33.000, Surakarta berkisar Rp31.000, karanganyar berkisar Rp.30.000, Boyolali berkisar Rp.28.800, Klaten berkisar Rp. 28.300, Sukoharjo berkisar Rp. 28.100 dan Sragen berkisar 26.100. Sehingga dapat disimpulkan bahwa harga jahe merah/kg disaat sebelum Covid-19 di level harga paling tinggi berada di Kab. Wonogiri sebesar Rp.33.000 dan terendah di Kab. Sragen yaitu sebesar Rp. 26.100 Tabel 3. Distribusi harga Jahe merah saat covid-19 Tabel 3. Distribusi harga Jahe merah saat covid-19 Tabel 3. Distribusi harga Jahe merah saat covid-19 Tabel 2. Distribusi harga jahe merah sebelum covid-19. Berdasarkan data diantara sebelum dan disaat Covid-19 yang telah dipaparkan diatas maka diperoleh prosentase kenaikan yang terjadi di daerah Surakarta dan Sekitarnya sebagai berikut yaitu, Wonogiri mengalami kenaikan harga sebesar 71.3%, Surakarta mengalami kenaikan harga sebesar 65,5%, Boyolali sebesar 61,7%, Sukoharjo sebesar 60,8%, Klaten sebesar 60,7%, Karanganyar sebesar 60% dan Sragen sebesar 53,2%. Sehingga dapat disimpulkan bahwa wilayah Wonogiri menjadi daerah dengan tingkat kenaikan harga komoditas jahe merah paling tinggi yaitu sebesar 71,3% dan Sragen menjadi Berdasarkan data diantara sebelum dan disaat Covid-19 yang telah dipaparkan diatas maka diperoleh prosentase kenaikan yang terjadi di daerah Surakarta dan Sekitarnya sebagai berikut yaitu, Wonogiri mengalami kenaikan harga sebesar 71.3%, Surakarta mengalami kenaikan harga sebesar 65,5%, Boyolali sebesar 61,7%, Sukoharjo sebesar 60,8%, Klaten sebesar 60,7%, Karanganyar b 60% d S b 53 2% Tabel 2. Distribusi harga jahe merah sebelum covid-19. 96 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 22, No. 2, Desember 2020 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 22, No. 2, Desember 2020 daerah dengan kenaikan harga komoditas jahe merah yang paling rendah yaitu sebesar 53,2%. Tingkat penjualan jahe merah seperti dalam table 4 diatas, diperoleh informasi bahwa tingkat penjualan Jahe Merah tertinggi berada di daerah Sukorharjo Dimana Sukoharjo menjadi daerah yang mengalami peningkatan penjualan terbesar yaitu dengan prosentase sebesar 81,75 % lalu di susul Klaten dengan Prosentase sebesar 79,20%, Wonogiri dengan Prosentase sebesar 69,16%, Boyolali dengan Prosentase sebesar 64,89%, Sragen dengan Prosentase sebesar 60,37%, Karanganyar dengan Prosentase sebesar 59,89% dan Surakarta dengan Prosentase sebesar 58,24%. Sehingga dapat di simpulkan bahwa wilayah wonogiri menjadi daerah dengan tingkat peningkatan penjualan tertinggi dan sedangkan Surakarta menjadi daerah dengan tingkat peningkatan paling rendah . Tabel 4. Tingkat Penjualan jahe merah Tabel 4. Tingkat Penjualan jahe merah Tabel 4. Tingkat Penjualan jahe merah Hasil analisis regresi linear berganda dapat di lihat pada tabel 5 di bawah ini. Tabel 5. Hasil Analisis Regresi Linier Berganda Variabel Independen Koefisien t-ratio Sig. Pemahaman dan pengetahuan tentang Covid-19 (X1) 0,107 1.720 .088 Persepsi dan sikap masyarakat untuk mengantisipasi Covid-19 (X2) 0,203 3.506 .001 Konstanta = 8.304 R2 = 0.140 F = 9.486 (Sig. 0,000) Sumber: Hasil Analisa Data (2020). Tabel 5. Hasil Analisis Regresi Linier Berganda Tabel 5. Hasil Analisis Regresi Linier Berganda Sumber: Hasil Analisa Data (2020). Tabel 3. Distribusi harga Jahe merah saat covid-19 Variabel Pemahaman & Pengetahuan tentang Covid-19 (X1) berpengaruh positif tetapi tidak signifikan pada alpha 5% terhadap penjualan jahe merah di kawasan kota Surakarta dan Hasil uji T untuk variabel Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 menunjukkan nilai T seebsar 3.506 dengan nilai sig 0, 01. Artinya variabel Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 (X2) berpengaruh positif dan signifikan pada alpha 5% terhadap penjualan jahe merah di kawasan kota Surakarta. Dari hasil analisis Variable Pemahaman & Pengetahuan tentang Covid-19 di atas berpengaruh positif dan tetapi tidak signifikan terhadap penjualan jahe merah di kota Surakarta dan sekitarnya. Hal ini di karenakan tingkat pemahaman responden belum sepenuhnya paham dan mengetahui tentang Covid-19. Sedangkan Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 berpengaruh positif dan signifikan terhadap penjualan jahe merah di kota Surakarta dan sekitarnya. Hal ini dikarenakan adanya dorongan persepsi masyarakat dalam melakukan keputusan pemilihan komoditas jahe merah tersebut. Menurut Kotler dan Keller (2016:153), keputusan pembelian seseorang dipengaruhi oleh faktor psikologi utama, antara lain. persespsi serta keyakinan dan pendirian. Pendapat di atas menunjukkan adanya hubungan antara persepsi konsumen dengan keputusan untuk membeli suatu produk. Sehingga jika tingkat pembelian masyarakat akan Jahe Merah meningkat maka akan berkorelasi dengan tingkat penjualan dari pedagang Jahe Merah tersebut. Pengujian asumsi klasik digunakan untuk memastikan bahwa data yang digunakan dalam kondisi yang baik. Uji ini meliputi uji normalitas, heterokedastisitas dan multikolinieritas. Hasil uji normalitas untuk mengetahui apakah data dari masing- masing variabel memiliki sebaran yang normal ataukah tidak. Uji kolmogorof- smirnov digunakan menguji normalitas data. (Ghazali, 2015). Hasil analisis menunjukkan bahwa semua nilai asymp. Sig dia ats 5% sehingga disimpulkan data dalam penelitian ini memiliki sebaran data yang normal. Uji Heteroskedastisitas digunakan untuk mendeteksi ada tidaknya heteroskedastisitas dilakukan dengan uji glejser, yaitu dengan melakukan regresi variabel independen terhadap absolut residual variabel dependen dalam regresi utama (Ghazali, 2015). Hasil analisis menunjukkan bahwa besarnya nilai signifikan untuk hasil uji heterokedastisitas terhadap semua variabel memiliki nilai lebih besar dari 0,05 sehingga disimpulkan bahwa semua variabel penelitian bebas dari masalah heterokedastisitas. Tabel 3. Distribusi harga Jahe merah saat covid-19 Berdasarkan tabel 5 dapat dibuat persaman regresi linear berganda sebagai berikut: (X2) sebesar 0,203 . Setiap kenaikan 1 satuan, maka tingkat penjualan jahe merah di kawasan kota Surakarta meningkat sebesar 0,203. 2 Y = 8.304 + 0,107 (X1) + 0,203 (X2) (0,88) (0,01) Nilai R2 sebesar 0.104 . Variasi tingkat penjualan jahe merah di kawasan kota Surakarta dijelaskan oleh variabel Pemahaman & Pengetahuan tentang Covid-19 dan Persepsi & Sikap Masyarakat untuk Hasil analisis regresi linear berganda ini di atas menunjukkan bahwa Koefisien regresi variabel Pemahaman dan Pengetahuan tentang Covid-19 (X1) sebesar 0,107. Setiap kenaikan 1 satuan pemahaman dan pengetahuan masyarakat terhadap covid19, maka tingkat penjualan jahe merah di kawasan kota Surakarta meningkat sebesar 0,107. Koefisien regresi Persepsi dan Sikap Masyarakat untuk mengantisipasi Covid-19 mengantisipasi Covid-19 sebesar 10,4 %, sedangkan sisanya 89,6 % dijelaskan oleh variabel lainnya diluar model yang diteliti. Hasil uji F menunjukkan nilai 9, 486 dengan signifikansi 0,000. Model penelitian ini adalah tepat (fit) dan Pemahaman & Pengetahuan tentang Covid-19 serta Persepsi & Sikap Masyarakat untuk mengantisipasi 97 Dampak Wabah Covid-19... (93-100) Covid-19 secara simultan mempunyai pengaruh signifikan terhadap tingkat penjualan Jahe merah di kawasan kota Surakarta. Hasil uji T untuk variabel dan Pemahaman & Pengetahuan tentang Covid-19 menunjukkan nilai T sebesar 1.720 dengan nilai sig 0.88. Variabel Pemahaman & Pengetahuan tentang Covid-19 (X1) berpengaruh positif tetapi tidak signifikan pada alpha 5% terhadap penjualan jahe merah di kawasan kota Surakarta dan Hasil uji T untuk variabel Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 menunjukkan nilai T seebsar 3.506 dengan nilai sig 0, 01. Artinya variabel Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 (X2) berpengaruh positif dan signifikan pada alpha 5% terhadap penjualan jahe merah di kawasan kota Surakarta. dengan melihat nilai VIF dari masing- masing variabel. Jika nilai VIF lebih rendah dari 10, maka disimpulkan bahwa tidak ada multikolinieritas yang serius antara variabel independen dalam model (Ghazali, 2015). Hasil uji multikolinearitas menujukkan bahwa nilai VIF pada uji multikolinearitas variabel penelitian memiliki nilai kurang dari 10 sehingga dapat disimpulkan bahwa semua variabel penelitian tidak terjadi multikolinearitas. Hasil uji T untuk variabel dan Pemahaman & Pengetahuan tentang Covid-19 menunjukkan nilai T sebesar 1.720 dengan nilai sig 0.88. Penutup Simpulan dalam penelitian ini adalah wabah Covid 19 memberi dampak terhadap tingkat penjualan jahe merah di wilayah Surakarta dan sekitarnya. Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 berpengaruh positif dan signifikan terhadap tingkat penjualan jahe merah dikota Surakarta dan sekitarnya. Selain adanya pengaruh akan Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 ada hal yang Uji Multikolinieritass adalah suatu keadaan di mana salah satu atau lebih variabel independen dapat dinyatakan sebagai kombinasi linier dari variabel independen lainnya. Uji multikolinieritas dilakukan 98 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 22, No. 2, Desember 2020 & Sikap Masyarakat untuk mengantisipasi Covid-19 adalah salah satu Variable yang mempengaruhi tingkat penjualan jahe merah di Kota Surakarta dan Sekitarnya di saat situasi Pandemi Covid-19. Implikasi terhadap manajerialnya adalah pentingnya dalam mengelola Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 yang di implementasikan ke dalam bentuk penjualan jahe merah di tengah situasi Covid - 19 . membuat permintaan dan harga jahe merah mengalami kenaikkan yang cukup signifikan yaitu karena dipengaruhi oleh cuaca yang berkorelasi melalui kondisi media tanam dan minat daya beli masyarakat disaat terdampak Covid-19 serta juga dipengaruhi oleh musim tanam dan panen. & Sikap Masyarakat untuk mengantisipasi Covid-19 adalah salah satu Variable yang mempengaruhi tingkat penjualan jahe merah di Kota Surakarta dan Sekitarnya di saat situasi Pandemi Covid-19. Implikasi terhadap manajerialnya adalah pentingnya dalam mengelola Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 yang di implementasikan ke dalam bentuk penjualan jahe merah di tengah situasi Covid - 19 . Implikasi teoritis penelitian ini adalah membuka penelitian terbaru terkait penjualan jahe merah, dimana Persepsi Daftar Pustaka Aryanta, I. W. R. (2019). Manfaat Jahe Untuk Kesehatan. Widya Kesehatan, 1(2), 39-43 Buana, D. R. (2020) ‘Analisis Perilaku Masyarakat Indonesia dalam Menghadapi Pandemi Virus Corona (Covid-19) dan Kiat Menjaga Kesejahteraan Jiwa’, SALAM: Jurnal Sosial dan Budaya Syar-i, 7(3). doi: 10.15408/sjsbs.v7i3.15082. Darmawan, D. et al. (2020) ‘Sikap Keberagamaan Masyarakat Muslim Menghadapi Covid-19’, Religious: Jurnal Studi Agama-Agama dan Lintas Budaya, 7249, pp. 116–124. Isa, M., Mardalis, A. and Mangifera, L. (2018) ‘Analisis Keputusan Konsumen Dalam Melakukan Pembelian Makanan dan Minuman di Warung Hik’, Jurnal Manajemen Dayasaing, 20(1), pp. 44–51. doi: 10.23917/dayasaing.v20i1.6590. Mangifera, L., Isa, M. and Wajdi, M. F. (2018) ‘Faktor-Faktor yang Mempengaruhi Konsumen Dalam Pemilihan Kuliner di Kawasan Wisata Alam Kemuning’, Jurnal Manajemen Dayasaing, 20(1). doi: 10.23917/dayasaing.v20i1.6415. Mauliansyah, R. (2015) ‘Analisis Pengaruh Motivasi, Persepsi Kualitas dan Sikap Konsumen terhadap Keputusan Pembelian Sepeda Motor Honda di Kota Langsa’, Jurnal Manajemen dan Keuangan Unsam, 4(1), p. 196986. Medika, J. M. (2020) ‘Hubungan Pengetahuan dan Sikap Masyarakat Terhadap Covid-19: A literature Review’, Jurnal Menara Medika https://jurnal.umsb.ac.id/index.php/ menaramedika/index JMM 2020 p-ISSN 2622-657X, e-ISSN 2723-6862, 3(1), pp. 46–53. Redi Aryanta, I. W. (2019) ‘Manfaat Jahe Untuk Kesehatan’, Widya Kesehatan, 1(2), pp. 39– 43. doi: 10.32795/widyakesehatan.v1i2.463. Yanti, N. P. E. D. et al. (2020) ‘Gambaran Pengetahuan Masyarakat tentang Covid-19 dan Perilaku Masyarakat di Masa Pandemi Covid-19’, Jurnal Keperawatan Jiwa, Vol. 8 No.(3), pp. 485–490. Pradana, A. A., & Casman, C. (2020). Pengaruh Kebijakan Social Distancing pada Wabah COVID-19 terhadap Kelompok Rentan di Indonesia. Jurnal Kebijakan Kesehatan Indonesia: JKKI, 9(2), 61-67. Kriswibowo, A., & Utomo, S. A. P. (2020). Ekonomi Politik Indonesia di Tengah Pandemi Covid- 19 Penerbit Cakradewa Ilmu. Ramadhani, V. A. (2018). Pengaruh Persepsi, Motivasi Dan Gaya Hidup Konsumen Usia Muda Terhadap Keputusan Pembelian Makanan Cepat Saji Di Mcdonald’s Sudirman Pekanbaru (Studi Kasus Pada Mahasiswa UIN Suska Riau Fakultas Sains dan Teknologi Jurusan Teknik Industri S1) (Doctoral dissertation, Universitas Islam Negeri Sultan Syarif 99 Dampak Wabah Covid-19... (93-100) Kasim Riau Dampak Wabah Covid-19... (93-100) Kasim Riau Dampak Wabah Covid-19... (93-100) Kasim Riau Febriani, Y., Riasari, H., Winingsih, W., Aulifa, D. L., & Permatasari, A. (2018). The Potential Use of Red Ginger (Zingiber officinale Roscoe) Dregs as Analgesic. Indonesian Journal of Pharmaceutical Science and Technology, 1(1), 57-64. Rahmadhani, A. N., Novayelinda, R., & Woferst, R. (2014). Efektifitas pemberian minuman jahe madu Terhadap keparahan batuk pada anak dengan ispa. Jurnal Online Mahasiswa (JOM) Bidang Ilmu Keperawatan, 1(2), 1-7. Kartini, P. R., & Pratama, E. B. (2017, November). POTENSI EKSTRAK JAHE MERAH SEBAGAI TERAPI ALAMI KEJADIAN ASMA PADA ATLET. In Prosiding Seminar Nasional Hasil Penelitian LPPM Universitas PGRI Madiun (pp. 284-290). Rofi, A. (2013). Karakteristik Demografi, Sosial, dan Ekonomi serta Pola Pendapatan Usaha Perdagangan di Sepanjang Jalan Jogja-Solo Kabupaten Klaten. Jurnal Bumi Indonesia, 2(1). Utama, I. G. B. R., Suamba, I. B. P., Sumartana, I. M., Waruwu, D., & Krismawintari, N. P. D. (2020). Dampak Himbauan Social Distancing Dalam Mengurangi Penyebaran Covid-19 Pada Masyarakat Bali. Jurnal Aplikasi dan Inovasi Iptek, 2(1), 46-59. Yunus, N. R., & Rezki, A. (2020). Kebijakan Pemberlakuan Lock Down Sebagai Antisipasi Penyebaran Corona Virus Covid-19. 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The Influence of Technology on Accounting Practices in India: A Survey of Accounting Software Utilization by Micro Enterprises in Delhi
European journal of theoretical and applied sciences
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Abstract: significant in urban areas like Delhi, where micro enterprises form a vital component of the local economy. The advent of various accounting software solutions has enabled these businesses to manage their financial transactions more Abstract: Suggested Citation Gupta, R. (2023). The Influence of Technology on Accounting Practices in India: A Survey of Accounting Software Utilization by Micro Enterprises in Delhi. European Journal of Theoretical and Applied Sciences, 1(6), 1041-1053. DOI: 10.59324/ejtas.2023.1(6).101 Suggested Citation Gupta, R. (2023). The Influence of Technology on Accounting Practices in India: A Survey of Accounting Software Utilization by Micro Enterprises in Delhi. European Journal of Theoretical and Applied Sciences, 1(6), 1041-1053. DOI: 10.59324/ejtas.2023.1(6).101 This research paper investigates the impact of technology adoption on accounting practices within micro enterprises in Delhi, India. The primary objectives of this study were to identify the factors influencing the selection of accounting software, examine the relationship between technology adoption and perceived improvements in financial efficiency, and uncover the specific challenges faced by micro enterprises in integrating technology into their accounting processes. To achieve these objectives, a comprehensive survey was conducted among a diverse sample of micro enterprises in Delhi. The survey assessed the criteria used by these businesses to choose accounting software, their level of confidence in data security, and their satisfaction with the software's functionality. It also explored the challenges encountered during software adoption and identified the features that micro enterprises deemed essential for software improvement. The key findings of this research indicate that micro enterprises in Delhi prioritize cost-effectiveness and functionality when selecting accounting software. While technology adoption positively impacts confidence in data security and satisfaction levels, it does not consistently lead to perceived improvements in financial efficiency. Challenges in software adoption are closely related to the nature of the business, emphasizing the need for tailored approaches. Desired software improvements include enhanced tax compliance features, cloud-based access, improved reporting tools, and scalability. The implications of this study extend beyond Delhi's micro enterprises, offering insights for policymakers, business owners, and the accounting profession. Policymakers can use these findings to design initiatives that support digital transformation in small businesses. Business owners can make informed decisions when selecting accounting software, and accountants can adapt their skills to the evolving landscape of technology integration in finance. gg Gupta, R. (2023). The Influence of Technology on Accounting Practices in India: A Survey of Accounting Software Utilization by Micro Enterprises in Delhi. European Journal of Theoretical and Applied Sciences, 1(6), 1041-1053. DOI: 10.59324/ejtas.2023.1(6).101 Keywords: technology adoption, accounting practices, micro enterprises, accounting software, data security, financial efficiency, digital transformation. The Influence of Technology on Accounting Practices in India: A Survey of Accounting Software Utilization by Micro Enterprises in Delhi Renu Gupta  Department of Commerce, Jesus and Mary College, University of Delhi, India Introduction The integration of technology into accounting practices, particularly in the context of micro enterprises in India, represents a profound transformation in the financial management landscape. This shift has been especially This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if they made any changes. This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if they made any changes. efficiently and effectively, marking a significant departure from traditional manual methods. adapting to new technologies and methodologies (Kamau and Ilamoya, 2023). This research will contribute to a better understanding of these changes in the context of Indian micro enterprises, highlighting the nuances of technology adoption in a sector that significantly differs from large corporations. The evolution from manual book keeping to sophisticated accounting software is a pivotal development in the accounting domain. It signifies not just a change in the tools used for accounting but also represents a fundamental shift in the skills and mindset required within the accounting profession. This technological revolution, often encapsulated in the term "accounting information systems" (AIS), has had a far-reaching impact on how accounting data is collected, processed, and reported (Gnana Prakash, 2023). Moreover, the relevance of this research extends beyond the academic realm; it has practical implications for business owners, policymakers, software developers, and accounting professionals. By examining the utilization of accounting software in micro enterprises, this study will provide valuable insights into the effectiveness of these tools in improving business operations and compliance with financial regulations. Furthermore, it can guide policymakers in developing strategies to support the digital transformation of small businesses, a crucial step in enhancing the overall economic growth of the country. In India, and particularly in its capital, New Delhi, the adoption of technology in accounting poses both opportunities and challenges for micro enterprises. While these small businesses typically operate with limited resources, making the adoption of technology a critical and strategic decision, the potential benefits of such adoption are significant. Introduction They investigated the extent to which these systems have been adopted and the consequent impact on the financial management practices of MSMEs (Cahyani & Amna, 2022). examining the challenges, benefits, and overall impact on their business operations. The findings of this study are expected to contribute significantly to the literature on technology in accounting and provide practical insights for various stakeholders in the accounting and business sectors. By delving into the specific context of Delhi's micro enterprises, this study will shed light on a critical aspect of India's economic fabric, offering a comprehensive understanding of the interplay between technology and accounting in the small business sector. Wicaksono, Kartikasary, and Salma (2020) delved into cloud accounting software implementation and security systems. Their research provided insights into the adoption of cloud-based accounting solutions by MSMEs and the security considerations associated with such implementations (Wicaksono et al., 2020). The work by Vysochan et al. (2021) focused on the selection of accounting software for small and medium enterprises using the Fuzzy Topsis Method. This study presented a novel approach to choosing appropriate accounting software, considering various factors critical for small and medium-sized businesses (Vysochan et al., 2021). Wicaksono, Kartikasary, and Salma (2020) delved into cloud accounting software implementation and security systems. Their research provided insights into the adoption of cloud-based accounting solutions by MSMEs and the security considerations associated with such implementations (Wicaksono et al., 2020). The work by Vysochan et al. (2021) focused on the selection of accounting software for small and medium enterprises using the Fuzzy Topsis Method. This study presented a novel approach to choosing appropriate accounting software, considering various factors critical for small and medium-sized businesses (Vysochan et al., 2021). Introduction Improved efficiency, accuracy, and compliance with financial regulations are among the foremost advantages. The use of popular accounting software like Tally and QuickBooks, which offer functionalities catering to the specific needs of small-scale businesses, has gained prominence in this segment (Utami & Yulianto, 2019). The study of technology's influence on accounting in micro enterprises is not just about the adoption of new tools; it's about understanding the broader context in which these businesses operate. Factors such as the ease of use of the software, its cost-effectiveness, and the training required for effective utilization are critical aspects that influence the decision- making process of small business owners. Additionally, the study will explore the impact of such technological adoption on the financial performance and growth prospects of these enterprises. The significance of studying the impact of technology on accounting practices in micro enterprises in Delhi lies in understanding how widespread these tools are and the consequent effects on accounting practices. As Fauzi et al. (2022) point out, the advancement of information technology has a profound influence on the development of accounting practices (Fauzi et al., 2022). This study aims to bridge the gap in literature by focusing specifically on micro enterprises in a developing economy, offering insights into how these businesses are navigating the technological landscape in accounting. This research is also significant in the context of the global shift towards digitalization. As economies around the world increasingly embrace digital technologies, understanding how this trend impacts the grassroot level of the business ecosystem in developing countries like India becomes crucial. The insights gained from this study could be instrumental in shaping policies and strategies not only for India but also for other developing nations facing similar challenges and opportunities in the digital era. The evolution of accounting practices under the influence of technology has broader implications for the accounting profession itself. As noted by Kamau and Ilamoya (2023), the accounting profession is undergoing significant changes, In summary, this research aims to explore the extent of technology adoption in accounting practices among micro enterprises in Delhi, www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1042 1042 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com Anindya, 2021). In their 2022 study, Cahyani and Amna analyzed the application of accounting information systems in micro, small, and medium enterprises (MSMEs). Literature Review Literature Review Literature Review of Scholarly Works Literature Review of Scholarly Works Literature Review of Scholarly Works The intersection of technology and accounting practices, particularly in the context of micro enterprises, has been the subject of extensive research over the past few years. This literature review examines following scholarly works that provide insights into the utilization of accounting software in micro enterprises, highlighting the evolution and impact of this trend. Apriyanti and Yuvitasari (2021) researched the role of digital utilization in accounting to enhance MSMEs' performance during the COVID-19 pandemic. Their case study highlighted the increased reliance on digital accounting tools during the pandemic and its positive impact on the performance of MSMEs in Semarang, Indonesia (Apriyanti & Yuvitasari, 2021). In a 2019 study, Suhayati and Riandani explored an accounting application tailored for small and medium enterprises. Their research contributed to understanding how tailor-made accounting applications can meet the specific needs of SMEs, facilitating more efficient financial management practices (Suhayati & Riandani, 2019). Ezenwoke (2017) examined the organizational determinants and implementation of e-accounting systems in micro and small enterprises in Nigeria. This study provided a comprehensive analysis of factors influencing the adoption of electronic accounting systems in small businesses in a developing country context (Ezenwoke, 2017). Sumarna (2023) examined the usage of computerized accounting systems in micro-small enterprises. The study emphasized the increasing trend of digitalization in accounting practices among smaller businesses, highlighting the benefits and challenges faced during the transition from traditional methods to computerized systems (Sumarna, 2023). In Shbeilat and Al-Hajaia (2022), the focus was on electronic accounting applications in micro- enterprises. The paper discussed the challenges and the impact of these applications on financial inclusion, underscoring the role of technology in enhancing accessibility and efficiency in accounting for micro-enterprises (Shbeilat & Al- Hajaia, 2022). Rahman and Anindya (2021) explored how the utilization of accounting software could increase the performance of MSMEs. Literature Review of Scholarly Works Their findings suggested that the adoption of such software directly correlates with improved business efficiency and financial performance (Rahman & These scholarly works collectively highlight the evolving landscape of accounting practices in the www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1043 1043 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com Sampling Technique Stratified Random Sampling Data Collection Time March – May 2023 Response Rate 60% [out of 90 firms response was received from 54 firms Data Collection Tool Structured Questionnaire (See Appendix: Questionnaire) Pilot Study Conducted on a group of 10 firms to pre-test the questionnaire for clarity, context of micro enterprises. The transition to digital and computerized systems has brought about significant changes in how these businesses manage their financial transactions. Despite the challenges, the overall impact has been positive, with improvements in efficiency, financial performance, and inclusion. Identification of Literature Gap The existing literature extensively explores the adoption and impact of accounting software in micro enterprises, with a global perspective that often includes developing countries. However, a noticeable gap exists in the specific study of the influence of technology on accounting practices within the context of micro enterprises in Delhi, India. While previous studies have addressed the general utilization of accounting software in small and medium-sized enterprises, there is a lack of focused research on the unique challenges and opportunities faced by micro enterprises in a densely populated and economically diverse city like New Delhi. Variables for the Study To effectively analyse the influence of technology on accounting practices in micro enterprises in Delhi, it is essential to categorize the variables into Independent, Dependent, and Controlled Variables, based on the questionnaire provided in the appendix. Table 2. Variables for the Study Variable Type Variables Independent Variables • Type of Micro Enterprise • Nature of Business (Goods, Services, Both) • Accounting Software in Use • Duration of Current Accounting Software Usage, Dependent Variables • Satisfaction Level with Current Software • Factors Contributing to Satisfaction • Confidence in Financial Data Security • Reasons for Dissatisfaction • Frequency of Accounting Software Updates • Challenges Faced in Accounting Software Adoption • Perceived Improvement in Financial Efficiency • Justification of Cost for Software Benefits Table 2. Variables for the Study Addressing this gap is significant for several reasons. New Delhi, as a major economic hub in India, presents a unique environment where traditional business practices and modern technological advancements converge. Understanding how micro enterprises in this region adopt and utilize accounting software can provide valuable insights into the effectiveness of these technologies in varied economic landscapes. Additionally, this research can guide policymakers and software developers in creating more tailored accounting solutions and support mechanisms for micro enterprises in similar urban settings. Research Methodology Table 1. Sampling & Data Collection Source Element Description Sample Size 90 firms Source of Data Online Surveys and In-Person Questionnaires Geographical Area Delhi, India www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1044 1044 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 Controlled Variables • Gender • Age • Education Level • Years in Business • Annual Turnover • Factors Considered in Software Selection • Desired Features for Software Improvement Controlled Variables • Gender • Age • Education Level • Years in Business • Annual Turnover • Factors Considered in Software Selection • Desired Features for Software Improvement Table 3. Results of Pilot Testing of the Questionnaire/Instrument or Reliability Analysis Variables Cronbach's Alpha Values No. of Items Type of Micro Enterprise 0.73 4 Nature of Business 0.78 3 Accounting Software in Use 0.82 5 Duration of Accounting Software Usage 0.75 4 Satisfaction Level with Current Software 0.81 5 Factors Contributing to Satisfaction 0.84 8 Confidence in Financial Data Security 0.77 5 Reasons for Dissatisfaction 0.79 7 Frequency of Accounting Software Updates 0.72 4 Challenges in Accounting Software Adoption 0.80 6 Perceived Improvement in Financial Efficiency 0.85 5 Justification of Cost for Software Benefits 0.74 5 The data collected through the survey was analysed using the following inferential statistical tests: 1. Frequency Count & Percentage: Applied to controlled variables to understand the demographic and business profile of the respondents. 2. Correlation Matrix: This tool was used to assess the relationship between all dependent and independent variables. It helped in understanding how variables such as the type of software used, duration of its use, and nature of the business relate to satisfaction levels, security confidence, and perceived efficiency improvements. 2. Correlation Matrix: This tool was used to assess the relationship between all dependent and independent variables. It helped in understanding how variables such as the type of software used, duration of its use, and nature of the business relate to satisfaction levels, security confidence, and perceived efficiency improvements. 3. Regression Analysis: This analysis was conducted to evaluate the predictive power of independent variables on all the dependent variables. The beta coefficient indicated the strength and direction of these relationships, while the p-value helped in determining the statistical significance. This analysis is crucial in understanding which factors most strongly predict satisfaction with accounting software, challenges in adoption, and perceived improvements in financial processes. Research Methodology To test the reliability, a pilot test was conducted on a subset of responses to the questionnaires. It was found that all the Cronbach's alpha values were above the threshold of 0.70, indicating a high level of internal consistency and reliability in the measures used in the survey. This result validates the reliability of the questionnaire for the larger study. Table 4. Socio-demographic Characteristics of the Respondents (n=54) Demographic Information Frequency Count Percentage (%) Gender Male 39 72.22 Female 15 27.78 Age 18-24 10 18.52 25-34 16 29.63 Table 4. Socio-demographic Characteristics of the Respondents (n=54) Results and Analysis This section presents the findings from the research, offering a comprehensive analysis of the data collected. It includes various tables and figures that illustrate the key points of the research. www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1045 1045 2023 | Volume 1 | Number 6 www.ejtas.com www.ejtas.com EJTAS 35-44 12 22.22 45-54 8 14.81 55 and above 8 14.81 Education High School or below 5 9.26 Bachelor's Degree 18 33.33 Master's Degree 20 37.04 Doctorate or Professional Degree 11 20.37 Type of Enterprise Sole Proprietorship 20 37.04 Partnership 12 22.22 Private Limited Company 16 29.63 Others 6 11.11 Business Nature Goods 15 27.78 Services 25 46.30 Both Goods and Services 14 25.93 Years in Business Less than 1 year 6 11.11 1-5 years 24 44.44 6-10 years 16 29.63 More than 10 years 8 14.81 Annual Turnover (Rs.) Less than 1 crore 12 22.22 1-2 crore 18 33.33 2-4 crore 16 29.63 4-5 crore 8 14.81 35-44 12 22.22 45-54 8 14.81 55 and above 8 14.81 Education High School or below 5 9.26 Bachelor's Degree 18 33.33 Master's Degree 20 37.04 Doctorate or Professional Degree 11 20.37 Type of Enterprise Sole Proprietorship 20 37.04 Partnership 12 22.22 Private Limited Company 16 29.63 Others 6 11.11 Business Nature Goods 15 27.78 Services 25 46.30 Both Goods and Services 14 25.93 Years in Business Less than 1 year 6 11.11 1-5 years 24 44.44 6-10 years 16 29.63 More than 10 years 8 14.81 Annual Turnover (Rs.) Less than 1 crore 12 22.22 1-2 crore 18 33.33 2-4 crore 16 29.63 4-5 crore 8 14.81 The socio-demographic characteristics of the respondents (n=54) provide a diverse overview of micro enterprises in Delhi. The majority of respondents are male (72.22%), highlighting a gender disparity in micro enterprise ownership. Age-wise, most respondents are within the 25-34 (29.63%) and 35-44 (22.22%) age brackets, indicating a relatively young entrepreneurial base. In terms of education, a significant proportion holds a Master's Degree (37.04%), followed by those with a Bachelor's Degree (33.33%). This suggests a relatively high level of educational attainment among the micro enterprise owners. The most common type of micro enterprise is a sole proprietorship (37.04%), followed by private limited companies (29.63%) and partnerships (22.22%). This distribution indicates a variety of business structures in the micro enterprise sector. Regarding the nature of business, a larger percentage deals in services (46.30%) compared to goods (27.78%) or both (25.93%). This aligns with the growing trend of service-oriented businesses in urban areas like Delhi. Most businesses have been operating for 1-5 years (44.44%), suggesting a substantial number of relatively new enterprises. this dataset, warmer colors (such as red and orange) denote stronger positive correlations, while cooler colors (like blue) represent weaker or negative correlations. The descriptive statistics for the key variables in the study are presented in Table 4.3. The mean values range from 2.15 (Type of Micro Enterprise) to 4.61 (Satisfaction Level with Current Software), indicating varied levels of these characteristics among the respondents. The standard deviation values, which measure the amount of variation or dispersion in the data, range from 0.54 to 1.31, suggesting a moderate spread in the responses. Notably, all the skewness and kurtosis values lie within the range of -1 to +1, confirming the normality of the data. This is crucial for the validity of further statistical analyses such as correlation and regression. A skewness or kurtosis value within this range indicates that the data distribution does not significantly deviate from a normal distribution, which is a key assumption in many statistical tests. Note: Correlation is significant at the 0.05 level (2-tailed). This table represents the diagonal correlation matrix which shows the relationship between all dependent and independent variables. The correlation values range from -1 (perfect negative correlation) to +1 (perfect positive correlation), with 0 indicating no correlation. Figure 1 presents the correlation matrix, which illustrates the relationships between various independent and dependent variables of the study. The correlations range between -1 and 1, where values close to 1 or -1 indicate a strong positive or negative relationship, respectively, and values around 0 suggest little to no correlation. For instance, a notable positive correlation between 'Duration of Accounting Software Usage' and 'Accounting Software in Use' (0.89) suggests that longer use is associated with specific types of software. Conversely, a strong negative correlation between 'Nature of Business' and 'Satisfaction Level with Current Software' (-0.62) indicates that the nature of the business significantly impacts satisfaction levels, possibly due to varying software needs. Overall, these descriptive statistics provide a foundational understanding of the data's distribution and central tendencies, setting the stage for more in-depth inferential analyses. Figure 1. Correlation Matrix Heat Map The matrix also highlights interesting relationships like the strong negative correlation between 'Confidence in Financial Data Security' and 'Perceived Improvement in Financial Efficiency' (-0.92), suggesting that higher confidence in data security might not always correlate with perceived improvements in efficiency. This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if they made any changes. www.ejtas.com In terms of annual turnover, the most common range is 1-2 crore INR (33.33%), followed by 2-4 crore INR (29.63%), indicating a moderate level of business scale among the respondents. Table 5. Descriptive Statistics for Key Variables Table 5. Descriptive Statistics for Key Variables Key Variables Mean Standard Deviation Skewness Kurtosis Type of Micro Enterprise 2.15 0.54 -0.79 0.17 Nature of Business 4.30 0.94 -0.72 0.89 Accounting Software in Use 3.96 0.60 -0.38 0.32 Duration of Current Accounting Software Usage 4.15 1.31 0.74 -0.29 Satisfaction Level with Current Software 4.61 1.30 0.81 0.11 Factors Contributing to Satisfaction 2.49 0.64 0.59 0.53 Confidence in Financial Data Security 4.51 0.87 0.34 -0.05 Reasons for Dissatisfaction 3.53 1.16 0.91 0.67 Frequency of Accounting Software Updates 2.78 0.90 -0.75 -0.31 Challenges Faced in Accounting Software Adoption 3.15 1.02 -0.93 -0.49 Perceived Improvement in Financial Efficiency 2.18 0.96 -0.63 -0.91 Justification of Cost for Software Benefits 3.93 0.58 -0.81 -0.97 www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1046 1046 2023 | Volume 1 | Number 6 Additionally, the 'Challenges in Software Adoption' show a notable positive correlation with the 'Nature of Business' (0.75), indicating that the type of business significantly influences the challenges faced in software adoption. Figure 1. Correlation Matrix Heat Map This figure presents a visual representation of the correlation matrix in the form of a heat map. The color intensity and the values in each cell indicate the strength and direction of the correlations between the variables studied. In These correlations provide valuable insights into how different aspects of business operations and characteristics interact with the adoption and satisfaction levels of accounting software, guiding further analysis and discussion in the study. a strong and statistically significant relationship with satisfaction levels. Frequency of Software Updates shows a very high positive beta coefficient (1.567), suggesting a significant influence on satisfaction, although the p-value (0.078) is slightly higher than the conventional significance level. The p-values, especially those below 0.05, indicate statistically significant relationships that can be confidently interpreted within the context of this study. Table 6. Regression Analysis Independent Variables Beta Coefficients p- Values Type of Micro Enterprise 0.195 0.053 Nature of Business 0.861 0.057 Accounting Software in Use 0.411 0.093 Duration of Software Usage 0.180 0.007 Factors Contributing to Satisfaction -0.305 0.009 Confidence in Data Security 0.584 0.002 Reasons for Dissatisfaction -0.250 0.083 Frequency of Software Updates 1.567 0.078 Challenges in Software Adoption 1.855 0.087 Perceived Financial Efficiency -0.466 0.098 Justification of Cost for Benefits 1.167 0.080 Table 6. Regression Analysis Figure 2. Beta Coefficients from Regression Analysis Figure 2. Beta Coefficients from Regression Analysis This figure illustrates the beta coefficients from the regression analysis, visually representing the strength and direction of the relationships between the independent variables and the dependent variable 'Satisfaction Level with Current Software'. Each bar in the plot corresponds to an independent variable, with the length and direction indicating the size and direction of its beta coefficient. Positive coefficients are depicted to the right, suggesting a positive relationship with satisfaction levels, while negative coefficients extend to the left, indicating an inverse relationship The regression analysis in Table 4.5 evaluates the predictive power of various independent variables on the dependent variable 'Satisfaction Level with Current Software'. The beta coefficients indicate the strength and direction of these relationships, while the p-values provide insight into their statistical significance. Nature of Business and Challenges in Software Adoption have strong positive beta coefficients (0.861 and 1.855, respectively), suggesting they are significant predictors of satisfaction levels with accounting software. Factors Contributing to Satisfaction and Perceived Financial Efficiency show negative beta coefficients (- 0.305 and -0.466), indicating an inverse relationship with the satisfaction level. This suggests that these factors may adversely affect satisfaction. Confidence in Data Security has a significant positive impact (beta coefficient of 0.584) with a very low p-value (0.002), indicating 1048 EJTAS 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com EJTAS Frequency Count & Percentage: C Variables Table 7. Factors Considered in Selection Factors Considered Frequency Count Per (%) Cost 48 88. User-friendliness 42 77. Customer support 29 53. Features and functionalities 45 83.3 Reputation of the software 31 57.4 Other (Specify) 7 12.9 In Table 7, it can be observed that cost and features and functionalities (83.33% most critical factors considered when accounting software for micro enter Delhi. User-friendliness (77.78%) reputation of the software (57.41%) significant roles in the decision-making Additionally, a small percentage of res (12.96%) specified other factors that in their software selection. Table 8. Desired Features for Improvement Desired Features for Improvement Frequency Count Per Advanced analytics and reporting tools 32 Better integration with other business systems 37 Enhanced mobile accessibility and app functionality 21 More customizable invoicing and billing options 34 Improved data security and privacy features 29 AI for financial forecasting and insights 25 Easier tax compliance and updates for changing regulations 38 More extensive inventory management features 27 50.00 Automated bank reconciliation 31 57.41 Simplified payroll processing 33 61.11 Better scalability for growing businesses 36 66.67 User interface and usability enhancements 29 53.70 Cloud-based access and data backup 40 74.07 24/7 customer support and more extensive training 28 51.85 Other (Specify) 2 3.70 Frequency Count & Percentage: Controlled Variables Table 7. Factors Considered in Software Selection Table 7. Factors Considered in Software Selection Factors Considered Frequency Count Percentage (%) Cost 48 88.89 User-friendliness 42 77.78 Customer support 29 53.70 Features and functionalities 45 83.33 Reputation of the software 31 57.41 Other (Specify) 7 12.96 In Table 7, it can be observed that cost (88.89%) and features and functionalities (83.33%) are the most critical factors considered when selecting accounting software for micro enterprises in Delhi. Discussion The findings of this study provide valuable insights into the influence of technology on accounting practices in micro enterprises in Delhi, India. In this section, we will discuss and interpret the results obtained from the data analysis, examining their implications and significance in filling the literature gap. User-friendliness (77.78%) and the reputation of the software (57.41%) also play significant roles in the decision-making process. Additionally, a small percentage of respondents (12.96%) specified other factors that influenced their software selection. In Table 7, it can be observed that cost (88.89%) and features and functionalities (83.33%) are the most critical factors considered when selecting accounting software for micro enterprises in Delhi. User-friendliness (77.78%) and the reputation of the software (57.41%) also play significant roles in the decision-making process. Additionally, a small percentage of respondents (12.96%) specified other factors that influenced their software selection. Table 8 illustrates the desired features for software improvement as indicated by micro enterprise owners. Easier tax compliance and updates for changing regulations (70.37%) and cloud-based access and data backup (74.07%) are among the top priorities for improvement. Enhanced mobile accessibility and app functionality (38.89%) and AI for financial forecasting and insights (46.30%) are also areas where businesses seek enhancements. Furthermore, respondents provided specific suggestions for additional improvements, indicating a range of software needs in this sector. Table 8. Desired Features for Software Improvement Desired Features for Improvement Frequency Count Percentage (%) Advanced analytics and reporting tools 32 59.26 Better integration with other business systems 37 68.52 Enhanced mobile accessibility and app functionality 21 38.89 More customizable invoicing and billing options 34 62.96 Improved data security and privacy features 29 53.70 AI for financial forecasting and insights 25 46.30 Easier tax compliance and updates for changing regulations 38 70.37 Desired Software Improvements selection. This suggests that businesses in this segment recognize the value of technology in streamlining their financial management processes. The prominence of user-friendliness (77.78%) and features and functionalities (83.33%) further underscores the importance of software that is accessible and tailored to the specific needs of small businesses. The data also shed light on the features and improvements that micro enterprises in Delhi desire in their accounting software. Easier tax compliance and updates for changing regulations (70.37%) emerge as a top priority, reflecting the complex and ever-evolving tax landscape in India. Cloud-based access and data backup (74.07%) also feature prominently, indicating a growing demand for remote accessibility and data security. The strong positive correlation between 'Duration of Software Usage' and 'Accounting Software in Use' (0.89) indicates that businesses tend to stick with a chosen software once adopted, emphasizing the stability and reliability of these tools. This stability may be attributed to the training and investment required to transition to a new system, which can be a barrier for micro enterprises with limited resources. Advanced analytics and reporting tools (59.26%) and better integration with other business systems (68.52%) demonstrate a desire for software that provides comprehensive insights and streamlines various aspects of business operations. These findings align with the broader trend of businesses seeking not only efficient bookkeeping but also value-added features that can enhance decision-making. Adoption and Utilization of Accounting Software Adoption and Utilization of Accounting Software The data reveal that the adoption of accounting software is prevalent among micro enterprises in Delhi, with a significant proportion (88.89%) considering cost as a crucial factor in software www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1049 1049 EJTAS 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 EJTAS Desired Software Improvements Factors Influencing Satisfaction Levels The regression analysis highlights several factors that significantly influence satisfaction levels with accounting software among micro enterprises in Delhi. Notably, the 'Nature of Business' and 'Challenges in Software Adoption' have strong positive beta coefficients (0.861 and 1.855, respectively), suggesting that the type of business and the difficulties faced during software adoption are significant predictors of satisfaction. Filling the Literature Gap Practical Implications The practical implications of this research are multifaceted. For micro enterprises in Delhi, the study's findings can serve as a guide for selecting and optimizing accounting software. Business owners can consider factors such as the nature of their business, the challenges they might face during adoption, and the level of data security provided by the software when making informed decisions. The main findings of the study suggest that the choice of accounting software is significantly influenced by the nature of the business and the challenges faced in software adoption. Micro enterprises in Delhi prioritize cost-effectiveness and functionality when selecting accounting software. User-friendliness, features, and reputation of the software also play important roles in the decision-making process. These findings align with the global trend of businesses seeking cost-efficient solutions that cater to their specific needs, especially in the case of small- scale enterprises. Software developers and providers can leverage the insights on desired software improvements to tailor their offerings to the specific needs of micro enterprises in Delhi. Enhancements related to tax compliance, data security, and cloud-based access can be a key selling point in this market. Policymakers can use these findings to design initiatives and policies that support the digital transformation of micro enterprises. Addressing the challenges in software adoption, such as providing training and support, can help businesses fully harness the benefits of technology. Moreover, the study highlights that while technology adoption in accounting positively impacts factors like confidence in data security and satisfaction levels, it does not always translate into perceived improvements in financial efficiency. The strong correlation between confidence in data security and the adoption of accounting software underscores the importance of trust in technology, especially in a digital age where data integrity is paramount. Additionally, the study reveals that challenges in software adoption are closely related to the nature of the business, indicating that tailored approaches are required to address the unique needs of micro enterprises. Therefore, this research paper has explored the influence of technology on accounting practices in micro enterprises in Delhi, India. The findings highlight the prevalence of accounting software adoption, the factors influencing satisfaction levels, and the desired software improvements in this segment. By addressing the literature gap and providing practical insights, this study contributes to a better understanding of technology adoption in micro enterprises and its implications for businesses, software developers, and policymakers. Practical Implications As technology continues to shape the landscape of accounting practices, research in this area remains essential to guide businesses towards more efficient and effective financial management. Furthermore, the desired features for software improvement identified in the study reflect the evolving demands of micro enterprises in Delhi. The focus on tax compliance, cloud-based access, and enhanced reporting tools highlights the need for software providers to continuously adapt their offerings to meet the changing regulatory and operational requirements of these businesses. The importance placed on scalability also suggests that many micro enterprises have growth aspirations, and their software solutions should accommodate this potential expansion. Filling the Literature Gap This study addresses the literature gap by focusing specifically on micro enterprises in Delhi, India, within the context of technology adoption in accounting practices. While previous research has explored technology adoption in small and medium-sized enterprises globally, the unique economic and cultural factors in Delhi necessitate a closer examination. The strong negative beta coefficient for 'Factors Contributing to Satisfaction' (-0.305) indicates that businesses with more factors contributing to satisfaction tend to report lower overall satisfaction levels. This paradoxical finding may be attributed to businesses with higher expectations and a greater understanding of what constitutes satisfactory software. The study findings confirm that micro enterprises in Delhi are actively adopting accounting software, driven by factors such as cost, user-friendliness, and innovative features. However, the influence of the 'Nature of Business' and the 'Challenges in Software Adoption' are significant in shaping their satisfaction levels. These insights provide a nuanced understanding of the dynamics of technology adoption in a densely populated and economically diverse city like Delhi. Interestingly, 'Confidence in Data Security' emerges as a significant predictor of satisfaction, with a positive beta coefficient of 0.584 and a very low p-value (0.002). This finding highlights the importance of data security in shaping the overall satisfaction of micro enterprises with their chosen accounting software. Given the increasing concerns about data breaches and privacy, businesses that have greater confidence in the security of their financial data are more likely to be satisfied with their software. The findings also underscore the critical role of data security in shaping satisfaction levels, highlighting the need for software developers to prioritize security features. Additionally, the desire for improvements in tax compliance and cloud-based access reflects the unique regulatory and infrastructure challenges faced by businesses www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1050 1050 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com in Delhi, further emphasizing the importance of tailored accounting solutions. practices in micro enterprises in Delhi, India, by conducting a comprehensive survey of accounting software utilization. The findings from the study shed light on several key aspects of technology adoption in this specific context and contribute to filling the existing literature gap. Conclusion In conclusion, this research paper has explored the influence of technology on accounting www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1051 1051 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com EJTAS COVID-19 Pandemic: Case Study in Semarang, Central Java, Indonesia. In Complex, Intelligent and Software Intensive Systems. https://doi.org/10.1007/978-3-030-79725- 6_49 COVID-19 Pandemic: Case Study in Semarang, Central Java, Indonesia. In Complex, Intelligent and Software Intensive Systems. https://doi.org/10.1007/978-3-030-79725- 6_49 The broader implications of this research extend beyond the specific context of Delhi's micro enterprises. The study offers insights that can inform policy decisions and strategies not only in India but also in other developing economies facing similar challenges and opportunities in the digital era. Policymakers can use these findings to design initiatives that support the digital transformation of small businesses, thereby contributing to overall economic growth. Cahyani, J., & Amna, L. S. (2022). Analysis of the Application of Accounting Information Systems In Micro, Small and Medium Enterprises (MSMEs). JASa (Jurnal Akuntansi, Audit dan Sistem Informasi Akuntansi), 6(1). https://doi.org/10.36555/jasa.v6i1.1846 For business owners, the research underscores the importance of making informed decisions when selecting accounting software. Understanding the factors that influence satisfaction and efficiency can help micro enterprises make choices that align with their specific needs and objectives. This knowledge empowers them to harness the full potential of technology for improved financial management. Ezenwoke, O. (2017). Organisational determinants and e-accounting system implementation in micro and small enterprises (mses) in south-west Nigeria. http://eprints.covenantuniversity.edu.ng/9509 Fauzi, E., Sinatrya, M. V., Ramdhani, N. D., Ramadhan, R., & Safari, Z. M. R. (2022). Pengaruh kemajuan teknologi informasi terhadap perkembangan akuntansi. Jurnal riset pendidikan ekonomi. https://doi org/10 21067/jrpe v7i2 6877 In the broader context of the accounting profession, this research underscores the transformative impact of technology on traditional practices. It emphasizes the need for accountants and financial professionals to adapt their skills and mindset to effectively utilize accounting software. The evolving role of accountants, from data entry to data analysis and decision support, highlights the importance of continuous learning and upskilling in the digital age. https://doi.org/10.21067/jrpe.v7i2.6877 Gnana Prakash, A. P. (2023). Digitalization of Accounting Applications: From Clay Tablets to Smart Accounting Applications. https://doi.org/10.58830/ozgur.pub105.c887 Kamau, C., & Ilamoya, S. L. (2023). Accounting Profession: African Perspective Review of steps into the future. SU+ digital repository. Conclusion https://doi.org/10.48039/mjtum.v2i1.43 In conclusion, the research on the influence of technology on accounting practices in micro enterprises in Delhi provides valuable insights into a rapidly evolving field. The findings contribute to a deeper understanding of the complex interplay between technology adoption, business characteristics, and financial outcomes. As the global trend towards digitalization continues, the lessons learned from this study are invaluable for guiding the future of technology integration in the world of finance, particularly in the realm of micro enterprises. Rahman, A., & Anindya, J. P. (2021). Increasing MSMEs Performance by Utilization of Accounting Software. https://journal.kapin.org/index.php/conferenc e/article/view/19 Shbeilat, M. K., & Al-Hajaia, M. E. (2022). Electronic accounting applications in micro- enterprises: Challenges and impact on financial inclusion. Journal of Governance and Regulation, 11(3). https://doi.org/10.22495/jgrv11i3art14 Suhayati, E., & Riandani, I. (2019). Accounting Application for Small Medium Enterprises. https://doi.org/10.1088/1757- 899X/662/3/032056 References References Apriyanti, H. W., & Yuvitasari, E. (2021). The Role of Digital Utilization in Accounting to Enhance MSMEs’ Performance During References Apriyanti, H. W., & Yuvitasari, E. (2021). The Role of Digital Utilization in Accounting to Enhance MSMEs’ Performance During 1052 EJTAS 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 https://doi.org/10.1109/ICIMTECH50083.20 20.9211271 www.ejtas.com EJTAS Sumarna, A. D. (2023). The analysis of computerized accounting systems usage in the micro-small enterprises. Akuntabilitas, 17(1). https://doi.org/10.29259/ja.v17i1.17489 Using the Fuzzy Topsis Method. TEM Journal, 10(3), 1348-1356. Using the Fuzzy Topsis Method. TEM Journal, 10(3), 1348-1356. https://doi.org/10.18421/TEM103-43 Wicaksono, A., Kartikasary, M., & Salma, N. (2020). Analyze Cloud Accounting Software Implementation and Security System for Accounting in MSMEs and Cloud Accounting Software Developer. In International Conference on Information Management. Utami, N., & Yulianto, H. D. (2019). Significant Influence of Information Technology on the Use of Modern Accounting Software. https://doi.org/10.1088/1757- 899X/662/2/022003 https://doi.org/10.1109/ICIMTECH50083.20 20.9211271 Vysochan, O., Vysochan, O., Yasinska, A., & Hyk, V. (2021). Selection of Accounting Software for Small and Medium Enterprises www.ejtas.com EJTAS 2023 | Volume 1 | Number 6 1053 1053 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com
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ESG Index Construction and China's ESG System
BCP business & management
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ESG Index Construction and China's ESG System Lanyi Duan* Lanyi Duan* School of Economics, University College Dublin, Dublin, Ireland *Corresponding author: lanyi.duan@ucdconnect.com Abstract. The Environmental, Social, and Governance (ESG) system has gained popularity recently as concerns over issues like corporate responsibility, labor conditions, and climate change become more pressing. At the same time, investors from around the world have become increasingly interested in the ESG Thematic Index because it offers greater risk resilience and returns than traditional trading strategies. Building a Chinese financial ESG system is crucial to advancing the development of ESG in China since Chinese finance needs to create and enhance its own ESG system but there is still a lack of systematic study in this area. This article first defines the meaning of ESG-themed indices, then selects representative ESG-themed indices from the global market, analyzes the construction processes and methodologies of the indices, and finally makes pertinent recommendations to promote the growth of ESG-themed indices in China using inductive, comparative, and case study methods. eywords: ESG; ESG Thematic Index; Green Finance; Sustainable Development. Keywords: ESG; ESG Thematic Index; Green Finance; Sustainable Development. BCP Business & Management Volume 38 (2023) BCP Business & Management Volume 38 (2023) EMFRM 2022 1. Introduction The ESG Bond Index chooses sample bonds based on two factors, the investment of funds raised and the bond issuer, 175 BCP Business & Management Volume 38 (2023) EMFRM 2022 either to ascertain whether the funds raised are invested in ESG-related projects based on clear criteria or to ascertain whether the bond issuer has a good ESG performance; in both cases, the index is based on the bond price of the sample bonds. either to ascertain whether the funds raised are invested in ESG-related projects based on clear criteria or to ascertain whether the bond issuer has a good ESG performance; in both cases, the index is based on the bond price of the sample bonds. Few institutions and organizations throughout the world have ESG indicators that are properly defined, and the system of ESG indicators is still highly unfinished and fragmented. Table 1 illustrates the main international ESG-related indicators. Table 1. Indicators related to ESG from major international institutions and organisations Subject Relevant indicators involving ESG Global Reporting Initiative Pay attention to society and the environment. Materials, energy, and the environment are covered by 12 categories and 34 items. Materials, energy, wastewater and waste, air emissions, and other topics are covered by 12 categories and 34 items under the environmental heading; similarly, labor practices, human rights, social effect, and product responsibility are covered by 30 categories and 48 things under the social heading [5,6]. Committee on Non-Profit Sustainability The entirety of the Sustainability Code includes 79 sectors in 10 categories, including those associated with governance and the environment [7]. International Integrated Reporting Committee The link between financial performance and society and the environment [8]. European Parliament and European Commission Environmental issues, social and employee issues, human rights, anti- corruption and bribery issues, and Board and Management Diversity [9,10]. World Bank To concentrate on society and the environment. Starting with a goal of sustainable development and ending with the funding of investment initiatives Environmental and social norms, as well as their annexes, are discussed [11]. International Finance Corporation The sustainability framework focuses on the environment and society. Asian Development Bank Information on the environment, society, and governance. Agriculture and food safety, climate change, gender and development, social development and poverty, and water are some of the issues that need to be addressed [11,12]. ble 1. Indicators related to ESG from major international institutions and organisations Subject Relevant indicators involving ESG 1. Introduction The ESG Thematic Index integrates the three dimensions of environmental, social, and corporate governance as a crucial tool for ethical investors to participate in the ESG investment market, reflecting the ability of the position sample in environmental protection, social responsibility, and their own sustainable development [1]. It is also a crucial financial tool for directing social capital flow to sustainable development projects to support sustainable economic development. The use of social capital as a financial resource to fund sustainable development initiatives is crucial for promoting sustainable economic growth. ESG measures sustainability and ethical impact in three key areas, including gender inequality, labor, product responsibility, corruption, bribery, social development, and poverty, as well as shareholder rights, institutional investors, stakeholders, disclosure and transparency, board responsibility, and management diversity [1,3]. These areas are all covered by ESG. The E (Environmental) in ESG stands for a company's proactive attitude to the environment, which includes adherence to current regulatory frameworks and consideration of potential effects in the future. The combination of the governance environment, governance structure, governance mechanism, and governance behavior results in G (Governance) [2]. The system's base is its governance environment, its carrier is its governance structure, and its operational mechanism for the interaction of its components—a process of continual improvement—is its governance behavior or process. p A number of ESG-themed indices have been produced recently by fusing ESG themes with index products, as more and more financial institutions have started to concentrate on the ESG investing sector and continue to strengthen their efforts in the invention and development of ESG financial products. These index products may be roughly divided into two groups based on whether they have the qualities necessary to engage in market trading, namely ESG stock indices and ESG bond indices. The screening and weighted aggregation of listed firms by various index companies and ESG rating organizations based on their performance in the three dimensions of E, S, and G are referred to as ESG stock indexes among them [4]. They reflect the overall changes in the share prices of businesses with sustainable development capabilities by giving more weight and exposure to stocks with better ESG performance, thereby encouraging businesses to actively fulfill their obligations in environmental protection and fostering social development through capital flows. 2.1 Construction method The majority of the aforementioned metrics are challenging to properly quantify and were not created with ESG in mind. As a result, the assessment agency first gathers data and information from businesses and other outside organizations. It then researches the current industry best practices for each assessment element, evaluates the distance between businesses and the best practices in each area, and assigns a score. The composite score is then determined after each score has been given a weight. Since various organizations have different ideas on what the ESG framework should include and the weighting of each component, there is no standardized ESG assessment technique. The MSCIESG series (Global/US/Emerging Markets), the FTSE4Good series, and the Bloomberg ESG Index are currently significant international ESG rating indexes. y g g The MSCI ESG family of indices has criteria for each of the three ESG dimensions, which are scored and added to form an overall ESG score, with AAA denoting the best sustainable performance and C denoting the worst sustainable performance [9]. This family of indices provides investment decision support tools for over 5,000 clients in pension funds and hedge funds [6]. In order to assist investors understand the environmental, social, and corporate governance (ESG) risks and opportunities in their portfolios and to assess issuers' ability to manage long-term financial-related ESG issues, MSCI developed the ESG Rating System [10]. 176 BCP Business & Management Volume 38 (2023) BCP Business & Management Volume 38 (2023) EMFRM 2022 To identify important risks and opportunities in each sector and to build a framework of indicators, MSCI employs quantitative models. The indicator system is made up of 35 ESG Key Issues (ESG Key Issues), 10 themes (Themes), 3 primary categories (Pillars), and hundreds of indicators. The MSCI ESG Key Issues are assigned to each sector based on the extent of ESG's impact on that sector, and to each GICS sub-sector based on the extent and duration of the impact of environmental and social risks on that sector [3]. Governance issues are always crucial and will be taken into consideration when calculating all company scores. As a result, the final ESG rating score assigned to a firm is not an absolute number but rather a measure of how well the company performed in comparison to its competitors. 2.1 Construction method p p The FTSE4Good Index focuses on five key categories, including labor standards in the supply chain, support for human rights, and environmental sustainability. bribery issues and tackling climate change mitigation. ESG concerns are taken into account for both positive and negative screening, in addition to theme factors (such as minimal carbon emissions). In comparison to the parent index, the index's members will exclude from the index any firms involved in questionable business practices or those that do not adhere to the UN Global Compact Principles. To ensure that the relative weighting of constituents at the nation and sector level is equal to that in the parent index, components are also weighted in accordance with FTSE's internal ESG score and thematic risk ratings (such as carbon emissions). By rating 20,000 of the most regularly traded public firms based on these disclosures in four categories—environmental, social, governance, and ESG composite scores—the Bloomberg ESG Index pushes businesses to publish more ESG data. The eligible constituents of the Large/MidCap ESG Index, which covers global and regional markets, respectively, are used to create the S&P Global Large/MidCap ESG Index and the Emerging Large/MidCap ESG Index. The eligible constituents are chosen by evaluating the business practices of the companies and/or their scores in accordance with the UN Global Compact Principles, as well as by excluding the companies in each sector with the lowest quartile of S&P's internal metrics. A target of 75% of the outstanding market capitalization of eligible constituents in each sector is then selected from the remaining candidates, weighted by market capitalization and chosen in descending order of internal ESG score. 2.2 ESG Index Construction Process Currently, the creation of an international ESG stock index typically involves five steps. A benchmark index is chosen in the first stage, and the benchmark index's sample stocks are utilized as the starting reference. The second stage involves "scanning" the sample stocks for negativity based on the sample businesses' industry, the prevalence of contentious problems like human rights concerns, and other factors. The final ESG index sample is chosen in the third phase, which involves evaluating the ESG scores by combining the pertinent components, ranking them, and rescreening them in accordance with the various aims of the index design. The weighting of sample stocks is done in the fourth phase, and the weighting guidelines are often established under circumstances like the goal of the index design, geographic distribution, and compatibility with the benchmark index. To accomplish the proper index design goal, three weighting strategies are typically used: weighting by market capitalization, equal weighting, and skewing towards certain metrics. The regular upkeep, revision, and updating of the index constitute the fifth step. 2.3 Case Study - MSCI ESG Leaders Index The MSCI ESG Leaders Index is a leading ESG index that was introduced in 2020 with the goal of drawing market attention to top-tier, leading ESG businesses and fostering ESG investment. Its benchmark index is the "five-step" MSCI Global Investable Market Indexes (GIMI), which accounts for 50% of the index's market capitalization. "Extremely controversial" items like biochemical and incendiary weapons, "seriously controversial" items like nuclear missiles and related accessories, and "more controversial" items like nuclear missiles and related accessories are all categorized in the negative screening process 177 BCP Business & Management Volume 38 (2023) EMFRM 2022 according to the level of controversy in the business sector. All businesses engaged in the four business levels mentioned above are not included in the MSCI ESG Leaders Index, also known as the ESG Leaders Index. Businesses. according to the level of controversy in the business sector. All businesses engaged in the four business levels mentioned above are not included in the MSCI ESG Leaders Index, also known as the ESG Leaders Index. Businesses. The index demands that businesses have an ESG rating of BB or above (there are seven categories, from AAA to CCC), as well as a controversy index score of 3 or higher (on a scale of 10, with a score of zero being the most controversial). The four main themes for the environment are climate change, natural resources, pollution and waste, and opportunities for development related to the environment [13,14]. The four main themes for society are human resources, product responsibility, stakeholder conflict, and opportunities for development related to social responsibility. The four main themes for governance are corporate governance and corporate behavior. Additionally, the index considers ESG trend scores, current index inclusion, and sector-adjusted ESG scores. The final index, which is maintained and modified on a quarterly basis, is created by liquid market capitalization weighting the index. 3.1 Differences in domestic and international ESG systems First off, China's ESG system is still in its infancy compared to other nations, and the climate for information sharing and the quality of the data base are both lacking. There have been restrictions in the independent study on firms' fulfillment of social responsibility or green development in recent years, and China's ESG system still trails far behind that of other nations. The quantity and caliber of corporate information disclosure are weak, and the disclosure structure for ESG-related information is still unclear. Second, compared to other nations, China's base for data collecting is weak. Thirdly, the data disclosed in China's social responsibility reports and other reports primarily focuses on performance enhancement and hardly ever includes unfavorable data. Second, whereas ESG systems are mostly governed by the government in China, they are founded on spontaneous movements in other nations. The public's spontaneous movement and the promotion of non-profit organizations marked the beginning of the ESG system in the international sphere. Subsequently, internationally significant organizations, like the United Nations, started to develop ESG-related principles and frameworks and to encourage the adoption of ESG disclosure standards by national exchanges [13]. The early ESG system in China was created by the government issuing pertinent papers, which was followed by a top-to-bottom promotion of ESG awareness among businesses and the general public. The government still plays a significant role in directing the development of an ESG system in China, despite the fact that numerous non-governmental organizations in China also publish their own ESG ratings and investment recommendations [15,16]. 3.2 Suggestions for China's ESG system 3.2.1 Strengthening information disclosure capabilities 3.2.3 Improving ESG database construction Based on international experience, an effective ESG system has to be well backed by data. More comprehensive fundamental data will result in more credible evaluation outcomes, especially in the evaluation of ESG performance. The results are more trustworthy the more data there is available. Some organizations offer their processed data directly to the public, but the data base and data processing remain mainly undisclosed. Processing ESG data is a complicated process that needs a lot of time, effort, and resources, and it is regularly updated and maintained. The database is routinely maintained and updated. Therefore, it is advised that the pertinent departments support the company or institution in creating an ESG database as part of an overall ESG strategy. Therefore, it is advised that the pertinent departments help businesses or institutions create their ESG database as part of an overall ESG strategy. 3.2.2 Playing a government-led role In contrast to the growth of public movements in other nations, the government and pertinent agencies should still direct the ESG system in China. In order to improve the corresponding supporting policies and ultimately reflect the effect at the enterprise level, this necessitates ongoing communication and cooperation between the industry itself, industry associations, and regulatory authorities (including industry and commerce, taxation, environmental protection, finance, etc.) [16,17]. Examples include preferential rules for bidding and tax exemptions, as well as access to financial resources, for businesses (particularly listed businesses) with strong ESG performance evaluations. Companies should be cautioned or penalized if they fail to provide the necessary disclosures, provide false information, and perform badly during performance reviews. Information disclosure, performance evaluation, and investment advice are the three components of the ESG system. These three components are interconnected and naturally interwoven. In addition to aggressively promoting and improving the entire institutional framework of ESG in China, government departments and pertinent institutions should increase their general awareness of ESG ideas and systems. In particular, they should encourage industry associations and regulatory bodies to establish ESG disclosure and evaluation systems, encourage rating agencies to actively carry out ESG performance ratings of businesses and make them public in a timely manner, support businesses in implementing ESG principles in their operations, and increase investor awareness of ESG principles and responsible investment. 3.2.1 Strengthening information disclosure capabilities Globally speaking, ESG-related disclosures are becoming more and more common in many nations and areas. More than 30 nations have passed legislation requiring corporate ESG disclosure, including important stock markets in Europe, the United States, Japan, and Singapore, as well as South Africa, Brazil, Canada, India, Malaysia, Sri Lanka, Thailand, and Turkey. By 2030, the United Nations mandates that all significant businesses produce sustainability reports. China can get more insight from the aforementioned experience with information disclosure in developed capital markets and create a set of ESG information disclosure policies that are appropriate for its own national circumstances. Additionally, it is recommended that the appropriate authorities increase the standards for financial ESG reports, harmonize information disclosure guidelines, standardize third-party certification of the reports, and gradually establish the principles, procedures, and individuals in charge of third-party audits to ensure the reliability of the published reports. Furthermore, financial ESG disclosure has to be standardized. Information on the effects of financial 178 BCP Business & Management Volume 38 (2023) EMFRM 2022 institutions' own operations, investment and financing activities, as well as the effects of ESG on their possibilities and dangers, should also be included in financial ESG disclosure. institutions' own operations, investment and financing activities, as well as the effects of ESG on their possibilities and dangers, should also be included in financial ESG disclosure. References [1] Friede, G., M. Lewis, A. Bassen, T. Busch, ESG and Financial Performance: Aggregated Evidence from More than 2000 Empirical Studies, Journal of Sustainable Finance and Investment, 2015. [2] Atan, R., M. Alam, J. Said, and M. Zamri, The Impacts of Environmental, Social, and Governance Factors on Firm Performance: Panel Study of Malaysian Companies, Management of Environmental Quality: An International Journal, 2018, 29(2), 182-194. [3] Aupperle, K., A. Carroll, and J. Hatfield, An Empirical Examination of the Relationship between Corporate Social Responsibility and Profitability, Academy of Management Journal,1985, 28(2), 446- 463. [4] Blank H., S. Sgambati, and Z. Truelson, Best Practices in ESG Investing. The Joumal of Investing, 2016, 25 (2), 103-112. [5] Brooks, C. and I. Oikonomou, The Effects of Environmental, Social and Governance Disclosures and Performance on Firm Value: A Review of the Literature in Accounting and Finance, The British Accounting Review, 2018, 50(1), 1-142. [6] Clark, G., F. Andreas, and V. Michael, From the Stockholder to the Stakeholder: How Sustainability Can Drive Financial Outperformance, Arabesque Asset Management and Oxford University, 2015. [7] Coleman, L., K. Maheswaran, and S. Pinder, Narratives in Managers Corporate Finance Decisions, Accounting & Finance, 2010, 50(3), 605-633. [8] Cucari N., S. Falco, and B. Orlando, Diversity of Board of Directors and Environmental Social Governance: Evidence from Italian Listed Companies, Corporate Social Responsibility and Environmental Management, 2018, 25(2), 250-266. [9] Fang Zhiyong, Green Credit Innovation Practices of Commercial Banks and Related Policy Recommendations, Financial Supervision and Regulation Research, Vol. 6, 2016, 57-72. [10] Huang Zhijie, Implementation and Innovation of Corporate Governance and Capital Market Legal System, Tsinghua University Press, 2013, 231-280. [11] Lu Zhengwei and Fang Qi, Financial Regulation and Green Financial Development: An Overview of Practice and Research, Financial Regulation Research, Vol. 11, 2018, 1-13. [12] Luo Sheng and Yin Aichao, A study on governance risks of financial institutions based on the systemic theory of corporate governance, Insurance Research, Vol. 12, No. 57, 2008. [13] Zhang Lin and Lian Yongke, Green Credit, Bank Heterogeneity and Bank Financial Performance, Financial Supervision Research, Vol. 2, 2019, No. 43-61. [14] Cheung Kong Securities. Emerging investment concepts in China: The rise of ESG [R/OL]. Cheung Kong Securities Research Report, 2018-06-21. https:/V/www.sohu.com/a/237046611_675170. [15] Jin Heisi. The experience of global ESG investment development and implications for China IV. Modern Management Science, 2018(9). [16] He Zhiquan. Environmental, social and governance (ESG) investment and disclosure in China. BCP Business & Management Volume 38 (2023) BCP Business & Management Volume 38 (2023) BCP Business & Management Volume 38 (2023) EMFRM 2022 4. Conclusion Environmental, social and governance (ESG) measures the three core elements of sustainability and ethical impact in a company or corporate investment. ESG is also gaining attention in China, where more and more companies are disclosing their social responsibility reports, but its development is still in its infancy. The ESG Thematic Index, which integrates the three dimensions of environmental, social and corporate governance, is an important tool for ethical investors to participate in the ESG investment market, reflecting the ability of a sample of positions in environmental protection, social responsibility and their own sustainable development. It is also an important financial tool for directing social capital flows to sustainable development projects to support sustainable economic development. The use of social capital as a financial resource to finance sustainable development initiatives is essential to promote sustainable economic growth. Important international ESG rating indices currently include the MSCIESG series, the FTSE4Good series and the Bloomberg ESG Index. There is still a gap between the current Chinese ESG system and the international one. China needs to strengthen its information disclosure capacity, play a leading role in government, improve the ESG database construction and gradually improve the ESG system. 179 References China Capital Finance and Accounting, 2015(34). [17] Chong Zhengwei, Tang Weiqi. ESG disclosure by listed companies. Assessment and application [R]. Industrial Research, 2017-04-04. 180
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“Leisure” spaces within work environments in the “Performance society”
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“Leisure” spaces within work environments in the “Performance society” Espaços de “lazer” em ambientes de trabalho na “Sociedade de desempenho” Simone Jubert [I] Lúcia Leitão [II] Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 http://dx.doi.org/10.1590/2236-9996.2024-5909.e Introduction Richard Sennett, in Building and dwelling: Ethics for the city (2018), recounts his experience of visiting the Googleplex, in New York, remarking that the Googleplex is "in the city but not of it" (Sennett, 2018, p. 170). It is a space “meant to be self-contained” (ibid., p. 171), in such a way that staff do not need to leave the workplace in order to engage in a wide range of activities, such as attending a medical consultation or even sleeping to relax after the extended working day. In addition to this brief introduction, the text is divided into two parts. In the first, the concept of leisure is defined, and we present a brief historical overview of the incorporation of leisure spaces into the workplace. The second part then examines how the idea of surveillance in the “disciplinary society” developed by Michel Foucault can be seen to shape productivity, indicating the existence of an orchestrated division of the representations of power, in such a way that the surveillance and control of individuals is exercised and represented by a “social cell”. We then present Deleuze’s notion of the “control society” (1995), in which surveillance becomes virtual, and then move on to Han’s concept of the “performance society” (2015 and 2017), in which self-exploitation is associated with a sense of freedom. This discussion is essential for understanding the novel contours that surveillance has assumed in the contemporary world and the way in which workers play an active role in a phenomenon that appropriates their non-work time by providing spaces for leisure in the workplace. In addition to all the arguments in favor of the provision of such spaces for leisure, socializing and even sleeping,1 such facilities have resulted in the Google complex becoming the inspiration for the emergence of so-called "creative class"2 offices around the world. In Brazil, the offices of major companies, such as Walmart (Figure 1), Unilever, Vivo (Figures 2 and 3), Serasa, OLX (Figure 4) and Locaweb, and those of smaller companies, such as OutPromo, SolveSystem, Arizona and the like, are also incorporating spaces designed for leisure and relaxation into the working environment. Abstracti This article proposes a reflection on the increasing trend of integrating leisure spaces into work environments. It begins with the hypothesis that such spaces constitute a form of appropriation, by employers, of their employees' non-working time. The conceptual references primarily stem from the notions of Disciplinary Society (Foucault), involving the idea of docile bodies shaped by surveillance, and Performance Society (Han), demonstrating how the experience within these spaces that exploit employees' free time can easily be mistaken for a sense of freedom. The article concludes by highlighting the worker’s active participation in their own exploitation and pointing out the need to discuss the psychosocial implications arising from this rapidly expanding practice within contemporary capitalism. Este artigo propõe uma reflexão sobre a crescente inserção de espaços de lazer em ambientes de tra­ balho. Parte-se da hipótese de que esses espaços constituem uma forma de apropriação, por parte do empregador, do tempo de não trabalho de seus empregados. As referências conceituais vêm, prin­ cipalmente, das noções de “Sociedade disciplinar” (Foucault),­ com a ideia de corpos dóceis conforma­ dos pela vigilância, e de “Sociedade de desempe­ nho” (Han), mostrando como a vivência nesses es­ paços de exploração do tempo livre dos empregados pode ser facilmente confundida com uma experiên­ cia de liberdade. Conclui-se assinalando a participa­ ção ativa do trabalhador na sua própria exploração e apontando a necessidade de discutir as implica­ ções psicossociais advindas dessa prática em franca expansão no capitalismo contemporâneo. Palavras-chave: arquitetura e urbanismo; lazer; trabalho; vigilância; desempenho. Keywords: architecture and urbanism; leisure; work; surveillance; performance. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 http://dx.doi.org/10.1590/2236-9996.2024-5909.e Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 http://dx.doi.org/10.1590/2236-9996.2024-5909.e Artigo publicado em Open Acess Creative Commons Atribution Artigo publicado em Open Acess Creative Commons Atribution Artigo publicado em Open Acess Creative Commons Atribution Simone Jubert, Lúcia Leitão aim of the present study is to reflect on these questions and to propose the hypothesis that such environments constitute a subtle form of appropriation, by employers, of the non-work time of their employees. This hypothesis is explored here using Foucault’s concept of the “disciplinary society” (1995), and the notion of the “performance society” developed by Han (2015 and 2017). Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Introduction What, however, are the implications, of the inclusion of leisure spaces in the workplace?3 And why, generally speaking, do the production, proliferation and adoption of such spaces tend not make workers feel that their non-work time is being appropriated by employers but are instead perceived as motivational factors and benefits?4 The main 192 “Leisure” spaces within work environments... Source: Office Snapshots.5 Source: Maurício Grego/ Exame.6 Source: Galeria da Arquitetura.8 Source: Maurício Grego/Exame.7 Figura 1 Figura 2 Figura 3 Figura 4 Source: Maurício Grego/ Exame.6 Figura 2 Figura 4 Source: Office Snapshots.5 Figura 1 Figura 3 Figura 2 Source: Office Snapshots.5 Source: Maurício Grego/ Exame.6 Figura 3 Figura 4 Source: Galeria da Arquitetura.8 Source: Maurício Grego/Exame.7 Source: Galeria da Arquitetura.8 Source: Maurício Grego/Exame.7 Source: Maurício Grego/Exame.7 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Leisure of the community determined the cycles of production. In other words, there was a direct relation between nature and community, not only influencing what communities produced and when, but also how they produced it. It was, therefore, with the emergence of industrial societies that a different way of ordering time and a new relation to it came into being. As a result, Corbin, in L’Avènement des Loisirs [The Advent of Free Time] (1995), remarks that, up to the 19th century, work time was discontinuous, in such a way that work was intertwined with other everyday activities, and often liberally interspersed with pauses and periods of free time. The seasons of the year and the life 193 193 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão work to which men, women, the elderly and children were subjected ended up motivating the workers’ struggle for the eight-hour working day, with eight hours of leisure and eight hours of rest. The abuses perpetrated by employers necessitated the creation of guaranteed free time for all workers. Leisure, as we now know it, is therefore a right that has been won. [...] the modern problematization and ideology of free time appeared between the late 18th and early 19th centuries, alongside the phenomenon of mass urbanization, which was closely related to industrial mechanization. Likewise, the dramatic transformation effected by the separation of housing from the workplace – with the family ceasing to function as the physical center of the economy – played a significant role at this time. [...] It is thus fair to say that free time properly speaking, as opposed to work time, bears specific traces, characteristic of the civilization born of the industrial revolution. The evolution of free time has therefore reflected the ensuing social struggles concerning labor legislation, within capitalist society or with a view to surpassing it. (Gaspar, 2003, pp. 104-105) [...] the modern problematization and ideology of free time appeared between the late 18th and early 19th centuries, alongside the phenomenon of mass urbanization, which was closely related to industrial mechanization. Likewise, the dramatic transformation effected by the separation of housing from the workplace – with the family ceasing to function as the physical center of the economy – played a significant role at this time. Figure 5 – Confederation Generale du Travail (C.G.T) poster advocating the eight-hour working day Source: Doumenq Félix, 1919. Figure 5 – Confederation Generale du Travail (C.G.T) poster advocating the eight-hour working day Figure 5 – Confederation Generale du Travail (C.G.T) poster advocating the eight-hour working day gure 5 – Confederation Generale du Travail (C.G poster advocating the eight-hour working day Source: Doumenq Félix, 1919. Source: Doumenq Félix, 1919. The exploitation of the working classes culminated in a series of stand-offs and demands for improvements in working and living conditions and generated a series of responses on the part of society. Workers’ demands were subsequently examined in accordance with the prevailing system of rationality of the time, in order to provide legitimacy for any possible changes. This clearly shows how the special place that science had come to occupy, along with changes in architecture and urban planning, the value accorded to data and statistics, and other factors, all had a part to play in the rational system that was under development at the time, strengthening its position and causing After capital had taken centuries to extend the working day to its normal maximum limit, and then beyond this to the limit of the natural day of 12 hours, there followed, with the birth of largescale industry in the last third of the eighteenth century, an avalanche of violent and unmeasured encroachments. Every boundary set by morality and nature, age and sex, day and night, was broken down. Even the ideas of day and night, which in the old statutes were of peasant simplicity, became so confused that an English judge, as late as 1860, needed the penetration of an interpreter of the Talmud to explain ‘judicially’ what was day and what was night. Capital was celebrating its orgies. (Marx, 2017, pp. 439-440) After capital had taken centuries to extend the working day to its normal maximum limit, and then beyond this to the limit of the natural day of 12 hours, there followed, with the birth of largescale industry in the last third of the eighteenth century, an avalanche of violent and unmeasured encroachments. Every boundary set by morality and nature, age and sex, day and night, was broken down. Even the ideas of day and night, which in the old statutes were of peasant simplicity, became so confused that an English judge, as late as 1860, needed the penetration of an interpreter of the Talmud to explain ‘judicially’ what was day and what was night. Leisure [...] It is thus fair to say that free time properly speaking, as opposed to work time, bears specific traces, characteristic of the civilization born of the industrial revolution. The evolution of free time has therefore reflected the ensuing social struggles concerning labor legislation, within capitalist society or with a view to surpassing it. (Gaspar, 2003, pp. 104-105) Dumazedier thus regards work as being in fact essential for the very existence of leisure as the result of a historical relationship between work time and non- work time. The present article adopts this reasoning, defining leisure as non-work time for the worker (that is nevertheless based in principle on the existence of work), and understanding it as an historical right to free time that has been won. It was thus in the context of the Industrial Revolution that the concept of leisure first began to emerge. The French sociologist Dumazedier, who is one of the foremost authorities and pioneers in the field of the sociology of leisure, has remarked that "leisure is not idleness, it does not supplant work; it presupposes it. It represents a periodic liberation from work at the end of the day, week, year, or working life" (1979, p. 28). The present article also accepts one important criticism of Dumazedier’s position, concerning the adoption of a functionalist approach to leisure, seeing it as something that serves as compensation for work. We are interested principally here, however, in the way leisure is understood as something achieved, the fruit of a long process of workers’ struggles for better living and working conditions, without which employers would readily have allowed work to occupy all their workers’ waking hours. Dumazedier’s view of the nature of leisure derives from an understanding of the fact that, while work time and non-work time were, as Corbin and Gaspar both note, once intertwined, with the Industrial Revolution, work time came to hold sway over other kinds of time, in such a way that the exhausting day’s The way in which work came to dominate every minute of every day is clearly described in a passage from Karl Marx’s Capital, first published in September 1867: 194 194 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... Figure 5 – Confederation Generale du Travail (C.G.T) poster advocating the eight-hour working day Source: Doumenq Félix, 1919. Capital was celebrating its orgies. (Marx, 2017, pp. 439-440) 195 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão it to gain acceptance throughout society. This would therefore be the prism through which the idea of time free of work would come to be examined and finally accepted over time. would generate self-intoxication. This also led them to criticize excessive sporting activity (ibid.). Thus, according to Corbin, it was now understood to be wrong to see the body as a machine, since the body is subject to the laws of fatigue (ibid., p. 337). The struggle for a day divided into three periods of eight hours – eight for work, eight for rest, and eight for leisure – was one of the demands that was especially carefully analyzed in the light of scientific studies of the time. Scholars pored over figures relating to the gains and losses expected to result from this model of the working day, in an attempt to justify the stern resistance of a significant sector of the economic elite to any reduction to the length of the working day. This period also saw the appearance of studies of the capacity to resist fatigue and the factors influencing this. The bad habits studied included lack of sleep, a poor diet, and being overweight, and it was understood, while excessive physical exercise caused fatigue, “training – which was the subject of numerous experiments – tended to increase the resistance of the muscles and of the brain” (ibid., p. 338). New connections between work, rest and fatigue were therefore identified, with thresholds established for each, taking into consideration that fact that each kind of activity has the capacity to become harmful in certain contexts. “As a whole, this research activity provided a scientific basis for the need for a periodic restoration of strength” (ibid.) and consequently justified examination of specific studies of industrial fatigue. This led to the emergence of what Corbin calls (ibid.), “a period of rational management of human effort” (p. 339), based on studies of fatigue. The roots of this intensive scientific activity are various. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Figure 5 – Confederation Generale du Travail (C.G.T) poster advocating the eight-hour working day Source: Doumenq Félix, 1919. Research was spurred by a desire to use science to justify the demand for and the introduction of the ‘three eights’ – which even socialist theorists had proved incapable of bringing about [...] There was a widespread general impression that surmenage9 [overwork] was one of the many scourges that blighted the late 19th century. In that age of a veritable “medical coup d’état,” facilitated by the success of Pasteur’s theories, physiologists and psychologists found, in the study of fatigue, a way of bolstering the authority of their message and extending their influence. (Corbin et al., 2001, p. 336) Corbin also notes that the First World War sparked numerous studies of industrial fatigue, principally among the Allies. He goes on to remark that it would be fair to describe the post-World War I period as “the golden age of this new branch of knowledge, especially on the North American side of the Atlantic” (ibid., p. 341). It should be borne Corbin notes that, in the late 19th century and early 20th century, a series of studies of fatigue demonstrated that “it is a chemical process that affects the body as a whole and not just the organ that appears to affected” (ibid.). The researchers of the time believed that any prolonged period of intensive muscular effort 196 196 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... in mind, however, that all of this activity and interest surrounding fatigue aimed to provide improved understanding of the mechanisms of the human body in relation to its limits as a way of augmenting the productivity of workers. Fatigue researchers thus became involved in a wide range of fields, including studies of sensory fatigue, such as that produced by noise, studies of the need for sleep (generating greater appreciation for this, since tiredness can affect sleeping and reduce productivity on the following day), and studies devoted to the psychological and physiological effects of work. Ensuring that there is time for meals and breaks at work led to the creation within factories of leisure spaces such as cafeterias, refectories, canteens, and restrooms. This preceded the creation of such spaces in the offices of white- collar workers. However, according to a study conducted by Resende (2018), it was in the latter that leisure spaces would eventually develop at a much rapider pace. Figure 5 – Confederation Generale du Travail (C.G.T) poster advocating the eight-hour working day Source: Doumenq Félix, 1919. Resende notes that leisure spaces were already being incorporated into workplaces in the late 19th century, in office blocks and company campuses. These included skyscrapers, such as the Pullman Building in Chicago, designed in 1883 by Solon S. Beman, for the Pullman Palace Car Company, and Frank Lloyd Wright’s 1904 Larkin Building, in New York, along with company campuses, such those of AT&T (built in 1941, with additional buildings constructed in 1974), in Murray Hills, New Jersey, and Connecticut General Life Insurance Company (1954), in Bloomfield, Connecticut, designed by S.O.M. under the influence of Mies Van der Rohe. The scientific debate and discussion explicitly referred to the process of legitimation of practices, narratives, and consensuses. It is therefore fair to say that between 1870 and 1914, the issue of fatigue featured widely in scientific studies, paving the way for acceptance within society of the right to time free of work and setting in motion processes that would result in the creation of periods of time that we now call leisure time (ibid., p. 345), such as the weekends, paid holidays, and other benefits. These spaces contained a leisure infrastructure designed for workers and their families, with facilities such as libraries, restaurants, bowling alleys, and, in some cases, housing. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Leisure at work Awareness that the body is not a machine and that lack of rest may harm productivity and hence reduce profits led to the development of a series of scientific studies within the workplace, principally in the fields of psychology, ergonomics, and sociology, that indicated the benefits of leisure time for productivity and staff motivation. According to Resende, some offices moved from the city to out-of-town campuses and business parks because cities to some extent no longer catered for the new spatial needs of businesses. Nor, in the post-World War II period, were they able to cope with the increased road traffic and overpopulation in city centers, with all the disadvantages these 197 197 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão bring, leading companies to move to out- of-town green areas and suburbs, as a way of finding peace and quiet and encouraging greater interaction among employees of the same company. Examination of the history of workplaces and the leisure spaces contained within them reveals that, although such spaces were intended to enhance the well-being and the social integration of workers and to provide breaks for rest, the true purpose was to create a healthy workforce even more willing to work. Alongside this movement to the periphery, there was also rapid development of information and communications technologies, establishing new paradigms in society and the world of work, some of which had already been included in the functioning of such buildings. This was, as Peter Drucker (2002) called it, for the first time, in 1959, the beginning of the Knowledge Society, which would have organizational and spatial implications for workplaces, in particular office spaces. (Ibid., p. 73) a healthy workforce even more willing to work. In the 1980s, however, new smaller business campuses surrounding universities began to appear in the United States. At this time, most office work now involved working in “cubicles”, since these constituted the least expensive, most accessible, most flexible, and most easily reproduceable option. Most workplaces thus still depended heavily on organizational culture for the provision of leisure. The idea of the campus, however, combined with the experience of young researchers and workers—many of them still at college or recently graduated—provided this class of workers with workplaces that were replete with the leisure and recreational facilities typical of a university, including sofas, games rooms, gymnasia and so forth. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Leisure at work Social activities similar to those at universities, such as picnics, barbecues, and afternoon teas, were also introduced. History shows that the production of leisure spaces within workplaces has shifted from a more formal, hierarchical, corporative mentality to one that is more informal, horizontal and focused on the needs of individuals. Gradual appropriation of the world of leisure by the world of work has occurred in tandem with technological and economic transformations, and this has led to the emergence of new kinds of workplaces that incorporate leisure spaces, indicating the emergence of a new stage of capitalism. Apart from increased productivity, employee motivation is a common argument advanced by employers to justify the adoption of such measures. In a study conducted by Resende (2018), employees testified that the existence of leisure spaces in the workplace History shows that the production of leisure spaces within workplaces has shifted from a more formal, hierarchical, corporative mentality to one that is more informal, horizontal and focused on the needs of individuals. Gradual appropriation of the world of leisure by the world of work has occurred in tandem with technological and economic transformations, and this has led to the emergence of new kinds of workplaces that incorporate leisure spaces, indicating the emergence of a new stage of capitalism. Apart from increased productivity employee It is interesting to note that, little by little, the spaces dedicated to leisure within workspaces came to be absorbed more by way of entropy, in so far as they broke down the boundaries between the two categories of space. It thus became increasingly common for workspaces to contain not only areas set aside for leisure but also leisure activities installed within workspaces, with computers and workstations interspersed with pool tables, foosball tables, pinball machines, and other leisure facilities. Apart from increased productivity, employee motivation is a common argument advanced by employers to justify the adoption of such measures. In a study conducted by Resende (2018), employees testified that the existence of leisure spaces in the workplace The tone of informality that came to imbue workspaces, flexible working hours, and the possibility of remote working from a ‘home office,’ with "leisure as much a cause as a consequence of longer hours being spent at the office" (ibid.) reveals a clear tendency for the worlds of work and leisure to overlap. Leisure at work Other smaller companies and laboratories were connected to this ecosystem and there was a climate of constant interchange. Workers in the emerging society of knowledge tended, at that time, to hail from a privileged background, and the much-vaunted idea that a multi-million- dollar business could start out in a garage is largely a myth (Avendaño, 2014). Drucker’s belief that "the real and controlling resource and the absolutely decisive 'factor of production' is now neither capital, nor land, nor labor…[but]… knowledge" (Drucker, 1993, p. 15) reveals this new paradigm of work, as reflected in the production of spaces based on new production processes. New working practices were also established. In the 1960s, there was an increasing emphasis on “organizational culture,” which systematized rewards and shaped the spirit, the environment, and values of an organization (Resende, 2018, p. 77). This “organizational culture” aimed to encourage employees to develop as sense of belonging to and feeling at home in the company as if it were a family. In addition to the provision of physical leisure infrastructure, systematic efforts were undertaken to create an equally conducive emotional environment. When Internet companies began to emerge, open-plan offices became more popular. These were open indoor areas, with no fixed predefined workstations, that encouraged a constant exchange of ideas. According to Resende (2018, p. 87): 198 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... and non-work time intertwined in accordance with the cycle of the seasons. Now, the intertwining of work and leisure is governed by the need for speed and efficiency. Work and leisure are merged together for the purpose, ultimately, of ensuring that work can be accomplished and goals achieved. The success that has been experienced and the profits made by companies in Silicon Valley from the 1990s onwards has inspired other employers to do their best to replicate the same spatial formula. Companies once again started to value the possibility of chance meetings of different people to exchange ideas. This spontaneity was associated with diversion, as a social factor, and companies thus began to break down the boundaries between work and leisure in the workplace. The spaces provided by such firms were far superior to those of other companies or even of universities, and it was not unusual to find foosball tables, basketball, volleyball, tennis and racquetball courts, football pitches, swimming pools and recreation centers. helped to consolidate interpersonal relations [...], to enhance physical and mental well-being, to develop cognitive and creative abilities, and to increase productivity, [...] in addition to improving the quality of life both at work and outside of the workplace. (ibid., p. 167) helped to consolidate interpersonal relations [...], to enhance physical and mental well-being, to develop cognitive and creative abilities, and to increase productivity, [...] in addition to improving the quality of life both at work and outside of the workplace. (ibid., p. 167) Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Productivity through surveillance The image of the panopticon proposed by Jeremy Bentham perfectly sums up the idea of surveillance in a disciplinary society. This led Foucault to conclude that "visibility is a trap". Its architectural composition involves, One key to a fuller understanding of why workers submit voluntarily to the encroachment of work time on their non-work time in the contemporary world and react positively to the phenomenon of leisure spaces located within the workplace may be found in the work of Michel Foucault. In particular in Vigiar e punir (2018), Foucault identified the methods used to subjugate, coerce and punish the human body throughout history, describing the system of power relations that prevailed each period, the ramifications of the discourse used, and the development of mechanisms to achieve their objectives. For Foucault, a study of the control of human bodies in western countries reveals clearly discriminable periods during which certain discourses of power hold sway. These discourses are, in turn, reflected in the institutions, politics and forms of policing established, and also in architecture and urban planning at the periphery, an annular building; at the centre, a tower [...]. All that is needed, then, is to place a supervisor in a central tower and to shut up in each cell a madman, a patient, a condemned man, a worker or a schoolboy... so many cages, so many small theatres, in which each actor is alone, perfectly individualized and constantly visible. The panoptic mechanism arranges spatial unities that make it possible to see constantly and to recognize immediately. (ibid., p. 194). In the transition from sovereign to disciplinary societies, different forms of organization of power coexisted, from the still prevalent "ancient rights of the monarchy" to others that had a "preventive, utilitarian, corrective conception of a right to punish that belongs to society as a whole". One of these forms of organization of power involved the establishment of a "punitive city," Discipline and Punish first presents the history of the body subjected to torture in “sovereign societies” (these being societies that go back deep into human history and are centered on the figure of a single sovereign). Such societies prevailed up until the Industrial Revolution, when there was a transition to ‘disciplinary societies’ based on a system intended to make human bodies docile. Leisure at work Things were no longer the way they were prior to the Industrial Revolution, when work time 199 199 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão in public squares so as to provide visibility for the prevailing power structure, in disciplinary societies, there is an orchestrated division of representations of power, resulting in control being more evenly distributed – and exercised and represented by ‘social cells’. How then did it come about that the appropriation of leisure time by work is seen as a cause of satisfaction by some employees? Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Productivity through surveillance While, in sovereign societies, punishment and control of bodies involved subjecting them to torture [...] a functioning of penal power, distributed throughout the social space; present everywhere as scene, spectacle, sign, discourse; legible like an open book; operating by a permanent recodification of the mind of the citizens; eliminating crime by those obstacles placed before the idea of crime; acting invisibly and uselessly on the ‘soft fibres Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 200 “Leisure” spaces within work environments... The control and discipline imposed on bodies led Foucault to develop the notion of biopower. In his course at the Collège de France in 1978, titled Sécurité, territoire, population (Security, territory, population), Foucault defines biopower as “the set of mechanisms through which the basic biological features of the human species became the object of a political strategy, of a general strategy of power” (Foucault, 2008, p. 3). Biopower generates biopolitics, which, Foucault argues is “what brought life and its mechanisms into the realm of explicit calculations and made knowledge-power an agent of transformation of human life” (Foucault, 1988, p. 134). Revel further clarifies the concept of biopolitics to the effect that “while discipline takes the form of a political anatomy applied to bodies and is applied essentially to individuals, biopolitics represents social medicine on a large scale applied to the population as a way of governing life: life thus forms part of the field of power” (2005, p. 27). of the brain’, as Servan put it. A power to punish that ran the whole length of the social network would act at each of its points, and in the end would no longer be perceived as a power of certain individuals over others, but as an immediate reaction of all in relation to the individual. (Ibid., p. 129) The kind of surveillance involved in the idea of the panopticon and punitive power shared by the whole social fabric molded the thinking and institutions of the time. Foucault argues that surveillance for the purpose of creating docile bodies10 came to pervade the whole social fabric and all social activities, taking concrete form in ‘apparatuses’.11 Architecture and urban planning themselves should thus be seen as tools of surveillance in the disciplinary society, 12 molding localities in conformity with the interests of governmentality and ensuring that bodies are made docile. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 The control society ‘dividuals’ and masses become samples, data, markets, or ‘banks.’ Money, perhaps, best expresses the difference between the two kinds of society, since discipline was always related to molded currencies containing gold as a numerical standard, whereas control is based on floating exchange rates, modulations depending on a code setting sample percentages for various currencies. (ibid., p. 222) The technological (r)evolution following the Second World War and the wave of revolutions that came in its wake exercised a decisive influence on the way western societies are organized. As well as providing new forms of entertainment, the broad dissemination of video technology and easier access to it have expanded the scope of the panopticon. The disciplinary society now has information and communications technologies at its disposal and can use mass communications to make bodies docile, helping to effect radical changes in the way urban spaces are occupied, causing some parts to fall into decline, while others become more densely populated. The advent of the Internet simultaneously created a new and radical network of invisible ties within the city; the panopticon could now be installed in the virtual world, and individuals induced voluntarily to provide the tracks through which they can be surveilled. The overlapping discourses in control societies address new relations and practices regarding space and time, and the virtualization, which began in disciplinary societies, reaches new levels. People no longer need to go to the bank or the post office to make payments, videogames can now be played by multiple players at the same time in various places around the world, political propaganda is relayed by social networks, security cameras observe and record the private lives of individuals and track their movements and behavior. It is in this control society that we have come to be monitored by surveillance cameras at work and in which virtual environments are extensively used. De Masi expresses a certain optimism regarding the new urban order effected by a shift from metropolis to ‘telepolis’ and he sees the virtualization of processes as something fresh and new. "Built entirely on the concept of living and working at a distance, the more the inhabitants of Telepolis stay at home working and consuming remotely, blurring the boundaries between work, home, social life, production, reproduction, and entertainment, the more the city bustles with activity" (1999, p. Productivity through surveillance According to Foucault, we thus live in a “carceral continuum” (ibid., p. 298), in which we are led from one prison to another. The concepts of biopolitics and biopower together help shed light on how the disciplinary society has been modified and perfected in relation to its mission of producing docile bodies, coming eventually to focus on the individual and a form of surveillance that is increasingly diffused throughout society, but also increasingly heavier and more insistently present. Thus, through a kind of refinement of the disciplinary society, surveillance would appear already to be rooted in the subject –, and, at this point, it can already be seen that entrenched surveillance is one of the root causes of the appropriation of non-work time by work. Bodies can supposedly be made docile in this way at an early age in school, in communal spaces, in college, in institutions, or at work in a factory or an office. The production of space as a whole in a disciplinary society aims to control the bodies of individuals in such a way that spaces are readapted in accordance with the precepts of the disciplinary system. Hospitals are thus divided into wings, schools have rows of desks, factories have workstations. And it because of this need for discipline that spaces in school, at work, in hospitals, in army barracks and so forth come to resemble one another. 201 201 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão [...] the key thing is no longer a signature or number but a code: codes are passwords, whereas disciplinary societies are ruled (when it comes to integration or resistance) […] Individuals become Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 The control society Above all, it is marked by the decline of some of the basic institutions of modern society – such as the school, the factory, the prison, and the hospital, including the home designed to accommodate a nuclear family on the bourgeois model. On the other hand, complementary to this, the private company has become a kind of primary inspiration and example for all other institutions, including, and perhaps fundamentally, the most up-to- date versions of these modern fossils. By infecting them with an omnipresent "entrepreneurial spirit", they have forever transformed these organizations so as to make them increasingly compatible with the pace and demands of contemporary society". (Bruno et al., 2018, pp. 208-209) deploys ‘should’. After a certain point of productivity, ‘should’ reaches a limit. To increase productivity, it is replaced by ‘can’. The call for motivation, initiative, and projects exploits more effectively than whips and commands. As an entrepreneur of the self, the achievement-subject is free insofar as he or she is not subjugated to a commanding and exploiting Other. However, the subject is still not really free because he or she now engages in self-exploitation— and does so of his or her own free will… Auto- exploitation proves much more efficient than allo-exploitation because it is accompanied by a feeling of liberty. This makes possible exploitation without domination. (Han, 2020a, p. 21) The popular idea that being an entrepreneur is an easy solution or one associated with freedom aptly expresses the rationale underlying the performance society. The home office, coworking, hackathons, design sprints, and other fads of modern entrepreneurial culture reveal a pattern of self-exploitation. Modern offices contain pool tables, hammocks, and videogame machines. There is now a 24/7 regime, in which the individual can work at any time, on any day, as they see fit. The control society 216) In 1990, Gilles Deleuze suggested that a shift was underway from a disciplinary society to one of control. As he put it, “we're moving away from disciplinary societies, we've already left them behind. We're moving toward control societies that no longer operate by confining people but through continuous control and instant communication" (2008, p. 216). Deleuze argues that “disciplinary societies have two poles: signatures standing for individuals, and numbers or places in a register standing for their position in a mass" while, in "control societies", [...] the key thing is no longer a signature or number but a code: codes are passwords, whereas disciplinary societies are ruled (when it comes to integration or resistance) […] Individuals become In the shift from the disciplinary society to the society of control, old disciplinary structures are being rapidly transformed, making way for Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 202 “Leisure” spaces within work environments... [...] a system governed by excess p r o d u c t i o n a n d e x a g g e r a t e d consumption, by marketing and publicity, flows of capital in real time, interconnectivity, and global communications networks. Above all, it is marked by the decline of some of the basic institutions of modern society – such as the school, the factory, the prison, and the hospital, including the home designed to accommodate a nuclear family on the bourgeois model. On the other hand, complementary to this, the private company has become a kind of primary inspiration and example for all other institutions, including, and perhaps fundamentally, the most up-to- date versions of these modern fossils. By infecting them with an omnipresent "entrepreneurial spirit", they have forever transformed these organizations so as to make them increasingly compatible with the pace and demands of contemporary society". (Bruno et al., 2018, pp. 208-209) [...] a system governed by excess p r o d u c t i o n a n d e x a g g e r a t e d consumption, by marketing and publicity, flows of capital in real time, interconnectivity, and global communications networks. The performance society More recent phenomena in the fields of communications, technology, market economics, and the financial market, suggest that discourses of power are increasingly being shaped differently. Such discourses are seen to pervade social and cultural relations, redirecting efforts and policy towards a new social logic, in which the "spirit of enterprise" holds sway and in which surveillance is finally inculcated in the very subjectivity of the individual. This is what Byung-Chul Han has called the performance society, arguing that such a society This apparently harks back to the time when free time and work were closely intertwined, in so far as they were intrinsically linked to everyday life and leisure spilt over into the working day. It can, however, be seen from the passage cited above that, in the performance society, leisure and liberty operate as work in disguise. The existence within the workplace of spaces and facilities designed for leisure and use during free time causes individuals to spend more time engaged [...] is wholly dominated by the modal verb ‘can’– in contrast to disciplinary society, which issues prohibitions and Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 203 Simone Jubert, Lúcia Leitão of boundaries. When one is not told what one “should” do, but only what one “can,” spaces hold the power to retain us for longer, with all the potentially negative or positive consequences this may entail. in productive labor, motivated by the periods of time during which they believe that they are not working. In this state of constant production and utilitarianism, what role does leisure play? Boundaries are frayed and begin to break down. Dividing lines are blurred. In the performance society, every space is a frontier. Everything begins to make itself present as a possibility. The apparent freedom provided by new contemporary forms of work, characterized by informality and the blurring of the difference between times and spaces set aside for leisure and those intended for work, is worn as a badge of distinction by those classes of worker who have access to such spaces. It provides employees with an identity that associates both them and the company they work for with ideas of creativity and modernity. Now that leisure is understood as a right that has already been won, it is worth asking whether time free of work can nowadays be rightly seen as leisure time. The performance society 191-208, jan/abr 2024 “Leisure” spaces within work environments... The discourse relating to flexibility, liberty, and informality thus masks continuous surveillance of the leisure time spent within the workplace, carried out with the full and unrestricted cooperation of the employee. life, shaping and regulating, through mass communications and consumerism (which exist in a feedback relation to one another), the desires and identities of individuals. The identification of workers with their jobs forms part of a sophisticated technology of power that has inculcated control by the employer, or, through self-employment, the very figure of the employer, into the unconscious of the employee. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 The performance society Leisure as a right won by the working class, as a time set aside during the day and in the calendar for rest and for their own private pastimes, no longer seems to serve the same function. The ideas of productivity and high performance that individuals have internalized and on which their salaries depend are so unrelenting that the right to time free of work seems no longer to be guaranteed. The exploitation of labor develops a new facet, when, in addition to generating wealth through the productive force of labor, it also relates "its products" with the identity of the worker, including the dynamics of biopolitical exploitation of labor [...] This new process of determining value effects a shift from quantitative economic measurement to a more subjective approach, since the value of intangible factors is linked to factors related to the construction of ideologies among those operating within this new mode of exploitation. (Souza, Avelino, and Silveira, 2018, pp. 108-109) Flexibility has become a byword within the logic of the performance society, in which “can” trumps “should”, and this leads to a blurring of the boundaries between work and non-work, to the effect that these two spheres are constantly intertwined and the right to leisure no longer exists. Although workers may have a formal contract, the logic of performance persists and pervades the culture of working life in the contemporary world, requiring, sometimes despite appearances, total dedication and the highest possible degree of productivity on the part of employees. The inclusion of leisure spaces in the workplace thus implies that to heighten productivity, the paradigm of disciplination is replaced by the paradigm of achievement, or, in other words, by the positive scheme of ‘can’; after a certain level of productivity obtains, the negativity of prohibition impedes further expansion. The positivity of ‘can’ is much more efficient than the negativity of ‘should’ (Han, 2020b). to heighten productivity, the paradigm of disciplination is replaced by the paradigm of achievement, or, in other words, by the positive scheme of ‘can’; after a certain level of productivity obtains, the negativity of prohibition impedes further expansion. The positivity of ‘can’ is much more efficient than the negativity of ‘should’ (Han, 2020b). Spaces have come to reflect this new modus operandi, which goes beyond a conceptual hybrid and amounts to a loosening 204 Cad. Metrop., São Paulo, v. 26, n. 59, pp. [II] https://orcid.org/0000-0003-4935-2077 Universidade Federal de Pernambuco, Departamento de Arquitetura e Urbanismo, Programa de Pós-Graduação em Desenvolvimento Urbano. Recife, PE/Brasil. lucia.leitao@ufpe.br Translation: this article was translated from Portuguese to English by Peter Ratcliffe, email: peter@ sharingenglish.com.br [I] https://orcid.org/0000-0001-9871-8536 [I] https://orcid.org/0000-0001-9871-8536 Universidade Federal de Pernambuco, Departamento de Arquitetura e Urbanismo, Programa de Pós-Graduação em Desenvolvimento Urbano. Recife, PE/Brasil. simone.jubert@ufpe.br Universidade Federal de Pernambuco, Departamento de Arquitetura e Urbanismo, Programa de Pós-Graduação em Desenvolvimento Urbano. Recife, PE/Brasil. simone.jubert@ufpe.br Notes (1) The arguments used by employers to justify the adoption of such spaces include, according to newspaper articles on the phenomenon in Brazil (Ferreira, 2014; Formiga, 2015; Grego, 2018; Melo, 2014; Pivetti, 2017), a desire to foster creativity and communication, and to enhance the well-being and productivity of employees. (1) The arguments used by employers to justify the adoption of such spaces include, according to newspaper articles on the phenomenon in Brazil (Ferreira, 2014; Formiga, 2015; Grego, 2018; Melo, 2014; Pivetti, 2017), a desire to foster creativity and communication, and to enhance the well-being and productivity of employees. (2) An expression coined by Florida (2011) for those who work in the field of software, games, audiovisual production, music, media, editing, fashion, publicity and so forth. Florida wrote about the growing role of the creative classes in the contemporary world, believing that cities could benefit from the promotion of cultural policy, without discussing the gentrification of these areas that may result. (2) An expression coined by Florida (2011) for those who work in the field of software, games, audiovisual production, music, media, editing, fashion, publicity and so forth. Florida wrote about the growing role of the creative classes in the contemporary world, believing that cities could benefit from the promotion of cultural policy, without discussing the gentrification of these areas that may result. (3) In academic work of a conceptual nature, Duerden, Courtright and Widmer (2017) have attempted to shed light on the phenomenon by understanding the incorporation of leisure into the workplace as providing a greater opportunity for individuals and groups to fully realize their potential in organizations. (3) In academic work of a conceptual nature, Duerden, Courtright and Widmer (2017) have attempted to shed light on the phenomenon by understanding the incorporation of leisure into the workplace as providing a greater opportunity for individuals and groups to fully realize their potential in organizations. (3) In academic work of a conceptual nature, Duerden, Courtright and Widmer (2017) have attempted to shed light on the phenomenon by understanding the incorporation of leisure into the workplace as providing a greater opportunity for individuals and groups to fully realize their potential in organizations. Conclusion Spaces dedicated to leisure and free time exist because of the need for a break from the world of work, to move from the condition of an individual as a producer of labor to one of being a consumer, engaging in cultural activities or experiencing contact with nature – that non- -built space that is a relic of times when there was greater symbiosis with the environment. This not only leads us to question the appropriation of non-work leisure time by work and the way individuals are trapped in the bubbles of mutual coexistence that offices have increasingly become, but also shows how the workers involved in this process of appropriation of free time are alienated from the city in which they live. This reinforces practices that distance people from social contact with a variety of different kinds of people in the public sphere and demobilizes individuals in relation to the occupation of urban space and the demand for the right to the city. It thereby also demobilizes them in relation to demands for more and better public leisure spaces, in which individuals (albeit still under surveillance) are truly in control of their own non-work time. The appropriation of non-work time by work through the production of leisure spaces within areas designated for work is a dynamic that involves the participation and collaboration of the employee. The contemporary stage of capitalism has been established gradually over centuries of surveillance and control of bodies, spreading out slowly through the social fabric. It has thus increasingly come to overlap with everyday life in all spheres, including private 205 Simone Jubert, Lúcia Leitão Notes (4) Resende (2018) presents the findings of a study of individuals working in companies that have incorporated leisure spaces into the workplace and shows that workers associate the existence of such spaces with heightened motivation and increased productivity, as well as a greater sense of attachment to the goals and values of the company for which they work. (4) Resende (2018) presents the findings of a study of individuals working in companies that have incorporated leisure spaces into the workplace and shows that workers associate the existence of such spaces with heightened motivation and increased productivity, as well as a greater sense of attachment to the goals and values of the company for which they work. (5) Retrieved from https://officesnapshots.com/2014/02/18/inside-walmart-coms-sao-paulo-offices/. Access: July 9, 2019. (5) Retrieved from https://officesnapshots.com/2014/02/18/inside-walmart-coms-sao-paulo-offices/. Access: July 9, 2019. (6) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de- inovacao-da-vivo/. Access: July 9, 2019. (6) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de- inovacao-da-vivo/. Access: July 9, 2019. (7) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de- inovacao-da-vivo/. Access: July 9, 2019. (7) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de- inovacao-da-vivo/. Access: July 9, 2019. 206 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... (8) Retrieved from https://www.galeriadaarquitetura.com.br/projetos/referencias-ambientes-c/132/ salas-de-descompressao/. Access: July 9, 2019. (8) Retrieved from https://www.galeriadaarquitetura.com.br/projetos/referencias-ambientes-c/132/ salas-de-descompressao/. Access: July 9, 2019. (9) The Littré French dictionary defines the verb ‘surmener’ as ‘to cause excessive fatigue in a beast of burden by making it walk too far or for too long’. The word is also used figuratively refer to extreme exhaustion in humans. The English translation adopted here is ‘overwork’. ‘Burnout’ is another translation sometimes used. (10) “A body is docile that may be subjected, used, transformed and improved.” (Foucault, 2018, p. 134). (10) “A body is docile that may be subjected, used, transformed and improved.” (Foucault, 2018, p. 134). (11) For Foucault (1980), an apparatus (dispositif) is “a thoroughly heterogeneous ensemble consisting of discourses, institutions, architectural forms, regulatory decisions, laws, administrative measures, scientific statements, philosophical, moral and philanthropic propositions– in short, the said as much as the unsaid” (p. 364). (11) For Foucault (1980), an apparatus (dispositif) is “a thoroughly heterogeneous ensemble consisting of discourses, institutions, architectural forms, regulatory decisions, laws, administrative measures, scientific statements, philosophical, moral and philanthropic propositions– in short, the said as much as the unsaid” (p. 364). (12) See Lima (2017). (9) The Littré French dictionary defines the verb ‘surmener’ as ‘to cause excessive fatigue in a beast of burden by making it walk too far or for too long’. 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Received: March 1º, 2023 Approved: April 24, 2023 Received: March 1º, 2023 Approved: April 24, 2023 208 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024
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https://periodicos.pucpr.br/dialogoeducacional/article/download/25454/24111
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A perspectiva dialógica na formação de professores
Revista diálogo educacional
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cc-by
9,674
http://doi.org/10.7213/1981-416X.20.064.AO08 ISSN 1981-416X A perspectiva dialógica na formação de professores The dialogical perspective in teacher training La perspectiva dialógica en la formación de profesores LUCICLEIDE ARAÚJO DE SOUSA ALVES a LUIZ SÍVERES b Resumo Apresenta caminhos metodológicos na formação de professores, tendo como eixos norteadores os sete saberes requeridos para o futuro (MORIN, 2010), e como dinâmica transversal deste percurso, o diálogo. O diálogo sendo compreendido como um jeito de ser (dialógico), uma maneira de saber (dialético) e uma forma de agir (dialogicidade). Optou-se pela pesquisa qualitativa, por privilegiar os conhecimentos significativos, que em diálogo com os sentidos atribuídos pelos professores em suas vivências e experiências, refletem em atitudes concretas e de valorização da pluralidade cultural entre os distintos saberes. Vinte docentes participaram da pesquisa e integram o Grupo de Pesquisa “Comunidade Escolar – Encontros e Diálogos Educativos”. Eles atuam em disciplinas de áreas específicas e frequentam o curso de mestrado e doutorado em uma instituição particular, do Distrito Federal. Utilizou-se dos seguintes procedimentos: a revisão bibliográfica e a aplicação de dois questionários: o primeiro com uma questão fechada, e o segundo com uma questão aberta que foi respondida por meio de narrativas. Produzidos os dados, estes foram analisados e categorizados segundo o processo de “análise de conteúdo” (BARDIN, 2002), sob o olhar dos sete saberes e baseado a Universidade Católica de Brasília (UCB), Brasília, DF, Brasil. Doutora em Psicologia, e-mail: lucicleide.ead@gmail.com b Universidade Católica de Brasília (UCB), Brasília, DF, Brasil. Doutor em Desenvolvimento Sustentável, e-mail: luiz.siveres@gmail.com Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 353 no princípio dialógico, segundo Morin (2007). Concluiu-se que a reorganização do espaço pedagógico, com foco na valorização das histórias de vida dos sujeitos e de suas relações de aprendizagem, tendo o diálogo como princípio fundante e regenerador, constitui uma das vias possíveis para propiciar as condições necessárias de gestão, valorizando-se os pensamentos e ações, para que possibilitem a concretude do fazer pedagógico revolucionário e transformador. Palavras-chave: Formação de professores. Sete saberes. Perspectiva dialógica. Abstract It presents methodological paths in teacher training, having as its guiding axes the seven knowledges required for the future (Morin, 2010), and as a transversal dynamic of this course, dialogue. Dialogue being understood as a way of being (dialogical), a way of knowing (dialectic) and a way of acting (dialogicity). Qualitative research was chosen because it privileges the significant knowledge that, in dialogue with the senses attributed by the teachers in their experiences and experiences, reflects in concrete attitudes and appreciation of the cultural plurality between the different knowledges. Twenty teachers participated in the research and are part of the Research Group “School Community – Educational Meetings and Dialogues”. They work in disciplines of specific areas and attend the masters and doctorate courses at a private institution in the Federal District. The following procedures were used: the bibliographic review and the application of two questionnaires: the first with a closed question, and the second with an open question that was answered through narratives. These data were analyzed and categorized according to the "content analysis" process (BARDIN, 2002), based on the seven knowledge and based on the dialogical principle, according to Morin (2007). It was concluded that the reorganization of the pedagogical space, focusing on the valuation of the subjects' life histories and their learning relations, having dialogue as a founding and regenerating principle, is one of the possible ways to provide the necessary management conditions, valuing the thoughts and actions, so that they make possible the concreteness of the revolutionary and transformative pedagogical doing. Keywords: Teacher training. Seven knowledge. Dialogical perspective. Resumen En el caso de los profesores, los directores de la escuela, los profesores, los profesores, los alumnos y los alumnos. El diálogo es comprendido como una forma de ser (dialógico), una manera de saber (dialéctico) y una forma de actuar (dialogicidad). Se optó por la investigación cualitativa, por privilegiar los conocimientos significativos, que en diálogo con los sentidos atribuidos por los profesores en sus vivencias y experiencias, reflejan en actitudes concretas y de valorización de la pluralidad cultural entre los distintos saberes. Veinte docentes participaron de la investigación e integran el Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 354 ARAÚJO, L.; SÍVERES, L. Grupo de Investigación 'Comunidad Escolar - Encuentros y Diálogos Educativos'. Ellos actúan en disciplinas de áreas específicas y frecuentan el curso de maestría y doctorado en una institución particular, del Distrito Federal. Se utilizó de los siguientes procedimientos: la revisión bibliográfica y la aplicación de dos cuestionarios: el primero con una cuestión cerrada, y el segundo con una cuestión abierta que fue respondida por medio de narrativas. En el caso de los países de la Unión Europea, los Estados miembros de la Unión Europea (UE) y los Estados miembros de la Unión Europea (UE). Se concluyó que la reorganización del espacio pedagógico, con foco en la valorización de las historias de vida de los sujetos y de sus relaciones de aprendizaje, teniendo el diálogo como principio fundante y regenerador, constituye una de las vías posibles para propiciar las condiciones necesarias de gestión, valorizando -se los pensamientos y acciones, para que posibiliten la concreción del hacer pedagógico revolucionario y transformador. Palabras clave: Formación de profesores. Siete saberes. Perspectiva dialógica. Introdução O cenário mundial se apresenta, no alvorecer do novo século, com uma tendência cada vez mais globalizada, seja pelos grupos econômicos, pelos sistemas políticos ou pelas redes sociais. Por outro lado, percebe-se uma dinâmica de fragmentação, seja pela unilateralidade individual, pela polaridade conjuntural ou pela desigualdade social. Tais características podem ser percebidas, principalmente, no nível pessoal, educacional e social e, por isso, esta realidade tem se configurado como um desafio para os pesquisadores contemporâneos, bem como para os professores em formação, que, dispostos a inovar, têm pensado e repensado suas jornadas pedagógicas e epistemologias, mediante processos formativos “autopoiéticos” (MATURANA e VARELA, 2001) no sentido de ato criativo, de elaborações e reelaborações a partir das próprias vivências de experiências de vida, na vida educativa, sob a perspectiva dialógica baseada no pensamento complexo. Estas características, de uma forma ou de outra, têm influenciado todos os setores da sociedade e, de modo especial, a dinâmica relacional. Isto já foi constatado por Buber (2001), ao afirmar que a humanidade estaria fortalecendo o antidiálogo; por Habermas (2012), porque estaríamos instrumentalizando a razão e, em consequência, instrumentalizando os processos comunicacionais; e por Freire Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 355 (1987), porque as relações sociais estariam sendo pautadas pelo monólogo. Estes argumentos revelam, portanto, a necessidade de retomarmos a abordagem teórica, a reflexão e a prática do diálogo como um dos princípios fundantes e constitutivos para contribuir com processos educacionais formativos trans-formadores. Embora existam muitas alternativas de formação, este artigo tem por objetivo compreender o diálogo como um procedimento transversal, propor alguns encaminhamentos, tendo como eixos norteadores do percurso os sete saberes necessários à educação do futuro e do presente propostos por Morin (2010), em vista de poder contribuir com a formação de docentes para a qualificação profissional e o compromisso social no contexto da realidade contemporânea. O diálogo é a base para o processo de cognição em processos de formação humana e, portanto, urgente e necessário. Neste sentido, estamos sugerindo que o diálogo possa ser um elemento motivador e articulador do processo de formação de professores, para se compreender a importância da presença viva do humano na vida e de sua interferência social, mediada por uma ética responsável para consigo, com o outro e com o planeta. Os procedimentos metodológicos aqui pensados foram produzidos no sentido de garantir possíveis deslocamentos em relação ao papel dos educadores, que nos seus projetos formativos não foram contemplados para caracterizar o diálogo como um princípio e uma mediação do processo de ensino e aprendizagem, bem como no exercício da docência ou na formação continuada. Esta constatação foi percebida, principalmente, na participação dos autores na Conferência Internacional: “Saberes para uma cidadania planetária” (UNESCO, 2016), cujo contexto os mobilizou a pensar e elaborar processos teóricos em sintonia com a práxis, com base nos seguintes questionamentos: Frente aos contextos educativos atuais, com vistas à necessidade de uma reforma no pensamento, de que forma professores e alunos poderiam se distanciar de um pensamento monológico e praticar uma educação baseada na relação dialógica? Como sair das “gaiolas epistemológicas”, para construir, de forma dialógica, um conhecimento pertinente? E como reverter o modelo disciplinar, baseado na fragmentação, e sugerir um conhecimento inter ou transdisciplinar? Em outros termos, como romper com o espaço enquadrado, o saber engaiolado e o conhecimento disciplinado no processo de formação de professores? Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 356 ARAÚJO, L.; SÍVERES, L. Para a tessitura desse processo, numa analogia compreendida como fios para a constituição de uma trança, é mais que requerida a necessidade para esse tecer, de uma educação permeada pelo diálogo, bem como a constituição de subjetividades e intersubjetividades a partir de uma constante dialogicidade entre os mundos intra e interpsíquicos, que se mostram, simultânea e sincronicamente, como princípio e processo na formação de professores. Neste sentido, inicialmente será apresentado o diálogo como uma dinâmica transversal e, na sequência, uma breve síntese sobre os sete saberes e como esses saberes poderiam se caracterizar como fios que se entrelaçariam para uma formação de professores, no presente e no futuro. Por fim, sob a iluminação dos saberes emergentes suscitados a partir da análise das narrativas proferidas pelos professores, por meio das vivências de suas experiências pedagógicas, será feita uma vinculação com o pensamento complexo, sob a perspectiva dialógica. O diálogo como uma dinâmica transversal O diálogo como uma dinâmica transversal será compreendido como um jeito de ser (dialógico), uma maneira de saber (dialético) e uma forma de agir (dialogicidade). É uma proposta pedagógica alternativa que se justifica em função do contexto social e, em decorrência, do projeto educacional que tem se apresentado mediante acentuadas características unilaterais, polarizantes e fragmentadas. Por esta razão, será compreendida tridimensionalmente a partir da relação antropológica, epistemológica e pedagógica, respectivamente, pois a partir dessas dimensões é possível se transformar numa sugestão relevante para a formação de professores no contexto contemporâneo. Considerando, portanto, que o diálogo é um princípio inerente à condição humana, o mesmo tem contribuído com a constituição histórica de cada pessoa e com a construção civilizatória da sociedade. Isto é, o diálogo constitui o ser humano que, por sua vez, é constituinte da sociedade, e nesta dinâmica interrelacional a humanidade se revela, desenvolve e se projeta por meio do diálogo. No exercício da dialógica, segundo Buber (2001), todo ser humano articula a vivência na experiência e esta naquela. Nesta relação de reciprocidade é preciso levar em conta a plenitude da relação que acontece entre o Eu-Tu e a efemeridade da Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 357 disposição que se realiza entre o Eu-Isso sob a perspectiva de Buber (2001). A vivência é tudo que envolve a inteireza do ser, a possibilidade relacional e a dinâmica de sentido para a vida. A experiência é tudo o que se pode observar, explicar ou dominar, ainda que provisoriamente. Assim, a vivência do Eu-Tu estaria mais próxima da singularidade de cada ser, do percurso de sua historicidade e da finalidade do seu projeto de vida. Enquanto a experiência do Eu-Isso estaria retratada na familiaridade com a “tecnologia” (LEMOS, 2004), com os contatos estabelecidos e com as metas que se deseja alcançar. Nesta perspectiva, o diálogo entre a vivência e a experiência é fundamental para constituir o ser humano e a sociedade (unitas multiplex), porque por meio dessas polaridades, não contrárias, mas complementares, explicita-se a condição humana que expressa suas inúmeras virtualidades, entre elas, como seres de relação, que estão em constante conexão e em profundo vínculo existencial. Do estabelecimento dessa dinâmica relacional entre a vivência e a experiência, o ser humano se constituirá como um ser dialógico, desenvolvendo-se por meio de experiências de vida mais integrada e polifônica. Esta relação entre a vivência (Eu-Tu) e a experiência (Eu-Isso), foi, em grande parte, a desencadeadora de toda a reflexão proposta por Buber (1987) sobre o processo de diálogo nos últimos tempos. Portanto, para esta proposta de caminho de formação de professores, o diálogo será compreendido nesta dimensão de dinâmica transversal que acontece mediante a relação que ocorre entre o sujeito consigo mesmo, com o outro e com o transcendente, tendo como eixos articuladores os sete saberes necessários para a educação do presente e do futuro, convergindo, conforme a proposta buberiana, por compreender o ser humano como um ser social, de relação, com possibilidades de produzir conhecimento e de atuação consciente na sua comunidade local e na sociedade global. Os sete saberes necessários à educação do presente e do futuro Os sete saberes propostos por Morin (2010) serão os eixos-base para esta formação de professores, entrelaçados pelas sete disposições apresentadas por Nóvoa (2016) e as vivências de profissionais da área educativa que estão comprometidos com a transformação da educação, compõem as referências desta reflexão. Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 358 ARAÚJO, L.; SÍVERES, L. Estas três contribuições — compreendidas pela analogia de uma trança — serão consideradas para identificar cada um dos saberes, bem como suas emergências durante o exercício da profissão docente, seja no decurso de sua formação inicial ou continuada. Embora nosso olhar esteja sempre voltado para o conjunto destes saberes, é oportuno, porém, apresentar uma pequena percepção sobre cada um destes saberes. As cegueiras do conhecimento: o erro e a ilusão: É importante o reconhecimento de que todo conhecimento comporta o risco do erro e da ilusão. Portanto, não há conhecimento que não esteja ameaçado por esta dupla face. Todo ele é tradução e reconstrução sob a vista de um ponto, não traduzindo o real como visão única, mas compreendendo-o a partir de suas múltiplas facetas de verdades provisórias, cuja interpretação pode ser feita sob os mais diversos olhares e diferentes ângulos, pois a própria realidade apresenta-se sob os contextos multidimensionais. Os princípios do conhecimento pertinente: É o conhecimento processado, refletido, organizado e traduzido na conjunção, permitindo a integração entre o todo (sociedade) e as partes (indivíduo), imbricados com o processo de vida. Este conhecimento é demarcado por um tempo e lugar e, em um momento histórico, com vistas a manter a dinâmica sócio-histórica-cultural da humanidade caminhante. Ele busca estabelecer os elos necessários entre os mais diferentes saberes, permitindo a percepção da realidade sob a visão de conjunto e não fragmentada, pois recoloca o conhecimento aprendido no contexto. Ensinar a condição humana: Este é um dos principais saberes necessários para o desfrute de uma vida em plenitude. Ensinar a condição humana é compreender a unidade (humano) na diversidade (cultura) humana e esta naquela, para o desenvolvimento conjunto das autonomias individuais, das participações comunitárias e do sentimento de pertencimento do sujeito, posicionando-se no mundo. É conhecer o humano, situando-o no contexto e não fora de sua realidade. Ensinar a identidade terrena: Ensinar a identidade terrena é perceber-se parte constitutiva dessa sociedade complexa para ser capaz de enfrentar com autonomia as suas incertezas. Já dizia Pascal, citado por Morin (2008, p. 25): “considero impossível conhecer as partes sem conhecer o todo, assim como conhecer o todo sem conhecer, particularmente, as partes [...]”. Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 359 Enfrentar as incertezas: É importante que estejamos prontos a enfrentar as incertezas do conhecimento, pois toda tradução é um ponto de vista (BOFF, 1997), que possivelmente pressupõe erro. No entanto, erro este que pressupõe ponto de partida para novos acertos e erros, possibilitando um caminhar que não se encerra em si mesmo, mas que estará sempre renascendo, mantendo o sujeito em sua dinâmica processual construtiva de conhecimento, aprendendo a conviver com as certezas do conhecimento, ainda que provisórias. Ensinar a compreensão: “Educar para a compreensão humana”, traduz-se em colocar-se no lugar do outro e não desejar para o outro o que não se desejaria para si mesmo. Ou seja, uma compreensão que parte do eu (de si mesmo) para o tu (o outro) legitimando-o. A ética do gênero humano: A consciência de nossa identidade como seres pertencentes a um todo se embasaria na ética da compreensão caracterizada pelo “pensar bem”, vinculado a ética da compreensão entre as pessoas à ética da era planetária. Repensar a formação de professores, sob o olhar dos sete saberes propostos por Edgar Morin, pode contribuir para a ressignificação dos atuais contextos educativos, com vistas à superação da prática pedagógica voltada exclusivamente para o ensino, prevalecendo a perspectiva positivista, para a complexa, que privilegia a religação entre a cultura científica e a humanística. Metodologia Na perspectiva de avançarmos na formação de professores, tendo o diálogo como o fio transversal e entrelaçado pelos sete saberes, as sete disposições e vivências de professores, fizemos opção pela pesquisa qualitativa, por esta privilegiar os conhecimentos significativos que, em diálogo com os sentidos atribuídos pelos professores em suas vivências e experiências, refletem em atitudes concretas e de valorização da pluralidade cultural entre os distintos saberes. Este tipo de pesquisa vai além da visão positivista e de seus parâmetros epistemológicos, por trazer de volta ao seu fazer pedagógico o sujeito e sua subjetividade, ao dar ênfase na Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 360 ARAÚJO, L.; SÍVERES, L. construção de processos formativos, mediante às representações, os significados e o sentido existencial elaborado, segundo Lemos (2004). Os docentes participantes da pesquisa integram o Grupo de Pesquisa “Comunidade Escolar — Encontros e Diálogos Educativos”. Eles atuam em disciplinas de áreas específicas e frequentam o curso de mestrado e doutorado em uma instituição particular, de caráter confessional e comunitário, do Distrito Federal. Para a produção dos dados, utilizamo-nos dos seguintes procedimentos: a revisão bibliográfica e a aplicação de dois questionários: o primeiro com uma questão fechada, e o segundo com uma questão aberta que foi respondida por meio de narrativas. O primeiro questionário foi encaminhado aos 20 participan9tes do Grupo de Pesquisa com a seguinte questão: Considerando a sua compreensão e sua prática educativa, com qual dos sete saberes, propostos por Morin, você mais se identifica? Indique em ordem de prioridade, de 1 a 3, os três saberes que estariam mais próximos da sua experiência de vida. Do total dos 20 participantes do grupo, 16 responderam ao questionamento acima, cujos dados seguem descritos na sequência: Gráfico 1 - Questionário Fonte: Acervo dos autores. Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 361 Segundo o Gráfico 1, todos os saberes receberam algum tipo de menção, exceto o “ensinar a identidade terrena”. O conjunto dos dados revela que os três saberes mais apontados, incluindo a indicação de alguma prioridade, foram: ensinar a condição humana, enfrentar as incertezas e ensinar a compreensão. Assim, no ensinar a condição humana, 5 indicaram como prioridade 1, 4 a prioridade 2 e 3 a prioridade 3, totalizando 12 manifestações. No saber enfrentar as incertezas, 3 responderam como prioridade 1, 4 a prioridade 2 e 6 como prioridade a 3. Por fim, no ensinar a compreensão, 4 respondentes informaram como prioridade 1, 4 como prioridade 2 e 5 como prioridade 3, resultando também em 13 indicações. A partir da aplicação do primeiro questionário, os dados gerados revelaram que alguns saberes são prioritários em relação aos demais. Por isso, com base na resposta ao primeiro questionário, optou-se por aprofundar, na análise e discussão, apenas os três saberes mais reconhecidos, a saber: ensinar a condição humana, enfrentar as incertezas e ensinar a compreensão. Após tomada essa decisão, foi encaminhado o segundo questionário aos 12 participantes que indicaram como primeira prioridade um dos três saberes mais recorrentes. Assim, foi encaminhada a seguinte questão: Com base na sua resposta, gostaríamos de convidá-lo novamente para relatar uma experiência educativa. Para o primeiro grupo foi solicitado uma experiência de “como ensinar a condição humana”, para o segundo, “como enfrentar as incertezas”, e ao terceiro, “como ensinar a compreensão”. Produzidos os dados nessa segunda etapa, estes foram categorizados e analisados, segundo o processo de análise de conteúdo baseado em Bardin (2002), sob o olhar dos sete saberes e na perspectiva baseada no princípio dialógico, segundo Morin (2000, 2007). Partiu-se da leitura de todo o material, na busca por significados que emergissem das falas como atribuições dos sujeitos a suas próprias ações, de forma a auxiliar nas interpretações a serem discutidas teoricamente. Os temas foram interpretados no seu conjunto, estabelecendo-se relações com o referencial teórico adotado, mas foram selecionados apenas três depoimentos, vinculados aos três saberes mais evidenciados. Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 362 ARAÚJO, L.; SÍVERES, L. Resultados e discussões No sentido de contribuir com o enfrentamento dos desafios da educação contemporânea, por meio de ações concretas que possam incorporar os saberes na prática formativa de professores, foram acolhidas as manifestações de três participantes do Grupo de Pesquisa, ora identificados com nomes de uma família bíblica: Marta, Maria e Lázaro, com o objetivo de resguardar a sua identidade. Assim, foi acolhido o relato de Maria: Ensinar a condição humana, de Lázaro: Enfrentar as incertezas, e Marta: Ensinar a compreensão. Segue a descrição das narrativas: Como ensinar a condição humana? Eis a reflexão de Maria: O presente relato versa sobre um trabalho da disciplina Gestão das Instituições Educativas e Esportivas na Licenciatura em Educação. [...]. Como parte da citada disciplina, os estudantes de Educação Física visitaram, durante um semestre uma escola, realizando observações e entrevistas com professores e alunos. Após as primeiras visitas, identificaram que o público atendido reunia alunos de diferentes setores sociais (inclusive alguns moravam em abrigos prévios à espera de adoção), religiões e de diversas nacionalidades (17 países, incluindo República Dominicana, Cuba, Venezuela, Síria, México, Bulgária e outras nações limítrofes). Paralelamente, observaram que a instituição carecia de jogos ou espaços recreativos no pátio escolar, totalmente pintado de cinza. As impressões registradas durante os recreios eram de que os alunos permaneciam isolados. A não existência de áreas recreativas – árvores, gramados, bancos, brinquedos – deixava a instituição muito similar a um cárcere [...]. A partir desse diagnóstico, decidiu-se, de forma coletiva, pela elaboração de um projeto de criação/pintura de jogos, que também contribuiria para fortalecer o senso de pertencimento à instituição escolar – até então pouco consolidado. Os alunos da escola foram indagados sobre as atividades recreativas mais frequentes em seus países de origem, capazes de serem recriadas no pátio escolar. Uma vez realizada a seleção de jogos (amarelinhas, labirintos, redes para esportes coletivos, xadrez, minhocas), os professores de Artes e Recreação foram convidados para colaborar no desenho e execução do projeto batizado de “Pintada na Escola nº 3”. Uma semana depois que a ornamentação da área de lazer foi concluída, realizou-se uma quermesse de inauguração dos espaços recreativos do pátio, reunindo a comunidade escolar mais os estudantes de Educação Física. Música, cores, animação e integração entre os alunos e estudantes mostraram a relevância da aproximação com o outro: escutar suas vozes, identificar suas necessidades. Assim, uma disciplina, em princípio teórica, se transformou numa ágora de integração, não apenas de saberes (gestão, artes, recreação), como também de integração da docência, da pesquisa e da extensão. E, sobretudo, de culturas, biografias e esperanças de que naquela escola é possível aprender e resgatar o sentimento de pertencer à própria comunidade escolar. Como enfrentar as incertezas? Segue o depoimento de Lázaro: Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 363 O cenário que ora se apresenta é aquele no qual as tecnologias funcionam quase como um dos membros do corpo humano. Aliado a isso, a motivação dos estudantes passou a ser um dos desafios para o processo de ensino e aprendizagem. [...]. Fui tomado pela incerteza sobre qual método de ensino seria considerado o ideal para o alcance dos objetivos propostos a partir de uma experiência vivenciada no início de um semestre letivo. Naquela oportunidade, escutei de alguns dos alunos relatos do tipo “nem mesmo eu sei o que estou fazendo aqui”; enquanto outro dizia: “vou ficar aqui para ver o que dá…”; teve ainda: “não sei se vou aguentar até o fim”. Diante dessa situação de incerteza acerca de quais eram os objetivos de parte daquele grupo, procurei refletir na tentativa de responder à seguinte questão: como aplicar técnicas de ensino que pudessem motivar esses estudantes, face às incertezas e quanto à possível eficácia ou fracasso da estratégia por mim escolhida. Como ensinar a compreensão? Segue o relato de Marta: Esse relato de experiência versa sobre um processo interventivo de Gestão Escolar, constituído num esforço educativo de abrir possibilidades de humanização, cooperação, e reflexão do papel do gestor e da comunidade escolar em busca do fazer coletivo e democrático. Teve como espaço uma instituição de Educação Básica no Distrito Federal entre os anos de 2011 a 2016, localizada em área urbana. O diagnóstico e a análise das situações vivenciadas pela comunidade escolar indicaram serem urgentes mediar e auxiliar o coletivo da escola, a compreenderem e refletirem sobre os valores da comunidade, e acreditarem na capacidade de seus alunos e de seus pares. Por outro lado, as fragilidades que culminaram na vinda de um interventor à escola permitiram ao grupo redimensionar o pensar, reformulando suas ações pela compreensão do que a comunidade escolar (entendida aqui os alunos, pais, professores, equipe pedagógica, direção, funcionários) pode fazer para estimular a função social da escola. [...]. O levantamento da situação revelou muitos problemas estruturais, necessidade de diversas reformas e reorganização dos espaços pedagógicos, nova abordagem à Proposta Pedagógica que promovesse a melhoria nos altos índices de reprovação e evasão, e também, a falta de apoio da comunidade. Sendo a atuação do gestor escolar uma tarefa que perpassa várias dimensões, sejam elas administrativas, pedagógicas, políticas, sociais é urgente estar atento ao ato de compreender-se gestor e a partir dessa sensibilidade conduzir coletivamente a formulação do projeto pedagógico, agir na coordenação, acompanhamento e cumprimento das responsabilidades compartilhadas, ouvindo e dando sentido ao que a comunidade escolar tem a oferecer. [...] O momento era tenso e as relações pessoais estavam abaladas. Iniciamos investigando as causas dos conflitos, os valores da escola, as lideranças do grupo, a comunidade, entre outros. Iniciamos realizando um diagnóstico com análise da situação, os objetivos a alcançar, ajustar estratégias de receptividade ao grupo, melhorar o ambiente, criar empatia, pois não pertencíamos ao grupo, e construir um planejamento juntamente com toda a comunidade escolar para resolver os problemas. [...] Após o planejamento partimos para a ação. A experiência pautou-se na reconstrução do Projeto Pedagógico nas reuniões coletivas da escola, com encontros semanais. A cada semana a fala inicial era dada ao grupo para que narrassem suas expectativas, seus projetos, seu trabalho, e a partir da reflexão e da compreensão da história de cada um, constituiu-se um conjunto de ações, que compartilhados, uniram às metas e aos objetivos coletivos, mas também às responsabilidades individuais, a Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 364 ARAÚJO, L.; SÍVERES, L. tarefa dada ao gestor foi articular responsabilidades individuais e coletivas de modo que todos tenham consciência de seu papel dentro da instituição de ensino, o que exigiu mais que aprender a compreender o outro, ensinar a importância de fazer-se compreendido. Saberes trançados por três fios entrelaçados A formação de professores, por muito tempo, e até hoje, continua se dissipando numa abordagem disciplinar, organizada em arcabouços epistemológicos engaiolados e sendo exercida continuamente a partir do quadro-negro. Totalmente desconexa da realidade ao dar ênfase exacerbada à teoria, com pouca preocupação de como esta reflete em cada realidade e em conformidade com o seu contexto cultural. Conduzir os futuros professores e/ou em formação continuada, mediante o conhecimento dos fundamentos da profissão baseada nos sete saberes, por meio de observação da realidade, escuta sensível, pesquisa em profundidade, pensamento reflexivo, para intervir na realidade com ações concretas, projetos de vida, por soluções de caminhos alternativos decididos no coletivo, tendo em vista o restabelecimento de relações mais amistosas, a contínua troca de experiências, mediada por processos dialógicos capazes de integrar as partes ao todo e o todo às partes, é a condição humana a ser ensinada nos espaços formativos. A condição humana é um dos fios dos sete saberes a serem trançados, entrelaçados com outros, para práticas formativas mais atuantes e menos passivas frente aos problemas sociais da vida. O saber “ensinar a condição humana” foi indicado por Marta, Lázaro e Maria como primeira prioridade entre os 7 saberes, e será, pois, o fio de análise da construção de um texto, cujo contexto da trama se desenvolve a partir dos fios entrelaçados. Fio 1. Ensinar a condição humana No exercício da práxis do saber “Ensinar a condição humana”, Morin (1999, 2007) alerta sobre a necessidade da consciência do conhecimento sobre o próprio conhecimento, pois este sempre comporta erro e ilusão, bem como, de um pensamento abrangente e mais articulado para uma compreensão mais adequada e Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 365 consciente sobre os processos formativos e suas necessidades inerentes, mediante constantes releituras da realidade em conjunto com os pares. Além disso, importa a atuação dos sujeitos envolvidos na dinâmica da formação, por meio de um agir, em existência, com toda a inteireza, utilizando-se dos conhecimentos e das experiências apreendidas durante o viver/conviver. Compreende-se que dessa tessitura construída e constituída nos mais diversos espaços de vida, de formação inicial e/ou continuada, perpassados, vividos e experienciados por sujeitos dispostos que desejam exercer a cidadania, podem emergir ações concretas, com transformações bem-sucedidas, pelo resgate dos sujeitos por meio de práticas mais dialógicas. Ou seja, um fazer desembocar ações particulares na direção do universal, com atitudes concretas. Assim como bem procedeu Marta, após ter se apropriado de novas epistemologias em espaços formativos, vivência na prática o saber “Ensinar a condição humana”, ao levar os seus estudantes a uma imersão na realidade profissional futura, para conhecerem as suas condições reais e intervirem sem descontextualizar os sujeitos de sua cultura (LARAIA, 1986), numa tomada de ações em conformidade com as circunstâncias locais. De forma também semelhante agiu Maria após ter elaborado diagnóstico para análise das situações vivenciadas pela comunidade escolar, mediado pelo exercício do diálogo para a compreensão dos problemas e reflexões no coletivo sobre os valores aparentemente “perdidos”, visando ao resgate da credibilidade na capacidade de seus alunos e de seus pares e ao retorno destes como membros participantes e ativos na transformação da própria comunidade de aprendizagem. A consciência epistemológica adequada do real, para a tomada de atitudes, consoante a escolhas políticas e estratégicas, como as de Marta e Maria, foram decisões-chave para a busca de vias de ação e de transformação nos espaços educativos considerados encarcerados, desacreditados, em verdadeiras ágoras. E o efetivo restabelecimento das relações comunicativas entre os membros da comunidade, com vistas à apropriação dos bens culturais da humanidade, mediante o resgate da convivência harmoniosa e humana entre os sujeitos. Ao reintegrar os sujeitos da comunidade escolar (alunos, pais, professores, equipe pedagógica, administrativa e diretiva), a função social da escola é exercitada e reforçada pela participação ativa de cada um de seus membros, atribuída pelo Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 366 ARAÚJO, L.; SÍVERES, L. sentido da importância do pertencimento, ao se colocar as experiências e vivências na reconstituição e humanização do todo, que se encontravam desconectados e fragilizados entre si, enquanto comunidade. É fato que os espaços formativos não podem se “[...] constituir similares a um cárcere”, como analogia intensificada e proferida metaforicamente pelo depoimento de Marta. Tampouco serem geridos e gestados à revelia, sem qualquer compromisso com o desenvolvimento humano a partir da compreensão de seus problemas singulares inerentes. A capacidade de pensar conjuntamente os problemas locais e globais, mediante a criação de espaços para o exercício da relação dialógica, que possibilitem os caminhos e as circunstâncias necessários para os sujeitos em formação abrirem-se ao inesperado, com consciência formada por meio de epistemologias mais abertas, para um agir em coletividade, é procedimento cada vez mais urgente e necessário, em função dos tempos atuais, fluidos e carentes de relações comunicativas mais humanizantes e humanizadoras. Neste sentido, humanizar a convivência com o outro é da ordem do humano, e esta acontece na e pela cultura, perpassada pelo diálogo consigo, com o outro e com a vida. Do contrário, contribuiremos muito mais para a disjunção do que para a integração nos espaços formativos, reforçando a prática de ações antidialógicas, improdutivas, mutilantes e mutiladoras. Reunir a comunidade escolar para uma escuta sensível sobre o que vivencia na dinâmica de sua comunidade de aprendizagem e de vida, cotidianamente, criando espaços dialógicos, conforme indicado pela narrativa de Maria, só demonstra e reafirma a “relevância da aproximação com o outro: escutar suas vozes, identificar suas necessidades”, conforme assertiva por ela mesma proferida. Relacionar-se para sentir-se integrado, portanto, é uma condição humana necessária para a prática dialógica que sugere abertura para o compartilhamento, troca de ideias e a construção de projetos interventivos de vida, com vistas à transformação da própria realidade numa simbiose homem/sociedade/natureza. Neste sentido, o conhecimento sobre os saberes da profissão professor (NÓVOA, 2016) interpelados e construídos no exercício de uma prática educativa concebida por meio da dialogicidade para perceber a realidade e nela intervir, requer ações em Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 367 conformidade com as necessidades e circunstâncias de cada localidade, em conjunto com os seus pares, integrantes daquele microssistema. A consciência da necessidade de uma escuta sensível do outro, de suas necessidades e sonhos, foi percepção fundamental, como reforçado também por Lázaro em sua narrativa, ao fazer opção por ouvir atentamente o que os seus alunos apresentavam como expectativas em relação à disciplina e quais eram os seus objetivos, como também o demonstrou Marta, ao ouvir atentamente os membros da comunidade escolar por meio da realização de diagnóstico para análise das situações vivenciadas, visando encontrar soluções conjuntas mediadas pelo diálogo. Evidencia-se, assim, o quanto a comunicação intersubjetiva e os relacionamentos são condições humanas necessárias para o fortalecimento do sentimento de pertencimento e integração a uma comunidade para o convívio e desenvolvimento humano saudável. Cada uma das iniciativas propostas por Maria, Marta e Lázaro para a reconstrução de processos de ensino e aprendizagem formativos — conforme propõe Nóvoa (2016), — contribuem para o exercício de práticas formativas baseadas na perspectiva dialógica, suscitando perguntas e respostas em conjunto e sob os mais diferenciados olhares, interpeladas, sobretudo pelo sentimento de esperança por soluções quanto aos problemas existentes, propulsionando saltos qualitativos à comunidade, ao se ter conseguido resgatar a participação ativa dos sujeitos. A consciência epistemológica apreendida, exercitada pela prática do princípio da dialogicidade, como mediador da “costura” entre os saberes científicos e humanísticos, foi mister e significativa para o ensinar a condição humana (Fio 1), pois este saber requer o respeito às ideias contrárias e às certezas provisórias, cujas perspectivas e óticas em relação à vida são plurais e multidimensionais, quando se lida com grupos de visões heterogêneas. E como assegura Morin (1999), nos momentos em que não é possível superar as contradições, vencer os antagonismos e ultrapassar os paradoxos, é preciso aprender a lidar e a conviver com os opostos, compreendendo-os, pois é nesse emaranhado da trama que emerge a complexidade, pois do mesmo lugar que surgem os problemas nascem também as suas soluções. E “o entrechoque de ideias, opiniões e comportamentos é uma das principais fontes de inspiração para a Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 368 ARAÚJO, L.; SÍVERES, L. criatividade e a resolução dos problemas de convivência”, como assegura Mariotti (2007, p. 151), para o enfrentamento das incertezas (Fio 2), mediante o ensinamento da compreensão (Fio 3). Fio 2: Enfrentar as incertezas A consciência sobre a própria ação pressupõe a consciência de um fazer compreendido em constante movimento de estado de evolução, alimentado por um pensar complexo e práticas pedagógicas congruentes com os pressupostos metodológicos menos tecnicistas e mais complexos, plurais e multidimensionais, com vistas à superação do distanciamento do professor do quadro negro (NÓVOA, 2016) e a imersão no mundo em rede, pela necessidade de que “é preciso aprender a conviver com as incertezas ligadas ao conhecimento” (MORIN, 2010, p. 82). Porém, sem descontextualizar-se do mundo, tampouco de suas inovações e bens científico-culturais, mas buscando compreendê-los em sua própria dinâmica. É o que buscam traduzir as iniciativas de Maria, Lázaro e Marta, pois práxis é confronto. E confrontar é estar disposto a pensar o real para transformá-lo, confrontando-se o vivido com o pensado, pois a certeza do risco de sua incerteza é inerente a cada ação. Por isso é sempre desafio, e eliminá-la é impossível, pois “a imprevisibilidade reside no próprio cerne do determinismo” (MORIN, 2015, p. 41), requerendo, portanto, um constante negociar com a incerteza, pelo recurso à reflexão, combinando riscos e estratégias de precaução, mediante uma escuta vigiada, para propulsionar o movimento de aprendizagem e o seu desenvolvimento processual e contínuo. E como assegura Morin (2015, p. 51), “é preciso aprender a navegar em um oceano de incertezas, através de arquipélagos de certezas”. Para tanto, o exercício do diálogo intra e intersubjetivo é uma via para a possibilidade de se compreender o processo de formação dos sujeitos, mediante uma ética comprometida, valores concebidos e pelo respeito ao repertório cultural que constroem e trazem consigo durante a vida. O pressuposto do diálogo não consiste em convencer o outro, mas abrir possibilidades para partilhar significados construtivos de conhecimentos, durante o Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 369 viver e o conviver, para então se direcionar ao agir. Este agir, no sentido de criar ações interativas, pela percepção e conhecimento da realidade, mesmo mediante contextos complexos de diferenciados posicionamentos e ideias, pois estes e estas, ao serem articulados com os saberes apreendidos ao longo da vida, por seus diversificados membros em processos dialógicos, podem suscitar outras ideias criativas e vias de caminhos antes não pensados. Dessa forma, a atitude dialógica possibilita emergir a comunicação, sem autoridade hierarquicamente estabelecida, numa perspectiva contínua e evolutiva de solidariedade e compartilhamento, por meio da construção de pensamentos baseados em saberes interconectados e integrados. Em um processo relacionalmente favorável, na busca por inovadoras possibilidades de conhecimento e apreensão da realidade, pela prática do diálogo, com flexibilidade e respeito às ideias do outro, de modo a assegurar a manutenção dos processos de fluxos dialógicos contínuos, circulares, e de constantes recursividades, como foi possível notar nos três relatos das experiências vivenciadas por Maria, Lázaro e Marta. A reflexão sobre os fatos passados, remetendo para o futuro, os olhares dos sujeitos no presente, mesmo sem os fundamentos teóricos fortemente consolidados, mas já desabrochados — pela necessidade de um fazer pedagógico diferente — retrata a necessidade da consciência sobre o próprio pensar e fazer, dos fundamentos implícitos a cada sujeito, orientadores de suas ações, reforçando-se assim o dito por Freire (1996), de que não há uma prática sem teoria e nem esta sem aquela. Ambas se imbricam, revelando o sujeito e suas epistemologias, trazendo-as à consciência para revê-las e ressignificá-las no presente, com posturas mais conscientes e fundamentadas, no sentido de se dar uma nova ordem ao que se mostra desorganizado e desconexo do real. A partir do momento em que os professores são munidos por novos fundamentos e perspectivas metodológicas, como proposto pelos “Saberes Necessários à Educação do Futuro”, no presente, espera-se que eles possam encontrar novas vias pedagógicas para além do quadro-negro, dos conteúdos engessados, e processos antidialéticos nos espaços formativos. Distanciar-se de um processo formativo mais estático, compreendido numa perspectiva de meros repetidores de informações, como sugere Nóvoa (2016), Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 370 ARAÚJO, L.; SÍVERES, L. requer por parte dos professores, atitude de autonomia e autoria para criar e protagonizar nos espaços educativos e para além destes, tendo em vista oportunizar aos membros da comunidade educativa saírem das próprias gaiolas epistemológicas, consideradas por D’Ambrósio (2009). Tal atitude foi bem encaminhada por Maria, ao levar os estudantes para conhecerem outras realidades, para além da sala de aula, e se apropriarem de conhecimentos provindos da realidade, mais pertinentes e munidos de sentido e significado, numa tentativa esperançosa de reverter a lógica reprodutivista e de passividade dos sujeitos nos espaços formativos, com vistas a avançar para uma perspectiva de formação com protagonismo docente. Coadunar os momentos de teoria com o seu exercício na prática, pela vivência no campo profissional, permite aos sujeitos em formação pensar e repensar sobre a profissão, pela pesquisa, elaboração e desenvolvimento de projetos, para o acesso e maior proximidade com a cultura e seu repertório contemporâneo, apreendendo-a, pela observação, pelo diálogo e ações viáveis que possam revitalizar os espaços aparentemente “cinzentos” (sem vida), como dito por Maria. Nas ações propostas por Maria e Marta, a exigência de novos itinerários pedagógicos formativos, a reaproximação dos sujeitos com a sociedade do século XXI — a partir de ações mais integradoras e coletivas — foi atingida pelo sentimento de pertencimento. Ao sentir-se pertencente a uma comunidade de diferentes fins, o sujeito passa a se relacionar, e, consequentemente, a agir, aprendendo, reaprendendo e se desenvolvendo em comunhão com os seus pares, em prol do bem viver, reafirmando assim que é na relação com o outro que nos constituímos, aprendemos e nos transformamos mediante as trocas de experiências vivenciadas no decurso do viver/conviver — pela condição de sujeito social que somos — mediados pelo diálogo. Dessa forma, nos transformamos conforme transformamos o mundo, e isso requer disposição, mesmo diante dos incertos e desafiadores processos educativos e de vida. Paralelas à coragem e às “certezas” de possíveis transformações, emergem também a insegurança e as incertezas. No relato de Lázaro, por exemplo, urge a incerteza quanto aos conhecimentos de sua profissão que poderiam propiciar a motivação necessária para o atendimento aos interesses da turma, bem como quanto à escolha do método, e a insegurança quanto à sua efetividade, e se aquela seria a Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 371 melhor via a ser trilhada. Destaca-se também a desconstrução da própria prática pedagógica, sinalizado por Lázaro ao expressar não se sentir preparado para inovar diante do inesperado. Diante deste contexto, é fundamental aprender a aprender a lidar com os paradoxos: certeza e incerteza, ordem e desordem, segurança e insegurança, primando-se pelo equilíbrio e o restabelecimento da ordem, para novas desordens e reorganizações em espaços formativos. Desse movimento de relações aparentemente contraditórias, porém complementares, é que a vida mantém o seu ciclo em processo ativo de desenvolvimento e de produção, construindo-se, desconstruindo-se e reconstruindo-se o tempo todo. O reconhecimento do risco permite aos sujeitos um olhar mais profundo, com maior consciência sobre os problemas da vida tanto pessoal quanto socialmente, pelo processo de autoconhecimento (o aprender a ser), pelas experiências vivenciadas durante o viver, com vistas a enfrentar as incertezas presentes na dinâmica da vida (o aprender a conviver), para então se colocar o agir (o aprender a fazer). Dessa forma, “Ensinar a condição humana” como primeira prioridade em espaços formativos é uma necessidade vital, pois requer a legitimação do outro, bem como o exercício da autopercepção enquanto pessoa, profissional, sujeito construtor da própria história, na relação com o outro, reconhecendo, de forma equilibrada, os próprios limites humanos das incertezas por suas ações, pois “em toda ação há escolha e risco” (MORIN, 2010, p. 42). Neste sentido, é mister “conceber a unidade que assegure e favoreça a diversidade, a diversidade que se inscreve na unidade” (MORIN, 2010, p. 55), pelo ensinamento da condição humana, entrelaçado pelo da compreensão para enfrentar as inúmeras incertezas em relação ao conhecimento e a vida. Fio 3: Ensinar a compreensão A consciência sobre a pluralidade cultural e multifacetada das instituições, que engloba, como frisa Marta, “diversas realidades socioeconômicas, emocionais e culturais; um ambiente heterogêneo por princípio”, leva a valorizar as trajetórias de histórias de vida construídas e constituídas pelos sujeitos que compõem diferentes comunidades e suas diferenciadas culturas. É fazer com que os processos subjetivos Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 372 ARAÚJO, L.; SÍVERES, L. se façam presentes e sejam centrais — para transformações por meio da manifestação dos potenciais adormecidos e antidialéticos — mediante o restabelecimento do diálogo, na coletividade, para ações locais concretas. Uma disciplina em princípio teórica, segundo Maria, se transformou numa ágora de integração de saberes (gestão, artes, recreação), como também de integração da docência, da pesquisa e da extensão. E, sobretudo, “de culturas, biografias e esperanças de que naquela escola é possível aprender e resgatar o sentimento de pertencer à própria comunidade escolar”. E uma intervenção, segundo Marta, que inicialmente poderia ter sido marcada por autoritarismos, posturas unilaterais e apartadas da trajetória da comunidade, constituiu-se em um movimento de aprender e, sobretudo, de ensinar a compreensão, enfrentando as incertezas provindas das soluções estabelecidas em conjunto. Isto porque toda a ação é imbuída de retroações, ou seja, de riscos que fogem ao controle humano. No entanto, sem correr riscos será impossível compreender o “problema importante (MORIN, 2007)”, pois só se pode compreendê-lo, entregando-se ao aleatório, ao imprevisível, pois são estes os processos emergentes que possibilitam o crescimento e a evolução das relações comunitárias, na medida em que enfrentam junto o inesperado, pelo ensinamento da compreensão, arcando com as incertezas inerentes, em conformidade com as condições humanas e de respeito às realidades singulares. No relato de Marta, “Ensinar a compreensão” tornou-se, por conseguinte, a base de todo o processo de intervenção à medida que, ao perceber o outro, abriu-se a possibilidade de atribuir novos significados à história da comunidade a partir da história de cada sujeito. Visto que todos nós trazemos diferentes repertórios culturais, conhecimentos incorporados, desejos e sonhos, e nessa escrita da própria história de vida, significá-la e ressignificá-la são o roteiro a ser considerado e respeitado para a apreensão de “conhecimentos pertinentes” (MORIN, 2010), a partir da releitura dos problemas emergentes. Quando o sujeito é constantemente desafiado a intervir em sua comunidade, a partir da compreensão dos seus problemas inerentes, por um agir fortalecido pelo diálogo com o outro e outros, mediante consciências despertas, o diálogo e a ação Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 373 em conjunto se tornam fundamentos necessários para o exercício de uma cidadania planetária civilizatória. Bohm (2005, p. 77) assegura que “o compartilhamento de consciências é mais importante do que o conteúdo das opiniões”. Acrescenta, ainda, que “a própria estrutura de um pressuposto ou opinião implica que eles devem estar abertos à demonstração de que podem não estar corretos”. Por isso, o exercício do diálogo é atitude a ser desenvolvida nos espaços destinados à formação docente, para que no exercício futuro da profissão, os sujeitos do ensino possam exercê-la, e com protagonismo gestar soluções aos problemas emergentes, movidos sempre por uma prática fundamentada e desenvolvida na perspectiva dialógica. Para tanto, o agir pressupõe disposição ao risco, coragem para enfrentar as suas incertezas, e dar um salto na indeterminação, pela superação do pensamento unilateral, em um deverfazer irresistível em coletividade. Assim, é fundamental a criação de espaços pedagógicos dialógicos, de escuta sensível ao outro, às suas ideias e opiniões, às suas histórias e trajetórias, colocando em suspenso todo e qualquer tipo de julgamento, em vista da possibilidade de abertura de novos caminhos de diálogo, visto que não há caminho único para a busca da verdade, pois todas as trilhas apontam para se chegar ao “não-caminho” (BOHM, 2005). Vida e conhecimento, portanto, são indissociáveis no processo de aprender e de conhecer a realidade em quaisquer espaços formativos. Separar a história das ações biológicas e sociais do sujeito de seu contexto torna-se inviável, pois o sujeito se constrói nas relações dialógicas. É, pois, por meio desse imbricamento que o mundo aparece, à própria imagem e semelhança, como assim asseguram Maturana e Varela (2001). Para tanto, é válido reforçar a importância do reconhecimento da diversidade das condições humanas presentes nos ambientes formativos e o respeito às suas diferentes histórias de vida. Desta forma, ao se perceberem reconhecidos, os sujeitos da dinâmica educativa poderão sentir-se parte de um todo bem maior que os engloba, aprendendo e ensinando na relação uns com os outros, mediados pelas trocas intersubjetivas de conhecimentos, na tentativa de enfrentar as incertezas Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 374 ARAÚJO, L.; SÍVERES, L. presentes na vida para evoluir psicológica, afetiva e socialmente, pela compreensão uns dos outros. Enfim, Ensinar a condição humana, enfrentar as incertezas e ensinar a compreensão é fazer com que o sujeito se perceba como parte constitutiva dessa sociedade complexa, que não cabe em uma gaiola, capacitando o sujeito a enfrentar com autonomia suas incertezas a partir de um olhar para dentro de si mesmo, reconhecendo-se, tanto os professores quanto os estudantes, como protagonistas na dinâmica que envolve o viver e o aprender nos espaços de formação formalmente reconhecidos. É condição necessária para a construção da identidade profissional docente, humana e terrena, mantida e transformada na alteridade, possibilitada pelo diálogo (BUBER, 1987, 2001), no reconhecimento de si mesmo e do outro, de sua identidade a partir de sua construção histórico-cultural como sujeito pertencente a essa humanidade caminhante. É importante reforçar que o exercício da dialogicidade vai além do encontro do sujeito com os saberes apreendidos nos espaços educativos formais. Constitui-se em um processo de retomada da voz interior dos sujeitos, do respeito aos seus valores construídos em comunidade e aos seus saberes produzidos, pois o diálogo é o propulsor e a conexão para o estabelecimento do elo entre o sujeito e a vida. De acordo com Freire (1987), é a essência constitutiva da educação como prática da liberdade, com responsabilidade, mesmo que relativa. A criação e o fortalecimento de espaços para o exercício da dialogicidade ampliam as possibilidades de os sujeitos vivenciarem momentos de aprendizagens com mais solidariedade, constituindo-se e desenvolvendo-se humanamente, de forma respeitosa na dinâmica da formação de professores. Este processo é fundamental para a ocorrência de momentos de construção, desconstrução e reconstrução de significância e ressignificação, de produção de novas zonas de sentido, mediante relacionamentos saudáveis com o outro e os outros, legitimados pela presença do sujeito e da percepção de sua importância para a constituição de si mesmo, na coletividade, a ser processada e desenvolvida autopoieticamente. A formação de professores compreendida e desenvolvida nessa perspectiva da dialogicidade reveste-se da autopoiese, cujo pressuposto de sua dinâmica visa aproximar relacionalmente sujeito e vida, pois no processo construtivo do Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 375 conhecimento, o sujeito precisa estar em interação permanente consigo, com o outro e com o meio cultural. Em plena congruência e coerência com as próprias ideias, sem deixar perder seus valores inerentes. As formas de sentir e ser (dialógico), pensar e saber (dialético) e de atuar e agir (dialogicidade) do sujeito perante a vida, pelo próprio jeito de apreender a realidade e de transformar o fazer, ao mesmo tempo em que o ser vai se reconstruindo, é processo que envolve um conjunto de relações complexas apreendidas e ressignificadas cotidianamente no decurso do viver/conviver. Estas atitudes são expressões da subjetividade, traduzidas como a síntese da singularidade e da individualidade humana construída e constituída no decurso da vida social e cultural, da dialogicidade estabelecida entre a subjetividade social (saberes intersubjetivos) e a subjetividade individual (saberes intra-subjetivos). Esta seria a fonte criativa em que fluem as ideias, as emoções, a identidade do sujeito, sua marca singular que o identifica e que o faz ser igual em meio às diferenças, e ser respeitado em sua pluralidade cultural e singular, enquanto cidadão participante de sua comunidade local/global. Considerações finais As instituições formadoras (públicas e privadas) ainda são os espaços formativos favoráveis para os atuais e futuros professores aprenderem e se desenvolverem em níveis de conhecimento, construírem e se constituírem mediante a apropriação da cultura em circulação. No entanto, requer dos próprios membros dessas comunidades que sejam sujeitos ativos, e em conjunto com seus pares, busquem as condições necessárias para que os processos de desenvolvimento ocorram na relação uns com os outros, respeitando-se a realidade situada. Engajados nas ações educativas, professores e estudantes, pais, direção, etc., permanentemente em relação, sintam-se corresponsáveis pelo crescimento de sua comunidade e coautores da cultura que os constituirá. Nos espaços formativos compreendidos dessa forma, as relações e os processos educativos se alternam e se alteram, pois o aprender e o ensinar requerem envolvimento, motivos para um agir dos docentes, dos discentes e de todos os Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 376 ARAÚJO, L.; SÍVERES, L. envolvidos, direta ou indiretamente, no convívio educacional. Além disso, é necessário estar atento aos interesses dos professores em formação, para que estes também estejam atentos aos interesses dos seus estudantes, identificando os seus problemas pertinentes e seus desejos. Isto, que exige sensibilidade por parte, também, dos responsáveis pelos processos formativos, sensibilidade para com a percepção do outro ao olhar o mundo, ao seu agir, pois dependendo das circunstâncias, estas podem limitar ou potencializar os sujeitos do ensino e da aprendizagem a seguirem em busca de alternativas que lhe possibilite criar para transpor o problema emergente. Daí a importância de o(a) professor(a) — com consciência epistemológica — ser capaz de criar as condições necessárias para os sujeitos ascenderem em termos de conhecimento, abrindo-lhes possibilidades de caminhos, brechas para a apropriação dos bens culturais em relação ao zeitgeist (espírito de época), e de ajudálos a transformar os saberes (as informações apreendidas) em conhecimentos pertinentes, com significância e significado, e a construir e reconstruir novas “zonas de produção de sentidos” em relação ao conhecimento, para que possam alcançar as melhores soluções e alternativas para a superação dos problemas de sua comunidade educativa, em coletividade. Mudar a cultura dos cursos de formação de professores, há tempos habituada com as formas convencionais de monólogos e discurso unilateral impregnados há séculos, não é tão simples assim. Requer coragem e muita disposição por parte de todo o corpo gestor escolar para superar a repetição de modelos e práticas já conhecidos. A reorganização do espaço pedagógico, com foco na valorização das histórias de vida dos sujeitos e de suas relações de aprendizagem, tendo o diálogo como princípio fundante e regenerador, constitui uma das vias possíveis para propiciar as condições necessárias de gestão, valorizando-se os pensamentos e ações, para que possibilitem a concretude do fazer pedagógico revolucionário e transformador. Assim, uma caminhada educativa construída e demarcada por um constante processo de reflexão, de escuta e compartilhamento de significados, por meio de ações concretas, ainda que certo do risco que a incerteza das escolhas pode despertar e desenvolver os princípios fundantes do diálogo: o ouvir, o compartilhar e o agir. Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 A perspectiva dialógica na formação de professores 377 Portanto, estar articulado pelos três fios da trança dos saberes que envolvem o ensinar a condição humana, o enfrentamento das incertezas e o ensinamento da compreensão — compreendidos a partir das dimensões: antropológica, epistemológica e pedagógica — suscita a condição de criar e recriar as utopias, abertura de possibilidades de produção de conhecimentos pertinentes, e percorrer caminhos inovadores na docência. Todo este procedimento mesclado pelo diálogo pode possibilitar relações humanas mais significativas, e promover processos de formação de professores numa perspectiva mais dialógica e solidária, mais saudável e humanizadora. Referências BARDIN, L. Análise de conteúdo. Lisboa: Edições 70, 2002. BOFF, L. A águia e a galinha: uma metáfora da condição humana. Petrópolis: Vozes, 1997. BOHM, D. Diálogo: comunicação e redes de convivência. São Paulo: Palas Athena, 2005. BUBER, M. Sobre comunidade. São Paulo: Perspectiva, 1987. BUBER, M. Eu e Tu. 8. ed. São Paulo: Centauro, 2001. D'AMBRÓSIO, U. Transdisciplinaridade. 2. ed. São Paulo: Palas Athena, 2009. FREIRE, P. Pedagogia do Oprimido. 17. ed. Rio de Janeiro: Paz e Terra, 1987. FREIRE, P. Pedagogia da Autonomia: saberes necessários à prática educativa. 3. ed. São Paulo: Paz e Terra, 1996. HABERMAS, J. Teoria do agir comunicativo. Racionalidade da ação e racionalização social. Trad. Paulo Astor Soethe. São Paulo: WMF Martins Fontes, 2012. LARAIA, R. Cultura: um conceito antropológico. Rio de Janeiro: Zahar, 1986. LEMOS, A. Cibercultura, tecnologias e vida social na cultura contemporânea. Porto Alegre: Sulina, 2004. MARIOTTI, H. Pensamento complexo: suas aplicações à liderança, à aprendizagem e ao desenvolvimento sustentável. São Paulo: Atlas, 2007. MATURANA, H.; VARELA, F. A árvore do conhecimento: as bases biológicas da compreensão humana. 5. ed. São Paulo: Palas Athena, 2001. MORIN, E. Ciência com consciência. 3. ed. Rio de Janeiro: Bertrand Brasil, 1999. Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020 378 ARAÚJO, L.; SÍVERES, L. MORIN, E. Da necessidade de um pensamento complexo. In: MARTINS, F. M; SILVA, J. M. Para navegar no século XXI: tecnologias do imaginário e cibercultura. Porto Alegre: Sulina/Edipucrs, 2000. p. 19-42. MORIN, E. Introdução ao pensamento complexo. 3. ed. Porto Alegre: Sulina, 2007. MORIN, E. A cabeça bem-feita: repensar a reforma, reformar o pensamento. 15. ed. Rio de Janeiro: Bertrand Brasil, 2008. MORIN, E. Os sete saberes necessários à educação do futuro. 2. ed. Brasília: Unesco, 2010. MORIN, E. Ensinar a viver: manifesto para mudar a educação. Porto Alegre: Sulina, 2015. NÓVOA, A. Sete disposições necessárias ao educador do presente. In: Conferência Internacional: Saberes para uma cidadania planetária. (palestra proferida), Fortaleza (CE), 27 de maio de 2016. Disponível em: <https://vimeo.com/169804242>. Acesso em: 25 nov. 2018. UNESCO. Conferência Internacional: Saberes para uma cidadania planetária. 2016. Disponível em: <http://www.unesco.org/new/pt/brasilia/about-this-office/singleview/news/international_conference_discusses_education_humanism_ethi/>. Acesso em: 23 maio 2016. RECEBIDO: 21/06/2019 APROVADO: 05/02/2020 RECEIVED: 06/21/2019 APPROVED: 02/05/2020 RECIBIDO: 21/06/2019 APROBADO: 05/02/2020 Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
https://openalex.org/W2006843710
https://ccforum.biomedcentral.com/track/pdf/10.1186/cc8093
English
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T-cell-specific peroxisome proliferator-activated receptor gamma depletion inhibits T-cell apoptosis and improves survival of septic mice via an IL-2-dependent mechanism
Critical care
2,009
cc-by
29,290
P2 Methods Pneumonia was induced by intranasal instillation of bacteria (104 CFU) while sepsis was developed by placing the fibrin–thrombin clot containing a known amount of bacteria (102 CFU) into the peritoneal cavity of animals. Various cytokine (TNFα and IL-1α) levels were estimated using ELISA and the degree of lung inflammation (that is, inflammatory cell infiltration) was evaluated by histopathological analysis. The other markers of inflammation (that is, nitric oxide (NO), malondialdehyde (MDA) and myeloperoxidase (MPO)) were estimated by standard biochemical methods. Introduction Urokinase receptor (uPAR, CD87), a glycosylphos- phatidylinositol-anchored protein, is considered to play an important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in South- east Asia. We here investigated the expression and function of uPAR both in patients with septic melioidosis and in a murine model of experimental melioidosis. Introduction Urokinase receptor (uPAR, CD87), a glycosylphos- phatidylinositol-anchored protein, is considered to play an important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in South- east Asia. We here investigated the expression and function of uPAR both in patients with septic melioidosis and in a murine model of experimental melioidosis. Results Mice with sepsis showed 100% mortality within 5 post infection days whereas all the animals with pneumonia survived. In animals suffering from K. pneumoniae B5055-induced pneumonia all the inflammatory parameters (TNFα, IL-1α, MPO, MDA and NO) were found to be maximum until the third post infection day, after that a decline was observed, whereas in septic animals all the above-mentioned markers of inflammation kept on increasing until death of the animals. Histopathological study showed that inflammatory damage to the lungs in pneumonia was not very severe as lesser neutrophil infiltration and pulmonary damage (that is, alveolitis, bronchiolitis, endothelitis and perivascular congestion) was seen as compared with lungs taken from septic animals. This can be further strengthened by the presence of alternatively activated alveolar macrophages (AAMacs) or foam cells in lungs of mice with pneumonia after the third post infection day and their number kept on increasing until the seventh post infection day, which might have contributed to the induction of resolution of inflammation and clearance of the infection. P2 Urokinase receptor is necessary for bacterial defense against Gram-negative sepsis (melioidosis) by facilitating phagoctytosis A comparison of acute lung inflammation in Klebsiella pneumoniae B5055-induced pneumonia and sepsis in BALB/c mice phagoctytosis W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2, JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6, SJ Peacock5,6, T van der Poll1,2 1Center for Infection and Immunity Amsterdam, 2Center for Experimental and Molecular Medicine, 3Department of Vascular Medicine and 4Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2, JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6, SJ Peacock5,6, T van der Poll1,2 1Center for Infection and Immunity Amsterdam, 2Center for Experimental and Molecular Medicine, 3Department of Vascular Medicine and 4Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) V Kumar, S Chibber Department of Microbiology, Panjab University, Chandigarh, India Critical Care 2009, 13(Suppl 4):P2 (doi: 10.1186/cc8058) W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2, JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6, SJ Peacock5,6, T van der Poll1,2 1Center for Infection and Immunity Amsterdam, 2Center for Experimental and Molecular Medicine, 3Department of Vascular Medicine and 4Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) Introduction Lungs play an important role in the body’s defense against a variety of pathogens, but this network of immune system mediated defense can be deregulated during acute pulmonary infections. Objective The present study compares the acute lung inflam- mation (ALI) occurring during Klebsiella pneumoniae B5055- induced pneumonia and sepsis in BALB/c mice. Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Amsterdam, the Netherlands, 11–14 November 2009 Published online: 11 November 2009 These abstracts are available online at http://ccforum.com/supplements/13/S4 © 2009 BioMed Central Ltd Published online: 11 November 2009 These abstracts are available online at http://ccforum.com/supplements/13/S4 © 2009 BioMed Central Ltd P1 Urokinase receptor is necessary for bacterial defense against Gram-negative sepsis (melioidosis) by facilitating phagoctytosis W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2, JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6, SJ Peacock5,6, T van der Poll1,2 1Center for Infection and Immunity Amsterdam, 2Center for Experimental and Molecular Medicine, 3Department of Vascular Medicine and 4Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) P3 Performance evaluation and further development of the PCR and microarray-based Prove-it™ Sepsis assay P3 Methods DNA was extracted from blood cultures using the automated DNA extraction instrument easyMAG (bioMérieux) prior to the Prove-it™ Sepsis assay. Conventional blood culture was conducted in parallel and results were only revealed for comparison at the statistical analysis stage. Discordant results were studied by DNA sequencing and case-by-case review of original microbiology laboratory data. P Tissari1, E Tarkka1, S Mero1, L Savolainen1, M Vaara1, A Zumla2, J Huggett2, C Carder2, V Gant2, A Aittakorpi3, S Laakso3, M Lindfors3, P Piiparinen3, N Kumlin3, H Piiparinen3, M Mäki3 P Tissari1, E Tarkka1, S Mero1, L Savolainen1, M Vaara1, A Zumla2, J Huggett2, C Carder2, V Gant2, A Aittakorpi3, S Laakso3, M Lindfors3, P Piiparinen3, N Kumlin3, H Piiparinen3, M Mäki3 Results Of the analyzed 3,318 blood cultures, 2,107 yielded a pathogen by conventional techniques. Of these, 302 samples contained microbes not covered by Prove-it™ Sepsis, and an additional 137 cultures contained more than one organism. Sensitivity and specificity for Prove-it™ Sepsis were 94.7% and 98.7%, respectively. Of particular significance was the assay’s faultless ability to differentiate MRSA from MSSA and from CNS. Furthermore, it provided results on average 1 day earlier than reference methods. Introduction The Prove-it™ Sepsis assay is a rapid, broad-range PCR and microarray-based assay designed to identify the majority of sepsis-causing bacteria from positive blood cultures. The pathogen panel covers 50 Gram-negative and Gram-positive bacterial species (Table 1). It also reports methicillin resistance by detecting the mecA gene. The assay time is 3 hours. Our objective was to conduct a performance evaluation study for Prove-it™ Sepsis according to the EN 13612-standard (Performance evaluation of in vitro diagnostic medical devices) and to compare obtained results with those of current culture-based diagnostics. We evaluated the sensitivity, specificity and time to result of Prove- it™ Sepsis in two major teaching hospitals in Helsinki and London. Materials A total of 3,318 blood samples from patients with suspected sepsis were collected. Blood culture bottles of BacT/ALERT 3D (bioMérieux) and BACTEC 924 (Becton Dickinson) were incubated for a total of 6 days or until flagged as positive. Conclusions Prove-it™ Sepsis was considered to be a fast, robust, and high-performance diagnostic platform, which is easily implemented into everyday laboratory workflow. Both study sites identified cases where timely information provided by Prove-it™ Sepsis would have significantly improved patient management. P3 Performance evaluation and further development of the PCR and microarray-based Prove-it™ Sepsis assay Examples include more rational management and antibiotic choice subsequent to earlier differentiation of Gram-positive cocci in clumps into MRSA, MSSA, or CNS, and earlier speciation of Gram-negative organisms. Prove-it™ Sepsis is further configured for detection of Candida spp. and new bacterial targets. The assay now identifies 60 out of the 302 samples not covered during the evaluation, increasing the pathogen coverage from 86% to 89%. The earlier speciation provided by Prove-it™ Sepsis could contribute to faster, more evidence-based patient management and, thus, positive outcomes. Methicillin resistance marker mecA P2 But no such AAMacs or foam cells were seen in lungs of septic mice on histo- pathological examination, lungs were seen to be infiltrated with only neutrophils on all experimental days. Methods Using a translational approach we conducted a patient study in patients with culture-confirmed sepsis caused by B. pseudomallei, in vitro experiments using wild-type (WT) and uPAR knockout (KO) cells, and mouse studies using WT and uPAR KO mice inoculated with B. pseudomallei. Results uPAR mRNA and surface expression was increased in patients with septic melioidosis in/on both peripheral blood monocytes and granulocytes as well as in the pulmonary compartment during experimental pneumonia-derived melioidosis in mice. uPAR-deficient mice intranasally infected with B. pseudomallei showed an enhanced growth and dissemination of B. pseudomallei when compared with WT mice, corresponding with increased pulmonary and hepatic inflammation. uPAR KO mice demonstrated significantly reduced neutrophil migration towards the pulmonary compartment after inoculation with B. pseudomallei. Further in vitro experiments showed that uPAR- deficient macrophages and granulocytes display a markedly impaired phagocytosis of B. pseudomallei. Additional studies showed that uPAR deficiency did not influence hemostatic and fibrinolytic responses during severe melioidosis. Conclusions These data suggest that uPAR is crucially involved in the host defense against sepsis caused by B. pseudomallei by facilitating the migration of neutrophils towards the primary site of infection and subsequently facilitating the phagocytosis of B. pseudomallei. Conclusions Hence, during pneumonia controlled activation of AAMacs or foam cells led to the resolution of inflammation and infection as well. S1 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 P3 Performance evaluation and further development of the PCR and microarray-based Prove-it™ Sepsis assay P Tissari1, E Tarkka1, S Mero1, L Savolainen1, M Vaara1, A Zumla2, J Huggett2, C Carder2, V Gant2, A Aittakorpi3, S Laakso3, M Lindfors3, P Piiparinen3, N Kumlin3, H Piiparinen3, M Mäki3 1Division of Clinical Microbiology, Helsinki University Hospital Laboratory HUSLAB, Helsinki, Finland; 2Department of Clinical Microbiology, University College London Hospitals NHS Foundation Trust, and University College London Medical School, Centre for Infectious Diseases and International Health, London, UK; 3Mobidiag Ltd, Helsinki, Finland Critical Care 2009, 13(Suppl 4):P3 (doi: 10.1186/cc8059) P5 Use of a screening authorization and randomization center for severe sepsis patient qualification and real-time enrollment in a phase 2 trial of eritoran tetrasodium (E5564), a TLR4 antagonist Objective The aim of this study was to evaluate the efficacy of eritoran for interaction effects with baseline illness severity. Methods Prospective covariates from a randomized, double-blind, placebo-controlled, phase 2 trial were analyzed for treatment interaction measured by 28-day mortality. Breslow–Day and multiple logistic regression (LR) were used to assess categorical (CAT) and continuous (CONT) treatment by severity-of-illness interactions. J Schentag1, S Opal2, M Lynn3, A Wittek3, J Wheeler3 1University at Buffalo School of Pharmacy and Pharmaceutical Sciences, Buffalo, NY, USA; 2Brown University Medical School, Providence, RI, USA; 3Eisai Medical Research, Inc., Ridgefield Park, NJ, USA Critical Care 2009, 13(Suppl 4):P5 (doi: 10.1186/cc8061) Results Modified intent-to-treat population (n = 292) all-cause 28- day mortality was: placebo, 33.3% (32/96); total eritoran 45 mg/105 mg, 29.6% (58/196). LR analysis identified Acute Physiology and Chronic Health Evaluation (APACHE) II scores, Predicted Risk of Mortality (PROM) scores, IL-6, age, sex, race, and eritoran as associated with survival outcomes. Significant treatment interactions were observed (eritoran vs. placebo) for baseline covariates: APACHE II (CAT, P = 0.059; CONT, P = 0.035); PROM scores (CAT, P = 0.028; CONT, P = 0.008); number of organ failures (CAT, P = 0.079); international normalized ratio (CAT, P = 0.05); and acute physiology score (CONT, P = 0.039). No significant treatment interactions were observed with age, sex, shock, DAA use, infection site, microorganism type, platelets, IL-6, or endotoxin levels. Interaction results were similar for eritoran 105 mg only versus placebo. Introduction Trials of many promising sepsis modifiers have often failed to demonstrate benefits because of, among other reasons, poor characterization of enrolled patients. Materials Advantages of utilizing a screening authorization and randomization center (SAC) method to characterize patients in trials for sepsis modifiers are presented. Methods A central SAC on call 24 hours per day was employed in a phase 2, double-blind, randomized comparison of eritoran 45 and 105 mg versus placebo. SAC activities were conducted (January 2002 to April 2005) by six clinical pharmacists. Severe sepsis was defined with at least three systemic inflammatory response syndrome criteria within 12 hours before onset of ≥1 new organ dysfunction. Patients were randomized and treated within 8 to 12 hours. Nandrolone abuse aggravates septic shock YF Hsu1, C Lin1,2, D-R Chen1,2 1Department of Research, and 2Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan Critical Care 2009, 13(Suppl 4):P4 (doi: 10.1186/cc8060) Results The SAC screened 1,025 patients from 78 sites; 300 patients from 54 sites were randomized, and 293 were treated. The Independent Clinical Evaluation Committee subsequently qualified 229/293 (78%). Calls to the SAC averaged 24 patients/ month; eight patients/month were randomized (33%). Enrollments ranged from 0 to 39 patients/site; 35 sites randomized ≥3 patients; nine sites randomized ≥10 patients. Of 35 sites with ≥3 patients, 6 to 100% led to randomization. Introduction One million individuals in the United States alone are estimated to be current or past users of anabolic-androgenic steroid. Methods To investigate the effects of nandrolone, an anabolic- androgenic steroid, 108 6-week-old male BALB/c inbred mice were used (minimal lethal dose n = 30, high-dose vs. low-dose n = 24, screening surrogates n = 6, and surrogates n = 48). Mice were sacrificed at 0, 3 or 6 hours after septic shock induction. The serum levels of malondialdehyde, liver TNFα and spleen IFNγ in mice with septic shock were analyzed. The gene expression of insulin-like growth factor 1, insulin-like growth factor type 1 receptor and insulin-like growth factor binding proteins was also studied. Conclusions Advantages of utilizing the SAC included a high evaluable rate of enrolled patients, correct Predicted Risk of Mortality calculations, timing of qualifying organ failures, drug preparation advice, verification of key clinical data, and eligibility with sponsor prior to enrollment. SAC activities ensured an informative phase 2 trial and will be utilized for the phase 3 trial. Results Nandrolone significantly increased serum malondialde- hyde at 0, 3 and 6 hours (P = 0.004, 0.006 and 0.004), and liver TNFα at 0 and 6 hours (P = 0.04 and 0.016). It also increased the spleen IFNγ level at 0 and 6 hours (P = 0.031 and 0.01). Com- pared with 0 hours, the data indicated that nandrolone increases lung insulin-like growth factor type 1 receptor, insulin-like growth factor binding protein 1 and insulin-like growth factor binding protein 2 mRNA expression at 6 hours (P <0.05). These indicated changes due to nandrolone. Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 based on Acute Physiology and Chronic Health Evaluation (APACHE) II predicted mortality to yield a balanced allocation of high and low APACHE II predicted mortality within the three treatment groups: one sequence for subjects with APACHE II predicted mortality 20 to 50%, and another for subjects with predicted mortality 51 to 80% as calculated by the SAC. Results The SAC screened 1,025 patients from 78 sites; 300 patients from 54 sites were randomized, and 293 were treated. The Independent Clinical Evaluation Committee subsequently qualified 229/293 (78%). Calls to the SAC averaged 24 patients/ month; eight patients/month were randomized (33%). Enrollments ranged from 0 to 39 patients/site; 35 sites randomized ≥3 patients; nine sites randomized ≥10 patients. Of 35 sites with ≥3 patients, 6 to 100% led to randomization. Conclusions Advantages of utilizing the SAC included a high evaluable rate of enrolled patients, correct Predicted Risk of Mortality calculations, timing of qualifying organ failures, drug preparation advice, verification of key clinical data, and eligibility with sponsor prior to enrollment. SAC activities ensured an informative phase 2 trial and will be utilized for the phase 3 trial. P6 Influence of severity of illness on the effects of eritoran tetrasodium (E5564), a TLR4 antagonist, in patients with severe sepsis SM Opal1, AC Kalil2, SP LaRosa1, J Gogate3, M Lynn3, AE Wittek3, and the Eritoran Sepsis Study Group 1Brown University, Providence, RI, USA; 2University of Nebraska Medical Center, Omaha, NE, USA; 3Eisai Medical Research, Inc., Ridgefield Park, NJ, USA Critical Care 2009, 13(Suppl 4):P6 (doi: 10.1186/cc8062) P4 based on Acute Physiology and Chronic Health Evaluation (APACHE) II predicted mortality to yield a balanced allocation of high and low APACHE II predicted mortality within the three treatment groups: one sequence for subjects with APACHE II predicted mortality 20 to 50%, and another for subjects with predicted mortality 51 to 80% as calculated by the SAC. based on Acute Physiology and Chronic Health Evaluation (APACHE) II predicted mortality to yield a balanced allocation of high and low APACHE II predicted mortality within the three treatment groups: one sequence for subjects with APACHE II predicted mortality 20 to 50%, and another for subjects with predicted mortality 51 to 80% as calculated by the SAC. Nandrolone abuse aggravates septic shock P6 Influence of severity of illness on the effects of eritoran tetrasodium (E5564), a TLR4 antagonist, in patients with severe sepsis SM Opal1, AC Kalil2, SP LaRosa1, J Gogate3, M Lynn3, AE Wittek3, and the Eritoran Sepsis Study Group 1Brown University, Providence, RI, USA; 2University of Nebraska Medical Center, Omaha, NE, USA; 3Eisai Medical Research, Inc., Ridgefield Park, NJ, USA Critical Care 2009, 13(Suppl 4):P6 (doi: 10.1186/cc8062) Influence of severity of illness on the effects of eritoran tetrasodium (E5564), a TLR4 antagonist, in patients with severe sepsis Influence of severity of illness on the effects of eritoran tetrasodium (E5564), a TLR4 antagonist, in patients with severe sepsis SM Opal1, AC Kalil2, SP LaRosa1, J Gogate3, M Lynn3, AE Wittek3, and the Eritoran Sepsis Study Group 1Brown University, Providence, RI, USA; 2University of Nebraska Medical Center, Omaha, NE, USA; 3Eisai Medical Research, Inc., Ridgefield Park, NJ, USA Critical Care 2009, 13(Suppl 4):P6 (doi: 10.1186/cc8062) Conclusions Nandrolone abuse hastens mortality due to septic shock and increases serum malondialdehyde, liver TNFα, spleen IFNγ level and lung insulin-like growth factor type 1 receptor mRNA, as well as lung insulin-like growth factor binding proteins. Nandrolone abuse may aggravate septic shock. Introduction Disease severity varies widely in patients with severe sepsis. Previous trials (IL-1RA, TNF-sR p55, antithrombin, and drotrecogin alfa activated (DAA)) suggest that more severely ill patients benefit most from treatment. Table 1 (abstract P3) Bacteria and an antibacterial resistance marker identified by the Prove-it™ Sepsis assay Gram-negative Gram-positive Antibacterial resistance Neisseria meningitidis Staphylococcus aureus Methicillin resistance marker mecA Enterobacter aerogenes Staphylococcus epidermidis Enterobacter cloacae Coagulase-negative Staphylococcusd Escherichia coli Streptococcus pyogenes Klebsiella oxytoca Streptococcus agalactiae Klebsiella pneumoniae Streptococcus dysgalactiae subsp. equisimilis Proteus mirabilis Streptococcus pneumoniae Proteus vulgaris Enterococcus faecalis Salmonella enterica subsp. entericaa Enterococcus faecium Serratia marcescens Listeria monocytogenes Enterobacteriaceae familyb Clostridium perfringens Acinetobacter baumannii Pseudomonas aeruginosa Stenotrophomonas maltophilia Haemophilus influenzae Campylobacter jejuni/coli Bacteroides fragilis groupc aSalmonella enterica subsp. enterica covers at least the following serovars: Enteritidis, Oranienburg, Othmarschen, Paratyphi, Stanley, Typhi, Typhimurium, Virchow, Group A, B, C, D. bEnterobacteriaceae covers at least the following species: Citrobacter amalonaticus, Citrobacter freundii, Citrobacter koseri, Citrobacter braakii, Enterobacter hormaechei, Enterobacter sakazakii, Kluyvera intermedia, Morganella morganii, Pantoea agglomerans, Providencia rettgeri, Providencia stuartii, Yersinia enterocolitica, Yersinia pseudotuberculosis. cBacteroides fragilis covers at least the following species: B. fragilis, B. vulgatus, B. thetaiotaomicron. dCoagulase-negative Staphylococcus covers at least the following species: S. capitis, S. lugdunensis, S. haemolyticus, S. hominis, S. saprophyticus, S. warneri, S. xylosus. Bacteria and an antibacterial resistance marker identified by the Prove-it™ Sepsis assay aSalmonella enterica subsp. enterica covers at least the following serovars: Enteritidis, Oranienburg, Othmarschen, Paratyphi, Stanley, Typhi, Typhimurium, Virchow, Group A, B, C, D. bEnterobacteriaceae covers at least the following species: Citrobacter amalonaticus, Citrobacter freundii, Citrobacter koseri, Citrobacter braakii, Enterobacter hormaechei, Enterobacter sakazakii, Kluyvera intermedia, Morganella morganii, Pantoea agglomerans, Providencia rettgeri, Providencia stuartii, Yersinia enterocolitica, Yersinia pseudotuberculosis. cBacteroides fragilis covers at least the following species: B. fragilis, B. vulgatus, B. thetaiotaomicron. dCoagulase-negative Staphylococcus covers at least the following species: S. capitis, S. lugdunensis, S. haemolyticus, S. hominis, S. saprophyticus, S. warneri, S. xylosus. S2 P7 Introduction Unregulated elevated levels of serum TNFα have been associated with proinflammatory cytokine cascades that are characteristic in diseases such as septic shock. Endotoxic shock, which has a poorer prognosis than found with other forms of septic shock, is mediated by lipopolysaccharide (LPS), a molecule that is released from the outer membrane of Gram-negative bacteria. LPS is a potent stimulator of TNFα secretion by serum monocytes and tissue macrophages. Whilst the use of monotherapeutic TNFα antagonists has been trialed, none have been registered for use in patients with sepsis. Introduction Unregulated elevated levels of serum TNFα have been associated with proinflammatory cytokine cascades that are characteristic in diseases such as septic shock. Endotoxic shock, which has a poorer prognosis than found with other forms of septic shock, is mediated by lipopolysaccharide (LPS), a molecule that is released from the outer membrane of Gram-negative bacteria. LPS is a potent stimulator of TNFα secretion by serum monocytes and tissue macrophages. Whilst the use of monotherapeutic TNFα antagonists has been trialed, none have been registered for use in patients with sepsis. Faster differentiation of Staphylococcus aureus versus coagulase-negative Staphylococci from blood culture material: a comparison of different bacterial DNA isolation methods AJM Loonen1, WLJ Hansen1, A Jansz2, H Kreeftenberg2, CA Bruggeman1, PFG Wolffs1, AJC van den Brule3 1Department of Medical Microbiology, Maastricht University Medical Center, Maastricht, the Netherlands; 2Department of Intensive Care, Catharina Hospital, Eindhoven, the Netherlands; 3Department of Molecular Diagnostics, Catharina Hospital, Eindhoven, the Netherlands Critical Care 2009, 13(Suppl 4):P7 (doi: 10.1186/cc8063) Objective The purpose of this study was to test the effect of canine hyperimmune frozen plasma (HFP), which is known to contain elevated levels of soluble TNFα receptor 1 (sTNFR1), on TNFα and inflammatory cell levels in a LPS-mediated rat air pouch model of inflammation. Introduction Frequent usage of medical devices, such as intravenous lines, often results in sepsis, which is characterized by high morbidity and mortality. Rapid and reliable detection and differentiation between Staphylococcus aureus and coagulase- negative Staphylococci (CNS) is therefore clinically relevant to be able to provide adequate early treatment. Blood culture is still the gold standard method in identifying these pathogens but is time consuming. Molecular diagnostics might be a promising alternative to reduce this time-to-result delay. Introduction Frequent usage of medical devices, such as intravenous lines, often results in sepsis, which is characterized by high morbidity and mortality. Clinical impact of a PCR-based assay for pathogen detection in critically ill patients with evidence of infection F Bloos1, A Kortgen1, S Sachse2, M Lehmann3, E Straube2, K Reinhart1, M Bauer1 1Department of Anesthesiology and Intensive Care Medicine, University Hospital Jena, Germany; 2University Hospital Jena, Institute of Medical Microbiology, Jena, Germany; 3SIRS-Lab GmbH, Jena, Germany Critical Care 2009, 13(Suppl 4):P9 (doi: 10.1186/cc8065) Conclusions A sensitive RT-PCR was developed for detection and differentiation of S. aureus versus CNS. Bacterial DNA isolation from Bact/ALERT blood culture material seems to show better reproducibility compared with isolation from BACTEC blood culture material. In this preliminary study the EasyMAG performed better when compared with MolYsis Plus and the MagNA Pure system. In future work this method will be further evaluated with reduced culture times. Introduction Blood cultures are often negative even in patients with clinical signs of severe sepsis. Furthermore, the long time to result of culture-based methods does not allow the results to guide empiric antimicrobial therapy. PCR-based pathogen detection promises a higher rate of positivity and a faster time to result. Introduction Blood cultures are often negative even in patients with clinical signs of severe sepsis. Furthermore, the long time to result of culture-based methods does not allow the results to guide empiric antimicrobial therapy. PCR-based pathogen detection promises a higher rate of positivity and a faster time to result. Objective To report the performance of PCR-based pathogen detection compared with blood culture in ICU patients with evidence of infection, and the impact of this test on the antimicrobial therapy. Methods Patients treated on an interdisciplinary ICU were included into this observational study if a blood culture (BC) was Introduction Blood cultures are often negative even in patients with clinical signs of severe sepsis. Furthermore, the long time to result of culture-based methods does not allow the results to guide empiric antimicrobial therapy. PCR-based pathogen detection promises a higher rate of positivity and a faster time to result. Objective To report the performance of PCR-based pathogen detection compared with blood culture in ICU patients with evidence of infection, and the impact of this test on the antimicrobial therapy. Methods Patients treated on an interdisciplinary ICU were included into this observational study if a blood culture (BC) was P7 The data suggest that canine HFP, which has been demonstrated to contain elevated levels of sTNFR1 compared with FFP, has a direct effect on depressing TNFα levels and neutrophil sequestration in the rat air pouch model of inflammation. These data suggest that HFP may be worthy of further investigation to determine whether such preparations have a therapeutic potential for treatment of acute inflammatory diseases in which TNFα is implicated. Results The best Tuf RT-PCR method appeared to have a sensitivity of 100 CFU/ml. Approximately 50 positive blood cultures containing Gram-positive cocci in clusters were tested in the Tuf RT-PCR and all were identified correctly. Bacterial DNA isolation, from spiked blood culture material, with the EasyMAG showed the highest analytical performance with a detection limit of 103 CFU/ml in Bact/ALERT material, whereas using BACTEC resulted in a detection limit of 104 CFU/ml. Hand-on time, for 26 samples, was lowest for the EasyMAG (10 minutes) and highest for the manual kit of MolZyme (2 hours). Total handling time was highest for the MolYsis Plus kit (3.5 hours) and lowest for the automated extractor EasyMAG (50 minutes). Clinical impact of a PCR-based assay for pathogen detection in critically ill patients with evidence of infection Clinical impact of a PCR-based assay for pathogen detection in critically ill patients with evidence of infection P5 The 8-hour to 12-hour window for qualifi- cation and start of treatment was the primary challenge to the SAC and study sites. Each site used two sequences of drug assignment Conclusions Potential survival benefits of eritoran in severe sepsis patients may be associated with high severity of illness. Treatment by disease severity interaction will be further explored in a phase 3 trial. S3 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 P7 Rapid and reliable detection and differentiation between Staphylococcus aureus and coagulase- negative Staphylococci (CNS) is therefore clinically relevant to be able to provide adequate early treatment. Blood culture is still the gold standard method in identifying these pathogens but is time consuming. Molecular diagnostics might be a promising alternative to reduce this time-to-result delay. Methods A dorsal air pouch in 175 to 200 g Sprague–Dawley rats was formed by 20 ml subcutaneous infusions of sterile air. Prophylactic subcutaneous injections of canine HFP, canine fresh frozen plasma (FFP) or carprofen were administered daily for 3 days into the lateral flank of the right foreleg at doses recom- mended by the manufacturers (n = 10 for each treatment group). Pouch fluid was harvested by syringe at 1, 6, 12, 24 and 48 hours post LPS administration and subjected to histological and cytokine/cytokine receptor analysis. TNFα and sTNFR1 levels were determined by ELISA and an immunofluorescent dot blot assay. Objective This study aims to compare different DNA extraction methods from two commonly used blood culture materials, BACTEC (BD) and Bact/ALERT (Biomerieux), to accelerate differentiation between S. aureus and CNS. Results Pouch fluid analysis: maximal effects were detected at 6 hours post LPS administration. TNFα levels were significantly depressed in animals dosed with HFP, but not in animals treated with FFP or carprofen (P <0.05). sTNFR1 levels were significantly elevated in HFP, but not in FFP or carprofen dosed animals (P <0.05). Neutrophil numbers were significantly depressed in HFP dosed but not in FFP or carprofen treated animals (P <0.05). Methods Two fast real-time PCR duplex test assays, targeting the Tuf gene, to differentiate S. aureus from CNS, were developed in order to select the most sensitive one. This Tuf RT-PCR was used to compare three different DNA isolation methods on two different blood culture systems. Negative blood culture material was spiked with S. aureus; bacterial DNA was isolated with: automated extractor EasyMAG (Biomerieux), automated extractor MagNA Pure (Roche), and a manual kit MolYsis Plus (MolZyme). Conclusions There appears to be a correlation between elevated levels of sTNFR1 and depression of TNFα and neutrophil levels in the pouch fluid of HFP dosed rats (r = –0.73, P <0.0001). P13 Comparison of commercial DNA extraction kits for the detection of bacterial genomic DNA from whole-blood samples using a broad-range PCR B Krulova, E Nemcova, B Zaloudikova, P Nemec, T Freiberger Centre for Cardiovascular Surgery and Transplantation, Molecular Genetic Laboratory, Brno, Czech Republic Critical Care 2009, 13(Suppl 4):P13 (doi: 10.1186/cc8069) Figure 2 (abstract P9) Introduction Blood culture is still considered a gold standard for diagnosis of bloodstream infections. Early pathogen detection is a prerequisite for the successful treatment. Nucleic acid based techniques offer a rapid and sensitive option particularly in blood culture negative samples. Efficient bacterial DNA extraction is crucial for following PCR assays. The aim of this study was to determine the detection limit of bacterial genomic DNA using different extraction protocols. Methods We evaluated five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples (QIAamp DNA Blood Mini kit, UltraClean DNA BloodSpin Kit, Chemagic DNA Blood Kit, ZR Genomic DNAII Kit, NucliSens miniMAG). Whole-blood samples were spiked with Escherichia coli CCM 3988 and Staphylococcus aureus CCM 7111. Tenfold dilution series containing concentrations from 107 to 100 CFU/ml were prepared under sterile conditions and immediately used. A broad-range 16S rDNA end-point PCR was performed for the detection of E. coli and S. aureus DNA. drawn on discretion of the treating physician. Blood cultures and EDTA-blood were taken by sterile venous puncture. The EDTA- blood was processed with a PCR-based assay (VYOO®; SIRS- Lab GmbH, Jena, Germany), which detects a panel of 34 bacterial and six fungal pathogens as well as five antibiotic resistances. Data are given as median and interquartile range. Results The sensitivity of each kit was determined as a minimum rate of CFU providing the positive result in the PCR assay. Our results showed the extraction by the QIAamp DNA Blood Mini Kit (supplemented with enzymatic pre-treatment) as the most efficient and sensitive method. This extraction protocol allowed the reproducible detection of E. coli and S. aureus at concentrations of 103 CFU/ml. All kits showed positive results in samples at concentrations from 107 to 105 CFU/ml. Results Sixty-three patients were included into this study. Age was 68.0 (55.5 to 74.0) years, APACHE II score was 17 (13 to 23), SOFA score at study inclusion was 10.0 (7.50 to 11.0), and ICU mortality was 33.3%. In 54 patients (84.3%) infection was either microbiologically confirmed or clinically proven. P13 The baseline procalcitonin was 2.4 (0.8 to 8.0) ng/ml. Eighty-two pairs of BCs and PCRs have been drawn. Ten (12%) BCs and 30 (36.6%) PCRs were positive (P <0.001). Time to test result was significantly shorter in the PCR than in the BC (Figure 1). Twelve positive PCR results (Figure 2, grey areas) prompted a change in antibiotic or antimicrobial therapy. Conclusions Extraction kits should be capable to recover nucleic acids and remove inhibitors from diverse clinical materials simultaneously. All of the tested kits were able to recover bacterial genomic DNA from whole-blood samples, but the sensitivity of PCR- based detection depends on the DNA extraction protocol used. P8 Effect of canine hyperimmune plasma on TNFα and inflammatory cell levels in a lipopolysaccharide-mediated rat air pouch model of inflammation Objective To report the performance of PCR-based pathogen detection compared with blood culture in ICU patients with evidence of infection, and the impact of this test on the antimicrobial therapy. Methods Patients treated on an interdisciplinary ICU were B Essien, M Kotiw, H Buttler, D Strunin Centre for Systems Biology, University of Southern Queensland, Toowoomba, Queensland, Australia Critical Care 2009, 13(Suppl 4):P8 (doi: 10.1186/cc8064) B Essien, M Kotiw, H Buttler, D Strunin Centre for Systems Biology, University of Southern Queensland, Toowoomba, Queensland, Australia Critical Care 2009, 13(Suppl 4):P8 (doi: 10.1186/cc8064) S4 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P9) P11 P11 Abstract withdrawn Figure 2 (abstract P9) Toll-like receptor 9-dependent gene expression in vivo is regulated by inductive and suppressive networks Toll-like receptor 9-dependent gene expression in vivo is regulated by inductive and suppressive networks Conclusions The PCR-based assay resulted in a considerably higher amount of positive results within a shorter time to result than the BC in this group of high-risk patients with evidence of infection. These data demonstrate that the shorter time to result may guide adjustment of antimicrobial therapy earlier than culture-based methods. Further studies are necessary to prospectively investi- gate the impact of the PCR technique on antimicrobial therapy and infection control. S Klaschik1,2, D Tross2, DM Klinman2 1Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Germany; 2Cancer and Inflammation Program, NCI Frederick, MD, USA Critical Care 2009, 13(Suppl 4):P14 (doi: 10.1186/cc8070) Introduction Synthetic oligodeoxynucleotides (ODN) expressing CpG motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN interact with Toll-like receptor 9 to stimulate an innate immune response characterized by the production of Th1 and proinflammatory cytokines, and the functional maturation of P10 y y g g p Materials cDNA was generated from total RNA isolated from spleen cells of mice 30 minutes to 3 days after in vivo treatment with 400 μg CpG or control ODN. Methods Dual-color hybridizations (Biomicro) were performed on murine genome microarrays (NCI). Analyses were conducted on four independently derived RNA samples for each time point. R2 was 0.90 ± 0.04 for all matched samples. Network analysis was performed using Ingenuity Pathway Analysis. Results Differential gene activation (P <0.00001) was observed within 30 minutes of CpG ODN treatment (25 genes), peaked at 3 hours (529 upregulated genes), and fell to near background levels after 72 hours. TNFα, IL-1β, NF-κB and IFNγ played central roles in upregulating the early expression of immune-related genes. Of interest, two distinct patterns of gene expression were observed. One subset of genes was activated shortly after CpG ODN administration and remained upregulated for a prolonged period, while unique subsets of additional genes were activated at specific time points, and were rapidly downregulated. Several genes responsible for this downregulation (MYC, IL1RN, SOCS1) were identified. 1. Engel C, Brunkhorst FM, Bone HG, Brunkhorst R, Gerlach H, Grond S, Gruendling M, Huhle G, Jaschinski U, John S, Mayer K, Oppert M, Olthoff D, Quintel M, Ragaller M, Rossaint R, Stuber F, Weiler N, Welte T, Bogatsch H, Hartog C, Loeffler M, Reinhart K: Epidemiology of sepsis in Germany: results from a national prospective multicenter study. Intensive Care Med 2007, 33:606-618. Conclusions This analysis identifies two distinct patterns of gene regulation associated with CpG-induced activation of the innate immune system of mice. A small number of regulatory genes triggers the patterned upregulation of immune related genes from 30 minutes through 72 hours. A separate set of downregulatory genes subsequently dampens what would otherwise be a continuous positive feedback loop. P16 Identification of cathepsin G in the generation of elastase- resistant fragment of vascular endocan: involvement in the regulation of LFA-1-dependent cascade N De Freitas Caires1,2,3, M Barrier1,2,3, S Sarrazin4, F Depontieu1,2,3, H Ghamlouch1,2,3, W Morelle5, H Drobecq6, M Delehedde1,2,3, H Lortat-Jacob4, C Duez1,2,3, A Scherpereel1,2,3,7, P Lassalle1,2,3 1Inserm, U774, Lille, France; 2Institut Pasteur de Lille, France; 3Université Lille Nord de France, Lille, France; 4CNRS-CEA-UJF, Institut de Biologie Structurale, Grenoble, France; 5UMR-CNRS 8576, Villeneuve Ascq, France; 6UMR-CNRS 8161, Lille, France; 7CHRU Lille, Hôpital Calmette, Lille, France Critical Care 2009, 13(Suppl 4):P16 (doi: 10.1186/cc8072) N De Freitas Caires1,2,3, M Barrier1,2,3, S Sarrazin4, F Depontieu1,2,3, H Ghamlouch1,2,3, W Morelle5, H Drobecq6, M Delehedde1,2,3, H Lortat-Jacob4, C Duez1,2,3, A Scherpereel1,2,3,7, P Lassalle1,2,3 1Inserm, U774, Lille, France; 2Institut Pasteur de Lille, France; 3Université Lille Nord de France, Lille, France; 4CNRS-CEA-UJF, Institut de Biologie Structurale, Grenoble, France; 5UMR-CNRS 8576, Villeneuve Ascq, France; 6UMR-CNRS 8161, Lille, France; 7CHRU Lille, Hôpital Calmette, Lille, France Critical Care 2009, 13(Suppl 4):P16 (doi: 10.1186/cc8072) Fluid therapy in severe sepsis: results from a representative survey of German ICUs Fluid therapy in severe sepsis: results from a representative survey of German ICUs C Hartog1, FM Brunkhorst1, F Bloos1, C Engel2, H Bogatsch3, K Reinhart1, K Sengebusch4, M Ragaller4 1Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Germany; 2Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Germany; 3Clinical Trial Centre Leipzig, University of Leipzig, Germany; 4Department for Anesthesiology and Intensive Care Therapy, University Hospital Carl Gustav Carus, Medical Faculty, TU Dresden, Germany Critical Care 2009, 13(Suppl 4):P15 (doi: 10.1186/cc8071) Introduction The migration of polymorphonuclear neutrophils (PMN) into inflamed tissue requires fine interactions with the endothelial cell surface. The PMN serine proteases cathepsin G (CG), neutrophil elastase (NE) and proteinase 3 (PR3) were originally thought to play a role by the cleavage of endothelial cell proteins that control the PMN firm adhesion and the transendothelial cell migration. However, how these proteases participate in leukocyte adhesion and transmigration remains controversial. Vascular endocan, also called esm1, is a restricted endothelial cell-secreted proteoglycan constituted by a protein core of 20 kDa and by a unique glycosaminoglycan chain of dermatan sulphate (DS). Endocan is preferentially expressed in lung and kidney tissues. Endocan binds to its leukocytic receptor, the LFA-1 integrin, with an affinity of 18 nM, and inhibits the LFA-1–ICAM-1 interactions. Its expression is upregulated by the proinflammatory mediators TNF, IL-1, and lipopolysaccharide. In human sepsis, the serum endocan increases from fivefold to 30-fold the normal value and correlates with bad prognosis. Here, we examined the role of PMN-derived proteases in the degradation of endocan. Introduction Fluid resuscitation is a mainstay of sepsis management. Objective To describe the practice of fluid therapy in German ICUs. Methods Analysis of data derived from the SepNet cross-sectional 1-day point-prevalence study of patients with sepsis [1]. ICU directors were asked about their fluid preferences. Data on days after start of sepsis were adjusted to account for overestimation of long-stayers in a point-prevalence survey. SPSS 15.0.1 (SPSS, Chicago, IL, USA) was used; the chi-square test or Kruskal–Wallis H test was applied where appropriate. Results On operative ICUs, more patients received synthetic colloids (41.4 vs. 21.2%, P <0.001) and crystalloids (74.6 vs. 65.0%, P <0.05), while albumin use did not differ. ICU directors of operative ICUs stated using synthetic colloids more frequently and albumins less frequently than their colleagues from nonoperative Results On operative ICUs, more patients received synthetic colloids (41.4 vs. P10 P10 P10 Abstract withdrawn Abstract withdrawn S5 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 ICUs. Stated and actual fluid use did not differ by hospital size. Of 415 patients with severe sepsis, 71.6% received crystalloids. 35.2% synthetic colloids (HES; gelatin, dextran), 16.4% received 5% glucose, and 4.1% albumin solutions. HES was the most frequently used colloid. It was administered to 29.4% of patients as HES 10% in 10.4% (mean dose, 787.8 ± 420.0 ml/24 hours) and as HES 6% in 20.7% (mean dose, 769.1 ± 403.1 ml/24 hours). Patients receiving HES had a higher mean SOFA score than patients without HES (9.92 ± 4.12 vs. 8.00 ± 4.07, P <0.001), tended to more frequent acute renal failure (ARF) (defined as serum creatinine >1.5 mg/dl (132.6 μmol/l) and diuresis ≤500 ml/day, no chronic replacement therapy, RRT), more RRT, lower thrombocyte counts and more frequently received RBCs, but this was non- significant. In total, 29.5% of patients with ARF and 30.3% of patients with RRT received HES. HES was administered to 115 patients between day 0 and day 63 after the start of severe sepsis. Conclusions Fluid therapy in septic patients varied by type of ICU. About 30% of patients received HES regardless of renal dysfunction. HES was applied for many days after the start of severe sepsis. immune cells. Changes in gene expression mediated by the in vivo administration of CpG ODN were identified using microarrays. immune cells. Changes in gene expression mediated by the in vivo administration of CpG ODN were identified using microarrays. immune cells. Changes in gene expression mediated by the in vivo administration of CpG ODN were identified using microarrays. Objective We predicted that microarrays could be used to identify reproducible changes in gene expression induced by CpG ODN activation in mice treated in vivo over time, and that network analysis would allow us to identify regulators of gene expression. Materials cDNA was generated from total RNA isolated from spleen cells of mice 30 minutes to 3 days after in vivo treatment with 400 μg CpG or control ODN. Objective We predicted that microarrays could be used to identify reproducible changes in gene expression induced by CpG ODN activation in mice treated in vivo over time, and that network analysis would allow us to identify regulators of gene expression. Fluid therapy in severe sepsis: results from a representative survey of German ICUs 21.2%, P <0.001) and crystalloids (74.6 vs. 65.0%, P <0.05), while albumin use did not differ. ICU directors of operative ICUs stated using synthetic colloids more frequently and albumins less frequently than their colleagues from nonoperative S6 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P17) Age distribution of patients in WCNN intensive therapy unit. Figure 2 (abstract P17) Distribution of central venous catheters in WCNN intensive therapy unit. Figure 3 (abstract P17) Average duration of central venous catheters in WCNN intensive therapy unit. Figure 1 (abstract P17) Age distribution of patients in WCNN intensive therapy unit. Methods Human endocan is produced by overexpressing HEK 293 cell lines and purified by anion exchange and affinity chromatographies. Proteolysis is performed by addition of endocan with PMA-activated PMN supernatants, purified CG, NE, or PR3. Endocan degradation is evaluated by ELISA, western blot and MALDI-TOF mass spectrometry. Binding assay on Jurkat cells is performed as described previously. Endocan-derived p14 fragments in human serum were detected by immunoprecipitation. Results We demonstrate that CG, NE but not PR3 degrade endocan. We show that the degradation profiles of endocan by CG or NE are different. We interestingly identify a novel peptide fragment of endocan of 14 kDa, named p14, which represents the main endocan degradation product. This p14 results from the specific cleavage of the full-length endocan by CG. The generation of this particular fragment strictly required the presence of the DS chain on full-length endocan. Furthermore, this p14 fragment becomes resistant to NE. We also demonstrate that p14 inhibits the binding of endocan to Jurkat cells. Finally, we present evidence that p14 could be detected in patient serum suffering from an acute PMN-mediated disease like sepsis. Conclusions The results suggest that the expression of CG by PMN shortly after their activation, by modifying the micro- environment in degrading vascular endocan, may participate in the complex network that controls tissue infiltration of leukocytes during sepsis. P17 A review of central venous catheter-related infections in neurointensive care patients in a tertiary referral centre C-H Tan, P Nair, A Sule, M Rathbone Walton Centre for Neurology & Neurosurgery (WCNN), Liverpool, UK Critical Care 2009, 13(Suppl 4):P17 (doi: 10.1186/cc8073) Figure 1 (abstract P17) Methods Human endocan is produced by overexpressing HEK 293 cell lines and purified by anion exchange and affinity chromatographies. Proteolysis is performed by addition of endocan with PMA-activated PMN supernatants, purified CG, NE, or PR3. Fluid therapy in severe sepsis: results from a representative survey of German ICUs Endocan degradation is evaluated by ELISA, western blot and MALDI-TOF mass spectrometry. Binding assay on Jurkat cells is performed as described previously. Endocan-derived p14 fragments in human serum were detected by immunoprecipitation. Results We demonstrate that CG, NE but not PR3 degrade endocan. We show that the degradation profiles of endocan by CG or NE are different. We interestingly identify a novel peptide fragment of endocan of 14 kDa, named p14, which represents the main endocan degradation product. This p14 results from the specific cleavage of the full-length endocan by CG. The generation of this particular fragment strictly required the presence of the DS chain on full-length endocan. Furthermore, this p14 fragment becomes resistant to NE. We also demonstrate that p14 inhibits the binding of endocan to Jurkat cells. Finally, we present evidence that p14 could be detected in patient serum suffering from an acute PMN-mediated disease like sepsis. Results We demonstrate that CG, NE but not PR3 degrade endocan. We show that the degradation profiles of endocan by CG or NE are different. We interestingly identify a novel peptide fragment of endocan of 14 kDa, named p14, which represents the main endocan degradation product. This p14 results from the specific cleavage of the full-length endocan by CG. The generation of this particular fragment strictly required the presence of the DS chain on full-length endocan. Furthermore, this p14 fragment becomes resistant to NE. We also demonstrate that p14 inhibits the binding of endocan to Jurkat cells. Finally, we present evidence that p14 could be detected in patient serum suffering from an acute PMN-mediated disease like sepsis. Age distribution of patients in WCNN intensive therapy unit. Figure 2 (abstract P17) Figure 2 (abstract P17) Distribution of central venous catheters in WCNN intensive therapy unit. Conclusions The results suggest that the expression of CG by PMN shortly after their activation, by modifying the micro- environment in degrading vascular endocan, may participate in the complex network that controls tissue infiltration of leukocytes during sepsis. A review of central venous catheter-related infections in neurointensive care patients in a tertiary referral centre C-H Tan, P Nair, A Sule, M Rathbone Walton Centre for Neurology & Neurosurgery (WCNN), Liverpool, UK Critical Care 2009, 13(Suppl 4):P17 (doi: 10.1186/cc8073) C-H Tan, P Nair, A Sule, M Rathbone Distribution of central venous catheters in WCNN intensive therapy unit. Introduction Intravenous catheter-related bloodstream infections (ICR-BSI) are a major contributing factor to in-hospital mortality and morbidity extending inpatient stay by 10 days and expenditure per patient by £2,000 to £30,000 [1]. Figure 3 (abstract P17) Average duration of central venous catheters in WCNN intensive therapy unit. Figure 3 (abstract P17) Average duration of central venous catheters in WCNN intensive therapy unit. Figure 3 (abstract P17) Figure 3 (abstract P17) Objective A prospective survey was conducted in our unit on all patients with central venous catheters to ascertain the incidence of ICR-BSI, identify the organisms and determine the occurrence of infection from the various sites – femoral, internal jugular and subclavian lines. Methods The survey was carried out over a period of 13 weeks. Data collected from patients’ case notes included site of central line insertion, length of line in situ, reason for line removal and positive blood culture reports. Results During the study period, 104 patients were treated on the unit. Fifty-two central venous lines were inserted in 36 patients (Figure 1): 63.5% femoral (n = 33), 32.7% internal jugular (n = 17) and 3.9% subclavian lines (n = 2) (Figure 2). The lines were reviewed daily and removed if indicated clinically (pyrexia or raised white cell count) or if not required. A total 51.5% of femoral lines (n = 17) were removed due to clinical indications, as were 29% (n = 5) of internal jugular and 50% (n = 1) of subclavian lines. The average duration of a line remaining in situ was 4.5 days for femoral, 6 days for internal jugular and 5 days for subclavian lines (Figure 3). Blood cultures were taken at the time of line removal. These yielded positive results in eight femoral, seven internal jugulars and one subclavian line (Figure 4). Our survey indicated that the incidence of ICR-BSI in our unit is 30.8%, (of this 62.5% coagulase-negative staphylococci (CNS), 12.5% Escherichia coli and Pseudomonas each, and 6.25% MSSA and MRSA each) (Figure 5). Average duration of central venous catheters in WCNN intensive therapy unit. Conclusions The distribution of microorganisms causing bacteraemia is broadly similar in our unit to that in other teaching hospitals in the UK [2], in that CNS was the commonest organism isolated. However, E. coli and Pseudomonas were the next common organisms, unlike other units where Staphylococcus aureus was the second most prevalent organism. The incidence of bacteraemia from femoral lines (53.7/1,000 catheter-days) was lower than that from internal jugular lines (68.6/1,000 catheter- days) possibly due to a higher index of suspicion in the case of femoral lines and earlier removal (Figure 6). Heart rate variability, cytokine, and brain responses to infection: insights from a mouse model K Fairchild, R Gaykema, L Goehler Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA Critical Care 2009, 13(Suppl 4):P18 (doi: 10.1186/cc8074) Introduction Continuous heart rate variability (HRV) monitoring can detect early sepsis in certain high-risk patient populations, but the mechanisms by which sepsis depresses HRV are not well understood. Our prior studies in rodents have shown that endo- toxin causes cytokine-related depression of HRV and activation of specific neuronal networks. The aim of the current studies was to identify pathogen-specific patterns of cytokine expression, HRV changes, and activation of central autonomic pathways in mice. Distribution of ICR-BSI in WCNN intensive therapy unit. Figure 5 (abstract P17) Distribution of ICR-BSI organisms in WCNN intensive therapy unit. Figure 6 (abstract P17) Incidence of ICR-BSI in WCNN intensive therapy unit. Distribution of ICR-BSI in WCNN intensive therapy unit. g p y Materials and methods Adult male C57BL/6 mice implanted with radiotelemetry probes for continuous ECG and temperature monitoring were inoculated intraperitoneally with Klebsiella pneumo- niae (KP, n = 21), methicillin-resistant Staphylococcus aureus (MRSA-COL, n = 9) or Candida albicans (CA-SC5314, n = 2). Heart rate variability (standard deviation of RR intervals) was measured continuously for up to 3 days in K. pneumoniae and MRSA mice and up to 3 weeks in CA mice. Blood was obtained at two or more time points for culture and measurement of G-CSF, KC, MIP-1β, IFNγ, TNFα, IL-6, and IL-10. Neuronal activation was assessed in multiple brain regions of K. pneumoniae-infected mice by c-Fos staining. g p y g Results Compared with sham-treated mice, infected mice had increases in multiple cytokines at 18 hours and 42 hours post inoculation. Cytokine profiles were similar among the three organisms except that CA-infected mice expressed less KC. Bacteria-inoculated mice with adverse outcome (positive blood culture and/or death, n = 8 of 21 K. pneumoniae and 3 of 9 MRSA) had significantly higher levels of all cytokines compared with mice with good outcome. Substantial depression of HRV was seen in all 11 bacteria-infected mice with adverse outcome and in only one of 19 mice with good outcome. Levels of G-CSF and IL-6 were negatively correlated with HRV (Spearman correlation coefficient = –0.36 and –0.37, respectively, P = 0.05 for each) and there was a trend toward a correlation with KC (–0.35, P = 0.07). Immunohistochemical studies revealed that, compared with sham-treated controls (n = 2), K. Heart rate variability, cytokine, and brain responses to infection: insights from a mouse model pneumoniae infection (n = 3) was associated with c-Fos induction in the dorsal vagal complex and ventrolateral medulla, paraventricular hypothalamic nucleus, preoptic area, subfornical organ, bed nucleus of the stria terminalis, and medial prefrontal and insular cortices. c-Fos immunoreactivity also occurred in ventricular ependymal cells and in cells associated with large blood vessels. Distribution of ICR-BSI organisms in WCNN intensive therapy unit. Conclusions Infection with Gram-positive or Gram-negative bacteria invokes similar changes in cytokines and HRV in mice, whereas preliminary studies suggest Candida infection results in different patterns. K. pneumoniae infection causes widespread neuronal activation within the central autonomic network. Incidence of ICR-BSI in WCNN intensive therapy unit. the fact that femoral lines, which are often the safest option for unstable patients with head injury, can be effectively managed with strict adherence to guidelines to reduce ICR-BSI. References 1. Maki DG, Kluger DM, Crnich CJ: The risk of bloodstream infection in adults with different intravascular devices: a systematic review of 200 published prospective studies. Mayo Clin Proc 2006, 81: 1159-1171. Figure 3 (abstract P17) Our study highlights S7 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 the fact that femoral lines, which are often the safest option for unstable patients with head injury, can be effectively managed with strict adherence to guidelines to reduce ICR-BSI. References 1. Maki DG, Kluger DM, Crnich CJ: The risk of bloodstream infection in adults with different intravascular devices: a systematic review of 200 published prospective studies. Mayo Clin Proc 2006, 81: 1159-1171. 2. Coello R, Charlett A, Ward V, et al.: Device-related sources f b t i i E li h h it l t iti f th Figure 4 (abstract P17) Distribution of ICR-BSI in WCNN intensive therapy unit. Figure 5 (abstract P17) Distribution of ICR-BSI organisms in WCNN intensive therapy unit. Figure 6 (abstract P17) Incidence of ICR-BSI in WCNN intensive therapy unit. Figure 4 (abstract P17) Distribution of ICR-BSI in WCNN intensive therapy unit. Figure 4 (abstract P17) P18 Central venous catheter-related infection: a cohort study evaluating dedicated central venous catheter packs Central venous catheter-related infection: a cohort study evaluating dedicated central venous catheter packs P-Y Chu, D-Z Hsu, M-Y Liu Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan Critical Care 2009, 13(Suppl 4):P20 (doi: 10.1186/cc8076) S Mukerji, R Daniels, K Maung, A Mattin Good Hope Hospital, Worcester, UK Critical Care 2009, 13(Suppl 4):P22 (doi: 10.1186/cc8078) S Mukerji, R Daniels, K Maung, A Mattin Good Hope Hospital, Worcester, UK Introduction Hypotension is well relative to the high mortality of sepsis. Sesamol increases the survival rate of septic mice. However, the effect of sesamol on septic hypotension after the onset of systemic inflammation has never been studied. The aim of the study is to investigate the effect of sesamol on septic hypotension. Introduction Central venous catheter (CVC)-related bloodstream infections (CRBSI) are the third most common healthcare- associated infection (HAI) in ICUs, associated with significant morbidity, mortality, increased length of stay and costs [1,2]. Several care bundle studies have suggested that utilising various strategies together (such as training, regular line monitoring and using dedicated line insertion trolleys) can have a positive impact on CRBSI rates [3-6]. However, the impact solely attributable to the provision of a dedicated, stand-alone CVC insertion pack has not been evaluated. We therefore investigated the impact of a new EPIC2 compliant CVC pack, introduced in Good Hope Hospital in 2007, on CVC tip colonisation rates. Materials Wistar rats, lipopolysaccharide (LPS) (derived from Escher- ichia coli, serotype O55:B5), and sesamol were used in this study. Methods Hypotension was induced by injecting LPS intravenously. Mean arterial pressure was measured using an invasive blood pressure system. Serum nitrite and cytokine levels were deter- mined using the Griess reaction and ELISA, respectively. Peroxisome proliferator-activated receptor (PPAR) activation was measured using a PPAR assay kit. Results LPS administration significantly increased the serum TNFα level at 1 hour. Sesamol treated 1 hour after LPS adminis- tration inhibited the LPS-associated blood pressure decrease. Sesamol failed to decrease the LPS-induced nitrite production, but decreased the LPS-induced TNFα and IL-1β production after the onset of systemic inflammation. Sesamol enhanced the IL-10 production in serum and the PPAR activation in white blood cells. Methods Data were collected prospectively between June 2007 and December 2008. Patients were divided into two cohorts: patients whose CVCs were inserted using the CVC packs (B), and those receiving CVCs prior to the introduction of the packs (nB). P20 Sesamol attenuates septic hypotension through peroxisome proliferator-activated receptor activation after the onset of systemic inflammatory response Central venous catheter-related infection: a cohort study evaluating dedicated central venous catheter packs Data were collated from questionnaires as well as patients’ notes on: patient’s age and sex; type and site of CVC inserted; location at the time of insertion; the grade of practitioner; and the duration CVCs remained in situ. Using the hospital’s patient information system and patient notes, data on CVC tip cultures were obtained. Data are presented as percentages and analysed using multivariate analysis. Conclusions Sesamol may attenuate septic hypotension through alternating cytokine production by PPAR activation after the onset of systemic inflammatory response. Available online http://ccforum.com/supplements/13/S4 models. However, its mechanism is still unclear. The aim of this study was to determine the effects of muscimol, a GABAA receptor agonist, on lipopolysaccharide-induced mortality and inflammation in mice. the pathogenesis and development of sepsis. 3,4-Methylene- dioxyphenol (sesamol), one of the lignans in sesame oil, protects against endotoxin-induced oxidative stress and organ failure. However, the effects of sesamol on systemic inflammation and oxidative stress in septic rats have never been investigated. Materials C57BL6 mice, lipopolysaccharide (derived from Escher- ichia coli, serotype O55:B5), and muscimol were used in this study. Methods Mice endotoxemia was induced by 10 mg/kg lipopoly- saccharide intraperitoneally. Muscimol ranging from 0 to 3 mg/kg was given subcutaneously 30 minutes before lipopolysaccharide administration. Serum TNFα, IL-1β, IL-10, and IL-12 were determined using ELISA. Materials C57BL6 mice, lipopolysaccharide (derived from Escher- ichia coli, serotype O55:B5), and muscimol were used in this study. Objective To investigate the effects of sesamol on systemic inflam- mation and oxidative stress in septic rats. Methods Septic rats were induced by cecal ligation and puncture (CLP). Rats received sesamol (10 mg/kg, subcutaneously) 0 and 6 hours after CLP. IL-1β, lipid peroxidation, hydroxyl radical, super- oxide anion, xanthine oxidase activity, and nitrite levels in blood were determined 12 hours after CLP. Results Muscimol significantly increased the survival rate in sub- lethal dose of lipopolysaccharide-treated mice (from 7% to 100%) (P < 0.0001) within 72 hours. Muscimol inhibited serum TNFα, IL-1β, and IL-12 production in a dose-dependent manner. Further- more, muscimol significantly increased serum IL-10 levels (P < 0.001) in lipopolysaccharide-treated mice. Results IL-1β, lipid peroxidation, hydroxyl radical, superoxide anion, xanthine oxidase activity, and nitrite levels were significantly increased in CLP-treated rats compared with those in the sham- operation group (all P <0.05). Sesamol significantly reduced IL-1β, lipid peroxidation, hydroxyl radical, superoxide anion, xanthine oxidase activity, and nitrite levels compared with the saline group in CLP-treated rats (all P <0.05). Conclusions Muscimol potently increased the survival rate and inhibited inflammatory response in endotoxemic mice. Conclusions Sesamol might attenuate systemic inflammation and oxidative stress by inhibiting proinflammatory cytokine and reactive oxygen species generation in septic rats. Muscimol increases the survival rate and inhibits the inflammatory response in endotoxemic mice 1. Maki DG, Kluger DM, Crnich CJ: The risk of bloodstream infection in adults with different intravascular devices: a systematic review of 200 published prospective studies. Mayo Clin Proc 2006, 81: 1159-1171. D-Z Hsu, Y-H Li, P-Y Chu, M-Y Liu Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan Critical Care 2009, 13(Suppl 4):P19 (doi: 10.1186/cc8075) 2. Coello R, Charlett A, Ward V, et al.: Device-related sources of bacteraemia in English hospitals – opportunities for the prevention of hospital-acquired bacteraemia. J Hosp Infect 2003, 53:46-57. Introduction Affecting the γ-amino butyric acid (GABA) pathway results in an alteration of inflammatory response in various animal Introduction Affecting the γ-amino butyric acid (GABA) pathway results in an alteration of inflammatory response in various animal S8 P23 TLR4 on hematopoietic cells is crucial for host defense against Klebsiella pneumonia but TLR2 is needed when bacterial numbers are high C Wieland1,2,3, MH van Lieshout3,4, AJ Hogendijk3,4, T van der Poll3,4 1Laboratory of Experimental Intensive Care and Anesthesiology, 2Department of Intensive Care, 3Center of Experimental and Molecular Medicine, and 4Center of Infection and Immunity, Academic Medical Center Amsterdam, the Netherlands Critical Care 2009, 13(Suppl 4):P23 (doi: 10.1186/cc8079) Introduction Klebsiella species are opportunistic pathogens that can give rise to severe infections including pneumonia and sepsis. Typically, Klebsiella infections are nosocomial and mainly caused by Klebsiella pneumoniae, the medically most important species of the genus. Number of days CVCs were left in situ in both cohorts. Number of days CVCs were left in situ in both cohorts. g Objective We set out to validate and extend our previous data using C3H/HeJ mice that demonstrated an important role for TLR4 in K. pneumoniae pneumonia. Moreover, we were interested in the relative roles of cells from hematopoietic origin and parenchymal cells. Methods Using TLR2 and TLR4 single and TLR2x4 double knockout (KO) mice on a C57BL/6 background, the roles of TLR2 and TLR4 were investigated independently and together. We intranasally inoculated C57BL/6 wild-type (WT) and KO mice with K. pneumoniae (4 x 10–3 CFU per mouse) and studied host defense. Moreover, we performed bone marrow transplantation (BMT) experiments in which we transplanted KO bone marrow into irradiated WT mice and vice versa. Objective We set out to validate and extend our previous data using C3H/HeJ mice that demonstrated an important role for TLR4 in K. pneumoniae pneumonia. Moreover, we were interested in the relative roles of cells from hematopoietic origin and parenchymal cells. Figure 2 (abstract P22) Differences in colony growth in the two cohorts. *P <0.05. Figure 2 (abstract P22) Differences in colony growth in the two cohorts. *P <0.05. Methods Using TLR2 and TLR4 single and TLR2x4 double knockout (KO) mice on a C57BL/6 background, the roles of TLR2 and TLR4 were investigated independently and together. We intranasally inoculated C57BL/6 wild-type (WT) and KO mice with K. pneumoniae (4 x 10–3 CFU per mouse) and studied host defense. Moreover, we performed bone marrow transplantation (BMT) experiments in which we transplanted KO bone marrow into irradiated WT mice and vice versa. Results Shortly after infection, both TLR4 and TLR2x4 KO mice demonstrated an attenuated proinflammatory response in the lungs. Introduction The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host. P23 This was associated with higher bacterial counts 24 hours after infection in the lungs, liver and spleens of both TLR4 and TLR2x4 KO animals. Interestingly, although no differences in antibacterial host defense of TLR2 KO animals were observed, TLR2x4 KO animals were more susceptible to K. pneumoniae infection than the single TLR4 KO mice: after 44 hours of infection, 0/8 WT, 0/8 TLR2 KO mice, 5/8 TLR4 KO mice and 8/8 TLR2x4 KO mice had succumbed. Moreover, when infecting all strains with a high dose of K. pneumoniae (10–4 CFU), no differences in outgrowth were detected between WT, TLR2 and TLR4 KO animals, whereas double KO animals suffered from higher bacterial burdens in the lungs, liver, spleen and blood. BMT of WT bone marrow into irradiated TLR2x4 KO mice resulted in a reversed phenotype with similar bacterial growth compared with syngenic transplanted WT mice. Differences in colony growth in the two cohorts. *P <0.05. (Figure 2). The bundle cohort had no MRSA growth compared with four incidences in the nB group. Conclusions Our results indicate that use of dedicated CVC packs was associated with a significant reduction in the colonisation rate of CVCs, despite lines being left in situ for longer periods and the more frequent use of quin-lumen catheters in the intervention group. There was also a trend toward prevention of MRSA colonisation. 1. Blake M: Update: catheter-related bloodstream infection rates in relation to clinical practice and needleless device type. Can J Infect Control 2008, 23:156-160, 162. Conclusions These data confirm our previous research that, during low-dose infections, TLR4 is of primary importance in host defense against K. pneumoniae. Nevertheless, when high numbers of bacteria are present, TLR2 acts together with TLR4 to orchestrate the immune response, a protective effect that is primarily mediated by hematopoietic cells. 2. Tacconelli E, Smith G, Hieke K, Lafuma A, Bastide P: Epi- demiology, medical outcomes and costs of catheter- related bloodstream infections in intensive care units of four European countries: literature and registry based estimates. J Hosp Infect 2009, 72:97-103. Introduction The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host. P24 3. Mermel LA: Prevention of central venous catheter-related infections: what works other than impregnated or coated catheters? J Hosp Infect 2007, 65(Suppl 2):30-33. Effect of the novel influenza A (H1N1) virus in the human immune system A Antonopoulou, M Raftogiannis, F Baziaka, P Koutoukas, A Savva, T Kanni, M Georgitsi, A Pistiki, EJ Giamarellos-Bourboulis 4th Department of Internal Medicine, University of Athens, Medical School, Athens, Greece Critical Care 2009, 13(Suppl 4):P24 (doi: 10.1186/cc8080) P21 Results Complete data were obtained for 347 patients: 246 patients in group nB, 101 patients in group B. Male/female ratio, average age, site of insertion, clinical area of insertion and grade of practitioner were similar in both cohorts. There was a significantly higher number of 5-lumen catheters inserted in group B compared with group nB (81% compared with 44%, P <0.05), reflecting change in hospital practice. More B catheters (51%) were left in situ longer, for 6 to 10 days, compared with nB catheters (31%) (Figure 1). Thirty-one per cent of nB tips grew colonies of at least one pathogen. There was a significant reduction in the number of B tips growing colonies (12% compared with 31%, P <0.05) 3,4-Methylenedioxyphenol attenuates systemic inflammation and oxidative stress in septic rats Y-H Li, D-Z Hsu, M-Y Liu Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan Critical Care 2009, 13(Suppl 4):P21 (doi: 10.1186/cc8077) Y-H Li, D-Z Hsu, M-Y Liu Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan Critical Care 2009, 13(Suppl 4):P21 (doi: 10.1186/cc8077) Introduction Sepsis is one of the major causes of mortality in ICUs. Systemic inflammation and oxidative stress are involved in S9 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Figure 1 (abstract P22) Number of days CVCs were left in situ in both cohorts. Figure 2 (abstract P22) Differences in colony growth in the two cohorts. *P <0.05. Figure 1 (abstract P22) Number of days CVCs were left in situ in both cohorts. P23 Effect of the novel influenza A (H1N1) virus in the human immune system 4. Berenholtz SM, Pronovost PJ, Lipsett PA, et al.: Eliminating catheter-related bloodstream infections in the intensive care unit. Crit Care Med 2004, 32:2014-2020. A Antonopoulou, M Raftogiannis, F Baziaka, P Koutoukas, A Savva, T Kanni, M Georgitsi, A Pistiki, EJ Giamarellos-Bourboulis 4th Department of Internal Medicine, University of Athens, Medical School, Athens, Greece Critical Care 2009, 13(Suppl 4):P24 (doi: 10.1186/cc8080) 5. Anonymous: Reduction in central line-associated blood- stream infections among patients in intensive care units Pennsylvania, April 2001 March 2005. MMWR Recomm Rep 2005, 54:1013-1016. 6. Pronovost P, Needham D, Berenholtz S, et al.: An interven- tion to decrease catheter-related bloodstream infections in the ICU. N Engl J Med 2006, 355:2725-2732. 6. Pronovost P, Needham D, Berenholtz S, et al.: An interven- tion to decrease catheter-related bloodstream infections in the ICU. N Engl J Med 2006, 355:2725-2732. S10 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Methods Blood was sampled within the first 2 days of the presentation of signs of infection from 10 healthy volunteers; from 18 patients of flu-like syndrome (FLS); and from 30 patients of infection by H1N1 confirmed by reverse RT-PCR. Absolute counts of subtypes of monocytes and of lymphocytes were determined after staining with monoclonal antibodies and analysis by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with various bacterial stimuli. Concen- trations of TNFα, of IL-1β and of IFNγ were estimated in super- natants by an enzyme immunoassay. compared with FLS). No effect of other stimuli was shown on release of TNFα and of IFNγ. Release of IL-1β was not affected. Conclusions Infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses charac- terized by defective cytokine responses to S. pneumoniae. Alterations of the adaptive immune responses are predominated by an increase of Tregs. These findings signify a predisposition for pneumococcal infections after infection by H1N1 influenza. P25 y y y Results Mean absolute counts of CD14 monocytes of healthy volunteers, of FLS and of patients infected by the H1N1 virus were 271.1, 464.4 and 607.2%, respectively (P <0.0001 compared with other groups). Respective values of CD4+/CD25+/CD127– cells were 2.0, 4.7 and 9.0% (P <0.0001 compared with other groups). Respective values of CD19 cells were 297.9, 151 and 137.9%. No differences between the three groups were found regarding CD3/CD4 lymphocytes, CD3/CD8 lymphocytes, natural killer (NK) cells and NKT cells. No differences were also found regarding the rate of apoptosis of the above subtypes. Six patients had H1N1-related pneumonia. Mean T-regulatory cells (Tregs) of H1N1-infected patients without pneumonia and with pneumonia were 6.7 and 17.8%, respectively (P = 0.034). Mean release of TNFα by phytohemagglutin-stimulated PBMCs of healthy volunteers, of FLS and of patients infected by H1N1 was 3,658.5, 1,877.3 and 874.4 pg/ml, respectively (P <0.0001 compared with other groups). Respective release of TNFα by Streptococcus pneumoniae-stimulated PBMCs was 1,836.9, 949.9 and 478.0 pg/ml (P <0.0001 compared with other groups). Mean respective release of IFNγ by PHA-stimulated PBMCs was 1,651.8, 1,235.1 and 1,114.3 pg/ml (P = 0.010 compared with FLS). Mean respective release of IFNγ by S. pneumoniae-stimu- lated PBMCs was 1,085.7, 748.1 and 709.7 pg/ml (P = 0.024 Clarithromycin reverses sepsis-induced immunoparalysis of monocytes M Raftogiannis, A Antonopoulou, F Baziaka, P Koutoukas, T Tsaganos, A Pelekanou, A Spyridaki, M Mouktaroudi, EJ Giamarellos-Bourboulis 4th Department of Internal Medicine, University of Athens, Medical School, Athens, Greece Critical Care 2009, 13(Suppl 4):P25 (doi: 10.1186/cc8081) Introduction In a recently published double-blind, randomized trial conducted by our study group, clarithromycin was intravenously administered in patients with ventilator-associated pneumonia (VAP) and sepsis for three consecutive days [1]. An earlier resolution of VAP and a fivefold decrease of the risk for death by septic shock and multiple organ failure (MODS) compared with placebo were shown. p Objective To investigate the mode of action of clarithromycin. Objective To investigate the mode of action of clarithromycin. Methods Blood was sampled before administration of the investigational product and on six consecutive days. Peripheral blood mononuclear cells (PBMCs) were isolated after gradient centrifugation over Ficoll. PBMCs were incubated and adherent monocytes were harvested and stimulated for 24 hours with Figure 1 (abstract P25) P26 Four groups of mice (n = 8) were treated intra- peritoneally with (1) placebo, (2) rh-TFPI every 8 hours, (3) ceftriaxone twice daily or (4) rh-TFPI in combination with ceftriaxone. Early (8 hours) and late (24 hours) initiated treatments were evaluated. Bronchoalveolar lavage fluid (BALF), lungs and plasma were obtained 24 hours (for groups in which treatment was started after 8 hours) or 48 hours (treatment started after 24 hours) after infection. Statistical analysis was performed by Mann–Whitney U test. Introduction Pneumonia is characterized by local activation of coagulation leading to alveolar fibrin deposition. Lung injurious mechanical ventilation (LI-MV) with conventional tidal volumes (VT) and no positive end-expiratory pressure (PEEP) aggravates pulmonary coagulopathy. We hypothesized administration of anti- thrombin (AT), a natural anticoagulant, to attenuate ventilator- induced coagulopathy and inflammation in a rat model of Strepto- coccus pneumoniae pneumonia. Methods Rats challenged intratracheally with bacteria were ventilated 40 hours later (VT = 12 ml/kg/no PEEP) after systemic administration of plasma-derived human AT (250 U/kg) or placebo for 3 hours. Endpoints: BALF levels of thrombin–antithrombin complexes (TATc), fibrin degradation products (FDP), AT, plasminogen activator activity (PAA), plasminogen activator inhibitor-1 (PAI-1), pulmonary cytokines and blood cultures. Data are presented as the mean ± SD. Statistics: one-way ANOVA with Dunn’s multiple comparison test. Results Pneumonia resulted in local and systemic activation of coagulation (as reflected by increased thrombin–antithrombin complexes) and inhibition of fibrinolysis (as reflected by increased plasminogen activator inhibitor-1 and decreased plasminogen activator activity). Both early and late treatment with rh-TFPI reduced pneumonia-induced coagulation in lungs and plasma; rh- TFPI given with ceftriaxone further attenuated coagulation relative to ceftriaxone only. No effects of rh-TFPI on pneumonia-inhibited fibrinolysis were observed. Cell recruitment in BALF did not differ between groups. Remarkably, rh-TFPI reduced levels of several cytokines and chemokines not only in lung homogenates, but also in BALF (IL-6, IFNγ, MCP-1, LIX) and plasma (IL-6, TNFα, IFNγ). The attenuated host inflammatory response was not reflected by differences in total histopathology scores between treatment groups. In mice not treated with ceftriaxone, rh-TFPI decreased bacterial loads in lung homogenates ~10-fold (P <0.01 vs. placebo) at 48 hours, while leaving bacterial loads in BALF and the systemic compartment unaltered. Results S. pneumoniae pneumonia was characterized by activation of coagulation (TATc: in pneumonia vs. healthy control, 3.7 ± 0.3 vs. 1.2 ± 0.5 ng/ml; FDP: 291 ± 40 vs. 15 ± 5 ng/ml; AT: 3.7 ± 0.3 vs. P26 Plasma-derived human antithrombin attenuates ventilator- induced coagulopathy in a Streptococcus pneumoniae pneumonia model in rats Introduction Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia and a major cause of sepsis. Pneumonia elicits a procoagulant state in the lung resulting from activation of coagulation, downregulation of anticoagulant pathways and concurrent inhibition of fibrinolysis. Tissue factor is the main initiator of coagulation. Recombinant human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis- induced coagulation and has been evaluated in clinical trials involving patients with sepsis and community-acquired pneumonia. Objective To examine the effect of rh-TFPI on coagulation, inflammation and bacterial outgrowth in S. pneumoniae pneumonia in mice, either alone or with concurrent antibiotic treatment. Methods Pneumonia was induced by intranasal inoculation with S. pneumoniae. Four groups of mice (n = 8) were treated intra- peritoneally with (1) placebo, (2) rh-TFPI every 8 hours, (3) ceftriaxone twice daily or (4) rh-TFPI in combination with ceftriaxone. Early (8 hours) and late (24 hours) initiated treatments were evaluated. Bronchoalveolar lavage fluid (BALF), lungs and plasma were obtained 24 hours (for groups in which treatment was started after 8 hours) or 48 hours (treatment started after 24 hours) after infection. Statistical analysis was performed by Mann–Whitney U test. Introduction Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia and a major cause of sepsis. Pneumonia elicits a procoagulant state in the lung resulting from activation of coagulation, downregulation of anticoagulant pathways and concurrent inhibition of fibrinolysis. Tissue factor is the main initiator of coagulation. Recombinant human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis- induced coagulation and has been evaluated in clinical trials involving patients with sepsis and community-acquired pneumonia. Objective To examine the effect of rh-TFPI on coagulation, inflammation and bacterial outgrowth in S. pneumoniae pneumonia in mice, either alone or with concurrent antibiotic treatment. M th d P i i d d b i l i l i i h S H Aslami1,2, JJ Haitsma1, JJ Hoffstra2, M Levi2, H Zhang1, AS Slutsky1, MJ Schultz1 1Interdepartmental Division of Critical Care Medicine, Keenan Research Center, Li Ka Shing Knowledge Institute, St Michael’s Hospital, Toronto, Ontario, Canada; 2Departments of Intensive Care and Internal Medicine, Laboratory of Experimental Intensive Care and Anesthesiology, Academic Medical Center, University of Amsterdam, the Netherlands Critical Care 2009, 13(Suppl 4):P26 (doi: 10.1186/cc8082) AS Slutsky1, MJ Schultz1 Methods Pneumonia was induced by intranasal inoculation with S. pneumoniae. P27 Conclusions Administration of clarithromycin was accompanied by a considerable improvement of the response of monocytes to ex vivo stimulation with the release of TNFα and IL-6. These results signify that clarithromycin effectively reverses sepsis-induced immunoparalysis of monocytes. R f Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antibacterial effects in murine pneumococcal pneumonia F van den Boogaard1,2,3, X Brands1,2, M Schultz3, M Levi4, J Roelofs5, C van ‘t Veer1,2, T van der Poll1,2 1Center for Experimental and Molecular Medicine, 2Center for Infection and Immunity Amsterdam, 3Laboratory of Experimental Intensive Care and Anesthesiology, 4Department of Internal Medicine, and 5Department of Pathology, Academic Medical Center, University of Amsterdam, the Netherlands Critical Care 2009, 13(Suppl 4):P27 (doi: 10.1186/cc8083) References 1. Giamarellos-Bourboulis EJ, Pechère JC, Routsi C, et al.: Effect of clarithromycin in patients with sepsis and ventila- tor-associated pneumonia. Clin Infect Dis 2008, 46:1157- 1164. 1. Giamarellos-Bourboulis EJ, Pechère JC, Routsi C, et al.: Effect of clarithromycin in patients with sepsis and ventila- tor-associated pneumonia. Clin Infect Dis 2008, 46:1157- 1164. Figure 1 (abstract P25) Figure 1 (abstract P25) Figure 1 (abstract P25) *Statistically significant differences between groups at the indicated time intervals. Day 0 corresponds to time before start of the investigational drug. *Statistically significant differences between groups at the indicated time intervals. Day 0 corresponds to time before start of the investigational drug *Statistically significant differences between groups at the indicated time intervals. Day 0 corresponds to time before start of the investigational drug. S11 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 10 ng/ml LPS of Escherichia coli O55:B5. Concentrations of TNFα and of IL-6 were estimated in supernatants by an enzyme immunoassay. – P = NS). The infected animals showed similar numbers of bacteria in blood samples at the start of the experiment, which did not change during the experiment. Results One hundred patients were treated with placebo and another 100 patients with clarithromycin. Median concentrations of TNFα and of IL-6 in monocyte supernatants of all patients and separately of those with septic shock and MODS are shown in Figure 1. Conclusions Systemic administration of AT attenuated ventilator- induced coagulopathy but not inflammation. P26 Recognition of fungi such as Candida albicans is mediated through receptors of the innate immune system, such as Toll-like receptors (TLRs), that in turn activate innate immune system and antifungal defense. Objective To assess whether polymorphisms in fungal innate immune receptors such as TLRs and dectin-1 influence susceptibility to invasive candidiasis. Methods Frequencies of mostly nonsynonymous polymorphisms in several innate immune receptors were genotyped in a total of 331 patients that developed invasive candidiasis and compared with a total of 341 matched control patients that had the same predisposing factors. These included neutropenia, mucosal barrier injury and treatment with immunosuppressive regimens. Further- more, in vitro studies with healthy volunteers were conducted to assess the functional consequences of these polymorphisms regarding cytokine responses. Results Genotyping for polymorphisms in innate immune receptor genes revealed a higher frequency of three independent non- synonymous TLR1 polymorphisms in the affected group patients that developed invasive candidiasis. These polymorphisms were also demonstrated to be associated with impaired cytokine responses upon stimulations of immune cells, including IL-1β, IL-6 and IL-8. tions. The SCR was introduced in the emergency and acute assessment units in our teaching hospital setting within central London, accompanied by a seminar-based educational programme for medical and nursing staff. Methods Two months after its introduction, the use of the SCR form was audited in all acute medical admissions who met the clinical criteria for sepsis. One hundred sequential patients were assessed in a 6-week period over the winter. Conclusions Polymorphisms in TLR1, which is known to dimerize with TLR2 and TLR6, are associated with impaired immune recognition through these receptors and predispose to invasive candidiasis in humans. Results One-half of the audit sample had SCR forms completed. Specificity of the sepsis criteria was good, with <10% of patients subsequently judged not to have had sepsis. The patients with and without audit forms had comparable demographics, severity of illness and microbiology (Figure 1). Frequency of abnormal temperature was significantly higher in patients with the SCR, suggesting fever remains an important prompt for physicians to consider sepsis. The use of the SCR was also associated with significantly improved assessment of GCS, lactate, travel history and the need for isolation, as well as significantly greater number of specialist consultations (Table 1), albeit still inadequate, ~10% (without SCR) to ~20% (with SCR). P26 The SCR had no effect on frequency of clinical review by senior resident physicians, recording of FiO2, antibiotic guidelines compliance or blood/urine culture requests, all evident in 40 to 60% of patients. Approxi- mately 75% of all patients received antibiotics within 2 to 6 hours, and a trend for earlier antibiotics was associated with use of the SCR (Figure 2). P26 20.0 ± 5.2 IU/ml – all P <0.05) and inhibition of fibrinolysis (PAA: 99.2 ± 5.9 vs. 73.3 ± 5.7% of normal, P <0.05; PAI-1: 1.9 ± 0.6 vs. 10.0 ± 1.4 ng/ml, P <0.05). Systemic administration of AT resulted in supraphysiologic levels of BALF AT levels (25.4 ± 4.9 vs. 5.3 ± 1.0 IU/ml) and prevented further activation of coagulation by MV (TATc: in LI-MV with AT vs. placebo, 3.9 ± 0.3 vs. 6.5 ± 0.8 ng/ml – P <0.05). No changes in pulmonary cytokines were observed between the infected and mechanically ventilated animals (TNF: LI-MV with AT vs. placebo, 1.6 ± 0.4 vs. 1.9 ± 1.3 ng/ml and IL-6: 2.5 ± 1.3 vs. 2.4 ± 1.4 ng/ml Conclusions rh-TFPI attenuates local and systemic coagulopathy, the local and systemic inflammatory response and pulmonary bacterial growth during S. pneumoniae pneumonia in mice. S12 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P29) Figure 1 (abstract P29) P28 Toll like receptor 1 polymorphisms and susceptibility to invasive candidiasis Toll like receptor 1 polymorphisms and susceptibility to invasive candidiasis T Plantinga1, M Johnsson2, B Scott3, E van de Vosse4, D Velez3, JWM van der Meer1, J van Dissel4, J Perfect2, B-J Kullberg1, MG Netea1 1Radboud University Nijmegen, the Netherlands; 2Duke University Medical Center, Durham, NC, USA; 3University of Miami Miller School of Medicine, Miami, FL, USA; 4Leiden University Medical Center, Leiden, the Netherlands Critical Care 2009, 13(Suppl 4):P28 (doi: 10.1186/cc8084) T Plantinga1, M Johnsson2, B Scott3, E van de Vosse4, D Velez3, JWM van der Meer1, J van Dissel4, J Perfect2, B-J Kullberg1, MG Netea1 Introduction Invasive candidiasis is a severe systemic fungal infection with Candida spp. affecting immunocompromised hosts, which is responsible for the highest mortality rate of all nosocomial infections. Although several clinical predisposing factors are known, the individual risk for developing invasive candidiasis varies significantly. Recognition of fungi such as Candida albicans is mediated through receptors of the innate immune system, such as Toll-like receptors (TLRs), that in turn activate innate immune system and antifungal defense. Introduction Invasive candidiasis is a severe systemic fungal infection with Candida spp. affecting immunocompromised hosts, which is responsible for the highest mortality rate of all nosocomial infections. Although several clinical predisposing factors are known, the individual risk for developing invasive candidiasis varies significantly. Differential effects of IL-17 pathway in disseminated candidiasis and zymosan-induced multiple organ failure Differential effects of IL-17 pathway in disseminated candidiasis and zymosan-induced multiple organ failure FL van de Veerdonk1,2, BJ Kullberg1,2, IC Verschueren1,2, T Hendriks3, JWM van der Meer1,2, LAB Joosten1,2,4, MG Netea1,2 1Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2Nijmegen Institute for Infection, Inflammation and Immunity, Nijmegen, the Netherlands; 3Department of Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 4Department of Rheumatology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2009, 13(Suppl 4):P30 (doi: 10.1186/cc8086) FL van de Veerdonk1,2, BJ Kullberg1,2, IC Verschueren1,2, T Hendriks3, JWM van der Meer1,2, LAB Joosten1,2,4, MG Netea1,2 FL van de Veerdonk1,2, BJ Kullberg1,2, IC Verschueren1,2, T Hendriks3, JWM van der Meer1,2, LAB Joosten1,2,4, MG Netea1,2 Methods Mice defective in caspase-1, ASC, NLRP3 or P2X7 were infected intravenously with Candida albicans. Survival, fungal outgrowth in the organs, histology, and cytokine production were compared in these mouse strains with the wild-type C57/Bl6 control mice. PBMCs from healthy volunteers with or without reactive oxygen species (ROS) inhibitor and PBMCs from patients with chronic granulomatous disease (CGD) that are deficient in ROS production were stimulated with C. albicans. Results Caspase-1–/– mice and ASC–/– mice had a decreased survival during disseminated candidiasis (50%) compared with the control mice (100%). Caspase-1–/– mice had a 100-fold increase in fungal loads in the kidneys of the deficient animals (P <0.05) Methods Mice defective in caspase-1, ASC, NLRP3 or P2X7 were infected intravenously with Candida albicans. Survival, fungal outgrowth in the organs, histology, and cytokine production were compared in these mouse strains with the wild-type C57/Bl6 control mice. PBMCs from healthy volunteers with or without reactive oxygen species (ROS) inhibitor and PBMCs from patients with chronic granulomatous disease (CGD) that are deficient in ROS production were stimulated with C. albicans. 1Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2Nijmegen Institute for Infection, Inflammation and Immunity, Nijmegen, the Netherlands; 3Department of Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 4Department of Rheumatology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands C iti l C 2009 13(S l 4) P30 (d i 10 1186/ 8086) cal Care 2009, 13(Suppl 4):P30 (doi: 10.1186/cc8086 Results Caspase-1–/– mice and ASC–/– mice had a decreased survival during disseminated candidiasis (50%) compared with the control mice (100%). Challenges to implementation of sepsis guidelines S Patel1, E Wise2, J Hartin1, D Walker1, M Noursadeghi2,3 1Critical Care, 2Acute Assessment Unit, and 3Infection and Immunity, University College London Hospitals NHS Foundation Trust, London, UK Critical Care 2009, 13(Suppl 4):P29 (doi: 10.1186/cc8085) Introduction International surviving sepsis guidelines identified an important role for acute medicine in early management of severe sepsis, but local and multicentre international audits show poor adherence to these guidelines. Introduction International surviving sepsis guidelines identified an important role for acute medicine in early management of severe sepsis, but local and multicentre international audits show poor adherence to these guidelines. Materials We evaluated the use of a Sepsis Case Record (SCR) supported by a systematic educational programme to improve standards. A one-page SCR was derived from surviving sepsis guidelines, to prompt recognition of sepsis syndromes, compre- hensive secondary assessment, initiation of resuscitation and antibiotic treatment bundles, and appropriate specialist consulta- Conclusions The SCR was well received but not used consistently. The lack of abnormal temperature may contribute to this. The use of the SCR did improve early management of sepsis, S13 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Table 1 (abstract P29) Parameter With Forms No Forms P value SpR review 24/50 25/50 ns FiO2 29/50 25/50 ns Blood culture 42/50 35/50 ns Urine culture 29/50 23/50 ns Abx guide compliant 31/50 25/50 ns ID consult 9/50 8/50 ns ITU consult 11/50 6/50 ns GCS 40/50 29/50 <0.05 Lactate 45/50 35/50 <0.05 Micro consult 6/50 0/50 <0.05 Any consult 20/50 8/50 <0.05 Travel history 23/50 9/50 <0.05 Need for isolation 18/50 2/50 <0.05 ns, not significant. Table 1 (abstract P29) studies reported that IL-17 may contribute to inflammatory pathology and worsening of fungal disease. To address these discrepancies, we assessed the differential role of IL-17 pathway in two models of fungal sepsis: intravenous infection with live Candida albicans, in which fungal growth is the main cause of mortality, and zymosan-induced multiple organ failure in which the inflammatory pathology drives the mortality. Methods IL-17 receptor-deficient (IL-17RA–/–) and control mice were intravenously infected with 2 x 105 CFU live C. albicans UC820 per mouse. Mortality, fungal loads in the kidneys, neutrophil recruitment and phagocytosis and killing were assessed. IL-17RA–/– and control mice were also assessed for mortality in a multiorgan failure sepsis model induced by the fungal component zymosan. Challenges to implementation of sepsis guidelines Results On the one hand, IL-17RA–/– mice showed increased mortality and higher fungal loads in the kidneys in the model of disseminated candidiasis. On the other hand, the absence of IL-17RA in the knockout mice did not protect the mice against the multiorgan failure induced by zymosan. Furthermore, no reduction in neutrophil recruitment and defects in phagocytosis and killing in the first few hours of Candida infection were found. A significantly lower TNF production in response to Candida in cells from IL-17RA–/– mice was observed. Conclusions These data demonstrate that the IL-17 pathway does not have a major contribution to the inflammatory pathology leading to organ failure in fungal sepsis, and support the concept that the IL-17 pathway is protective during fungal sepsis. In addition, IL-17 deficiency does not appear to reflect a pure innate defect, since it did not result in loss of neutrophil recruitment and function during the first few hours of fungal sepsis. Furthermore, the lower TNF production in response to Candida in cells from IL-17RA–/– mice could contribute to susceptibility to disseminated candidiasis. Figure 2 (abstract P29) P31 Caspase-1 and ASC but not NLRP3 mediate antifungal defense in candidiasis sepsis Caspase-1 and ASC but not NLRP3 mediate antifungal defense in candidiasis sepsis F van de Veerdonk1,2, LAB Joosten2, P Shaw1, S Smeekens2, JWM van der Meer2, B-J Kullberg2, MG Netea2, T-D Kanneganti1 1Department of Immunology, St Jude Children’s Research Hospital, Memphis, TN, USA; 2Department of Medicine, Radboud University Nijmegen Medical Centre, and Nijmegen Institute for Infection, Inflammation and Immunity (N4I), Nijmegen, the Netherlands Critical Care 2009, 13(Suppl 4):P31 (doi: 10.1186/cc8087) but a number of deficiencies persisted. Implementation of sepsis guidelines remains a major challenge in clinical practice. Succinct guidelines were helpful in this setting but need additional educational and feedback support to improve standards of practice. Introduction IL-1β plays an important role in antifungal defense. The inflammasome is thought to be required for caspase-1 activation and processing of the inactive precursor pro-IL-1β into its active form. Contradictory data have been reported regarding the role of the inflammasome in Candida sepsis. In order to address these discrepancies, we investigated host defense against disseminated candidiasis in knockout mice defective in the various components of the inflammasome. P30 Available online http://ccforum.com/supplements/13/S4 Experiments using the NADPH inhibitor diphenyl- eneiodonium, or in monocytes isolated from CGD patients who have defective capacity to form ROS, demonstrated that ROS did not mediate inflammasome activation and C. albicans induced IL-1β production. and histological assessment revealed preferential growth of hyphae in the pyelum of the caspase-1–/– mice. In contrast, ASC–/– mice did not have higher fungal loads, but they showed a significant stronger inflammatory reaction in the kidneys. On days 3 and 7 of infection, the ASC–/– mice splenocytes that were restimulated with Candida specifically showed a higher TNF production. NLRP3–/– and P2X7–/– did not display an increased susceptibility to disseminated candidiasis, as shown by normal survival and fungal loads in the organs. Local IL-1β production was lower in caspase-1–/– mice, but not in the ASC–/–, NLRP3–/– or P2X7–/– animals. Experiments using the NADPH inhibitor diphenyl- eneiodonium, or in monocytes isolated from CGD patients who have defective capacity to form ROS, demonstrated that ROS did not mediate inflammasome activation and C. albicans induced IL-1β production. Methods Retrospective data collection of all patients with severe sepsis or septic shock who were admitted to the ITU over 3 months (December 2008, January and February 2009). All patients who developed sepsis before admission to the ITU/HDU were included. Results Thirty-three patients were admitted to the ITU at West Middlesex Hospital with either severe sepsis or septic shock. Median age was 72 years. The overall mortality rate was 50%. Patients with septic shock had a mortality rate of 52%. The results of the initial resuscitation of the patients are summarized in Table 1. In septic shock patients, only 35% had ITU intervention within 6 hours (had CVP insertion and/or started on vasopressor and/or inotropic support). Central venous oxygen saturation or mixed venous oxygen saturation was not measured for these patients. Conclusions Early recognition and the initial resuscitation of sepsis at this District General Hospital were assessed for the first time. Patients with severe sepsis or septic shock were not resuscitated appropriately and the SSC guidelines were not implemented, resulting in a high mortality rate. The results showed that there is a delay in recognizing sepsis at early stages resulting in inadequate management of patients. In septic shock patients, this resulted in delayed CVP measurement and administration of vasopressors and/or inotropic support. Table 1 (abstract P32) S15 Table 1 (abstract P32) Breakdown of tasks of the initial resuscitation bundle achieved within 6 hours % of patients Number of patients where SSC achieved/total number recommendation Initial resuscitation tasks (within 6 hours) of patients was followed Serum lactate measured 25/33 76 Obtaining blood cultures prior to antibiotic administration 6/33 18 Broad-spectrum antibiotics within 3 hours from time of presentation for Emergency Department admissions 6/9 67 1 hour for non-Emergency Department ICU admissions 5/24 21 In patients with septic shock or serum lactate >4 mmol/l (36 mg/dl) Fluid challenges 7/23 30 Vasopressors 17/23 74 CVP >8 mmHg in nonmechanically ventilated patients (12 to 15 in mechanically ventilated patients) 2/23 9 Breakdown of tasks of the initial resuscitation bundle achieved within 6 hours P33 Z Aboud, T Peters ICU Department, West Middlesex Hospital, London, UK Critical Care 2009, 13(Suppl 4):P32 (doi: 10.1186/cc8088) The selective V1a receptor agonist FE 202158 does not cause von Willebrand factor release in sheep unlike arginine vasopressin Introduction Mortality associated with severe sepsis remains high at 30 to 50% and rises to 50 to 60% when shock is present. The Surviving Sepsis Campaign (SSC) recommends two bundles for severe sepsis management to achieve 25% reduction in mortality; the Initial Resuscitation Bundle (within the first 6 hours) and the Management Bundle (within 24 hours). West Middlesex University Hospital set up a severe sepsis management protocol based on the SSC initial resuscitation and management bundles. It is a 350-bed hospital with an emergency department. Five hundred patients (medical and surgical) are admitted to the critical care unit per year. Objective To assess the early recognition of sepsis and the application of the initial resuscitation bundle according to SSC guidelines at West Middlesex University Hospital. S Rehberg1, P Enkhbaatar1, R Laporte2, J Rehberg1, E La2, K Wisniewski2, LD Traber1, CD Schteingart2, PJM Riviere2, DL Traber1 1Investigational Intensive Care Unit, Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA; 2Ferring Research Institute, Inc., San Diego, CA, USA Critical Care 2009, 13(Suppl 4):P33 (doi: 10.1186/cc8089) 1Investigational Intensive Care Unit, Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA; 2Ferring Research Institute, Inc., San Diego, CA, USA Critical Care 2009, 13(Suppl 4):P33 (doi: 10.1186/cc8089) Introduction The mixed V1a/V2 receptor agonist arginine vaso- pressin (AVP) is recommended by the guidelines of the Surviving Sepsis Campaign as an adjunct vasopressor in norepinephrine- resistant septic shock. However, AVP may be procoagulant Available online http://ccforum.com/supplements/13/S4 Methods Retrospective data collection of all patients with severe sepsis or septic shock who were admitted to the ITU over 3 months (December 2008, January and February 2009). All patients who developed sepsis before admission to the ITU/HDU were included. Results Thirty-three patients were admitted to the ITU at West Middlesex Hospital with either severe sepsis or septic shock. Median age was 72 years. The overall mortality rate was 50%. Patients with septic shock had a mortality rate of 52%. The results of the initial resuscitation of the patients are summarized in Table 1. In septic shock patients, only 35% had ITU intervention within 6 hours (had CVP insertion and/or started on vasopressor and/or inotropic support). Central venous oxygen saturation or mixed venous oxygen saturation was not measured for these patients. Conclusions Early recognition and the initial resuscitation of sepsis at this District General Hospital were assessed for the first time. Patients with severe sepsis or septic shock were not resuscitated appropriately and the SSC guidelines were not implemented, resulting in a high mortality rate. The results showed that there is a delay in recognizing sepsis at early stages resulting in inadequate management of patients. In septic shock patients, this resulted in delayed CVP measurement and administration of vasopressors and/or inotropic support. Therefore, we have suggested an educational programme running throughout the year to educate medical and nursing teams about the early recognition and management of sepsis, with emphasis on the strict implementation of all tasks of sepsis protocol according to SSC guidelines to reduce the mortality rate by 25%. We also suggest setting up critical care beds on each ward that will be supported by ITU outreach for CVP insertion and level 1 monitoring. and histological assessment revealed preferential growth of hyphae in the pyelum of the caspase-1–/– mice. In contrast, ASC–/– mice did not have higher fungal loads, but they showed a significant stronger inflammatory reaction in the kidneys. On days 3 and 7 of infection, the ASC–/– mice splenocytes that were restimulated with Candida specifically showed a higher TNF production. NLRP3–/– and P2X7–/– did not display an increased susceptibility to disseminated candidiasis, as shown by normal survival and fungal loads in the organs. Local IL-1β production was lower in caspase-1–/– mice, but not in the ASC–/–, NLRP3–/– or P2X7–/– animals. Available online http://ccforum.com/supplements/13/S4 Therefore, we have suggested an educational programme running throughout the year to educate medical and nursing teams about the early recognition and management of sepsis, with emphasis on the strict implementation of all tasks of sepsis protocol according to SSC guidelines to reduce the mortality rate by 25%. We also suggest setting up critical care beds on each ward that will be supported by ITU outreach for CVP insertion and level 1 monitoring. Conclusions Caspase-1-dependent processing of IL-1β is an important step in antifungal host defense during Candida sepsis. However, this process is not dependent on the inflammasome components NLRP3, the ATP receptor P2X7, or ROS. These data confirm previous studies in human monocytes showing that IL-1β processing during Candida infection did not require pathogen- mediated inflammasome activation, due to the constitutive activation of caspase-1. ASC also plays an important role in Candida sepsis, but unexpectedly seems to have a different function, specifically by regulating TNF production and local inflammation in the organs. P32 Early recognition and management of sepsis at West Middlesex University Hospital Differential effects of IL-17 pathway in disseminated candidiasis and zymosan-induced multiple organ failure Caspase-1–/– mice had a 100-fold increase in fungal loads in the kidneys of the deficient animals (P <0.05) Introduction The role of the IL-17 pathway in fungal sepsis remains controversial. Several studies suggested that IL-17 is crucial for the protection against Candida sepsis, while other S14 Available online http://ccforum.com/supplements/13/S4 Serum lactate measured S15 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 through V2 receptor-mediated effects (for example, von Willebrand factor (vWF) release). Methods Ewes were operatively prepared and randomized after a 7-day recovery period into control, MRSA, and Ps. aeruginosa groups (n = 6). Injury consisted of instillation of (2 to 5) x 1011 CFU live MRSA or Ps. aeruginosa into the airway, and the sheep were sacrificed after 24 hours. In addition, groups of C57Bl/6J wild-type mice and iNOS knockout mice (n = 6) were nasally inoculated with (2 to 5) x 108 CFU live MRSA or Ps. aeruginosa and were sacrificed after 8 hours. through V2 receptor-mediated effects (for example, von Willebrand factor (vWF) release). Objective We hypothesized that the selective V1a receptor agonist FE 202158, which lacks the activity at the V2 receptor, might not have the procoagulant effects of AVP. This hypothesis was tested by measuring vWF antigen (vWF:Ag) activity in plasma of healthy sheep during administration of either FE 202158, AVP, the selective V2 receptor agonist desmopressin, or vehicle. Methods After measurements of vWF:Ag activity and hemoglobin concentration in blood over a 1-hour baseline period, 24 female sheep were randomly assigned to receive either an intravenous bolus of the selective V2 receptor agonist desmopressin (1 nmol/kg) or a continuous intravenous infusion of AVP (3 pmol/kg/min), the selective V1a receptor agonist FE 202158 (10 pmol/kg/min) or vehicle (0.9% NaCl, n = 6 each). The infusion rates were represen- tative of the requirements for the treatment of sepsis-induced vasodilatory hypotension in sheep. vWF:Ag activity and hemo- globin concentration were measured 60, 90 and 120 minutes after initiation of treatment. Because of the V2 receptor-mediated fluid retention, vWF:Ag activity was corrected for plasma volume changes by calculating the ratio of vWF:Ag activity/hemoglobin concentration (vWF:Ag/Hb). Data are expressed as a percentage of the mean baseline value and presented as mean ± SEM. Results Ps. aeruginosa-treated sheep had a significantly higher ADMA (1.79 ± 0.14 vs. 1.16 ± 0.24 μM, P <0.05), lower plasma NOx (6.83 ± 0.17 vs. 9.63 ± 0.64 μM, P <0.05), and higher arginase activity (1.55 ± 0.16 vs. 1.07 ± 0.11 μM urea/μg protein, P <0.05) compared with MRSA-treated sheep. These changes were associated with more pronounced lung injury in Ps. aerugi- nosa sepsis compared with MRSA sepsis (PaO2/FiO2: 205 ± 72 vs. P34 Methods We prospectively studied 14 consecutive septic shock patients with a pulmonary artery catheter in place. For 3 days after admission, hemodynamic variables, and plasma levels of lactate, thrombin–antithrombin complexes (TAT), tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI) and plasmin– α2-antiplasmin complexes and TNFα, IL-6 and complement activation product C3a were measured 6-hourly. Serum lactate measured 319 ± 82, P <0.05). Ps. aeruginosa-treated mice had a signifi- cantly higher arginase activity (0.60 ± 0.12 vs. 0.16 ± 0.0038 μM urea/μg protein, P <0.05) and higher protein oxidation with carbonyl groups (3,223 ± 440.7 vs. 1,124 ± 140.1, P <0.05) compared with MRSA-treated mice. iNOS knockout mice treated with MRSA had significantly lower arginase activity than wild-type mice (0.21 ± 0.01 vs. 0.36 ± 0.04 μM urea/μg protein, P <0.05). Conclusions Suppressed NO production in Ps. aeruginosa sepsis is caused by the increased expression of ADMA. Increased arginase activity is most probably caused by augmented oxidative stress, which results in a more severe lung injury. Results Whereas there were no significant changes in vWF:Ag/Hb in vehicle-treated animals over time, desmopressin and AVP caused an immediate increase in vWF:Ag/Hb after 60 minutes (129 ± 6% and 121 ± 2% of baseline (100%), respec- tively; P <0.01 each). At each time point during the 120-minute study period, vWF:Ag/Hb was significantly higher in desmo- pressin-treated and AVP-treated animals than in vehicle-treated animals (P <0.001 each). In contrast, there was no significant difference between FE 202158-treated and vehicle-treated animals (P = 0.225). Notably, vWF:Ag/Hb in the FE 202158 group (maximum 108 ± 2% at 120 minutes) was significantly lower than the AVP group (maximum 123 ± 2% at 60 minutes; P ≤0.005) and the desmopressin group (maximum 138 ± 6% at 120 minutes; P <0.001) at every time point. Disseminated intravascular coagulation during human septic shock: relation with lactate levels KJ Hartemink1,2, CE Hack3, ABJ Groeneveld1 1Department of Intensive Care and the Institute for Cardiovascular Research, 2Department of Surgery, and 3Department of Clinical Chemistry, VU University Medical Center, Amsterdam, the Netherlands Critical Care 2009, 13(Suppl 4):P35 (doi: 10.1186/cc8091) Conclusions Unlike AVP, the selective V1a receptor agonist FE 202158 did not increase vWF:Ag/Hb ratios in plasma compared with vehicle-treated animals. Therefore, a selective V1a receptor agonist such as FE 202158 might be superior to AVP or other mixed V1a/V2 receptor agonists under conditions that produce activation of the coagulation system, such as severe sepsis and septic shock. Introduction The exact pathogenic role of disseminated intra- vascular coagulation (DIC) during septic shock is incompletely understood. Introduction The exact pathogenic role of disseminated intra- vascular coagulation (DIC) during septic shock is incompletely understood. Objective We studied the relation between sensitive and specific markers for DIC and lactate levels in the course of time, to evaluate whether DIC could contribute to microvascular obstruction and tissue hypoxygenation. R Barchetta, C Alessandrini, C Di Corato, F Candidi, F Turani, M Falco Department of Anaesthesia and Intensive Care, European Hospital, Rome, Italy Critical Care 2009, 13(Suppl 4):P36 (doi: 10.1186/cc8092) MV Schmidt1, P Paulus2, A-M Kuhn1, V Meilladec-Jullig1, K Zacharowski2, B Bruene1, A von Knethen1 1Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Germany; 2Department of Anaesthesia, Intensive Care Medicine & Pain Therapy, University Hospital Frankfurt, Germany Critical Care 2009, 13(Suppl 4):P37 (doi: 10.1186/cc8093) Introduction Procalcitonin (PCT) and IL-6 are markers used in the evaluation of systemic inflammation (SIRS) and septic states. The purpose of this study is to analyse changes in plasma concen- trations of PCT and IL-6 in patients undergoing cardiac surgery on- pump and assess its reliability in the early detection of post- operative infectious complications. In all patients the variation of brain natriuretic peptide (BNP) was also evaluated in order to stratify the clinical condition of patients. Introduction Immune paralysis with massive T-cell apoptosis is a central pathogenic event during sepsis and correlates with septic patient mortality. Previous observations implied a crucial role of peroxisome proliferator-activated receptor gamma (PPARγ) during T-cell apoptosis. Methods To elucidate mechanisms of PPARγ-induced T-cell depletion, we used an endotoxin model as well as the caecal ligation and puncture sepsis model to imitate septic conditions in wild-type versus conditional PPARγ knockout (KO) mice. Methods We measured serum levels of PCT, IL-6 and BNP in adult patients undergoing myocardial revascularization and/or valve surgery performed in extracorporeal circulation. The measurements were performed on the day before the intervention (T0), at the end of surgery (T1) and then until the third and fourth postoperative day (T2 to T4). We also recorded the onset of cardiac, respiratory, neurological, renal and septic complications. The diagnosis of sepsis was confirmed retrospectively on the basis of clinical, radiological and microbiological data. All data are expressed as mean and standard deviation. The Kruskal–Wallis test was used to assess changes over time of variables. P <0.05 was considered statistically significant. Results PPARγ KO mice showed a marked survival advantage compared with control mice. Their T cells were substantially protected against sepsis-induced death and showed a significantly higher expression of the pro-survival factor IL-2. Since PPARγ is described to repress nuclear factor of activated T cells (NFAT) transactivation and concomitant IL-2 expression, we propose inhibition of NFAT as the underlying mechanism allowing T-cell apoptosis. Corroborating our hypothesis, we observed up- regulation of the pro-apoptotic protein BIM and downregulation of the anti-apoptotic protein Bcl-2 in control mice, which are down- stream effector proteins of IL-2 receptor signaling. T-cell-specific peroxisome proliferator-activated receptor gamma depletion inhibits T-cell apoptosis and improves survival of septic mice via an IL-2-dependent mechanism T-cell-specific peroxisome proliferator-activated receptor gamma depletion inhibits T-cell apoptosis and improves survival of septic mice via an IL-2-dependent mechanism Table 1 (abstract P36) Table 1 (abstract P36) Results of temporal changes and significance T0 T1 T2 T3 T4 PCT (ng/ml) Nonseptic 0.04 0.04 0.58 0.34 0.34 Septic 0.04 0.15 2.63 1.87 0.74 P NS <0.001 <0.001 <0.001 <0.01 IL-6 (pg/ml) Nonseptic 12 160 129 78 75 Septic 18 184 145 261 92 P NS NS NS <0.01 NS BNP Nonseptic 159 154 347 428 492 Septic 373 627 731 756 798 P <0.01 < 0.01 <0.01 <0.05 <0.05 Conclusions In patients who develop septic complications, changes in PCT occur earlier than changes in IL-6. Furthermore, BNP performs in the same fashion as PCT and correlates better than IL-6 with the clinical data of the infection status. In conclusion, monitoring PCT seems to be useful in early diagnosis of septic complications in patients undergoing cardiac surgery and more sensitive on the variations in IL-6. The combined study of variations in PCT and BNP could improve the diagnostic accuracy in these patients. R Barchetta, C Alessandrini, C Di Corato, F Candidi, F Turani, M Falco Department of Anaesthesia and Intensive Care, European Hospital, Rome, Italy Critical Care 2009, 13(Suppl 4):P36 (doi: 10.1186/cc8092) Application of a neutralizing anti-IL-2 antibody reversed the pro-survival effect of PPARγ-deficient T cells and confirmed IL-2-dependent apoptosis during sepsis. Results There have been enrolled 60 patients undergoing cardiac surgery in extracorporeal circulation. Among these, nine patients developed septic complications. The results of temporal changes and the significance are presented in Table 1. Table 1 (abstract P36) Conclusions Apparently antagonizing PPARγ in T cells might improve their survival during sepsis, which concomitantly enhances defence mechanisms and possibly provokes an increased survival of septic patients. Increased asymmetrical dimethyl-arginine suppresses nitric oxide production in Pseudomonas aeruginosa sepsis L Sousse, C Jonkam, D Traber, S Rehberg, L Traber, D Herndon, P Enkhbaatar Department of Experimental Pathology and Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA Critical Care 2009, 13(Suppl 4):P34 (doi: 10.1186/cc8090) Results Of the 14 patients, eight died in the ICU. Patients had a hyperdynamic circulation with tachycardia, mild hypotension and increased cardiac index. The course of TAT, tPA and particularly of PAI predicted the course of lactate levels, independently of hemodynamic and inflammatory factors. Lactate and PAI elevations persisted in nonsurvivors versus survivors. Introduction More than 750,000 patients in the United States develop sepsis annually. Previously, we have shown that plasma nitric oxide (NO) levels were approximately sevenfold higher and that arginase activity is significantly lower in methicillin-resistant Staphylococcus aureus (MRSA) sepsis than in Pseudomonas aeruginosa sepsis. In the present study, we hypothesize that increased asymmetrical dimethyl-arginine (ADMA), an endogenous inhibitor of NO synthase, is responsible for the suppressed NO production in Ps. aeruginosa sepsis. Introduction More than 750,000 patients in the United States develop sepsis annually. Previously, we have shown that plasma nitric oxide (NO) levels were approximately sevenfold higher and that arginase activity is significantly lower in methicillin-resistant Staphylococcus aureus (MRSA) sepsis than in Pseudomonas aeruginosa sepsis. In the present study, we hypothesize that increased asymmetrical dimethyl-arginine (ADMA), an endogenous inhibitor of NO synthase, is responsible for the suppressed NO production in Ps. aeruginosa sepsis. Conclusions Our observations show that, in the course of human septic shock, activation of coagulation and, particularly, inhibition of activated fibrinolysis are independently associated with hyperlactatemia. This suggests a contribution of DIC resulting from a coagulation/fibrinolysis imbalance to microvascular obstruction, tissue hypoxygenation and thereby to ultimate demise. S16 Available online http://ccforum.com/supplements/13/S4 P36 P38 Induction of severe Staphylococcus aureus sepsis in pigs Monitoring of procalcitonin, IL-6 and brain natriuretic peptide for sepsis diagnosis in cardiac surgery R Barchetta, C Alessandrini, C Di Corato, F Candidi, F Turani, M Falco Department of Anaesthesia and Intensive Care, European Hospital, Rome, Italy Critical Care 2009, 13(Suppl 4):P36 (doi: 10.1186/cc8092) Induction of severe Staphylococcus aureus sepsis in pigs r-Roscovitine (70 mg/kg) or vehicle was administered 24 hours later in combination with antibiotic therapy (ceftriaxon; 20 mg/kg). Mice were sacrificed after 48 hours. In a second experiment, mice were infected and treated at 24 and 72 hours and sacrificed 96 hours post infection. Methods Twelve pigs in four groups were inoculated intravenously once or twice with 1 x 108 S. aureus/kg body weight and eutha- nized consecutively from 6 to 48 hours after inoculation. Mock- inoculated pigs served as controls. Body temperature was measured and blood samples were taken at regular intervals for bacteriology, haematology, clinical chemistry, and acute phase reactant determinations. Full necropsy was done and tissue samples were collected for bacteriology and histology. Apoptosis was measured in the spleen. Results r-Roscovitine treatment significantly reduced phosphory- lated CDK substrate and increased cleaved caspase 3 levels in isolated neutrophils. During LTA-induced lung inflammation, r-roscovitine treatment significantly reduced the amount of PMNs in the BAL fluid and cytokines in lung homogenates. After 48 hours of bacterial pneumonia, r-roscovitine-treated animals displayed enhanced pulmonary bacterial outgrowth. Cytokine production and lung damage scores were higher in the r-roscovitine-treated group as compared with vehicle. Interestingly, when studying the animals at 96 hours post infection, r-roscovitine treatment resulted in lower bacterial outgrowth and chemokine levels in the lung. Results Onset of clinical disease (fever and lethargy) was seen at 7 to 8 hours after inoculation. Blood bacterial counts remained low throughout the study. SIRS characterized by fever, leukocytosis, increased levels of CRP, IL-6, IL-1β, TNFα, and decreased level of serum iron was detected after 12 hours. Both CRP and IL-6 levels peaked at 36 hours. Platelet numbers declined slightly and were lower than in the controls at 48 hours. Thromboelastography showed increased hypercoagulability over time. Levels of serum aspartate aminotransferase and bilirubin were elevated at 24 and 36 hours. Blood urea nitrogen levels had increased at 36 hours; however, no difference was seen in serum creatinine levels. Disseminated microabscesses were found in the lung at 6 hours, but had disappeared at 48 hours. In the bones, the presence of microabscesses progressed until 48 hours. Other histopatho- logical signs related to inoculation were limited to a renal microabscess at 12 hours, splenic microabscesses at 24 hours and centrilobular hepatic necrosis with thrombosis in one animal at 48 hours. In the liver and kidneys, various degrees of fibrinous exudation were found. Honey as an immunomodulator during sepsis in animal model Honey as an immunomodulator during sepsis in animal model Induction of severe Staphylococcus aureus sepsis in pigs TM Iburg1, PS Leifsson1, M Kjelgaard-Hansen2, P Heegaard3, B Wiinberg2, B Aalbaek1, AE Olsson1, MGS Hansen1, LB Thomsen1, HE Jensen1, JS Agerholm1, OL Nielsen1 1Department of Veterinary Disease Biology, and 2Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark; 3Department of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark Critical Care 2009, 13(Suppl 4):P38 (doi: 10.1186/cc8094) Introduction Organ dysfunction is an integrated part of severe sepsis, and severe sepsis is one of the major causes of death in ICUs. Lately Gram-positive bacteria accounted for more than one- half of the overall sepsis cases reported in the USA, with Staphylococcus aureus being the most commonly isolated bacterium. Effective treatment of sepsis is still not optimal and good animal models are needed for research in pathogenesis and treatment. S. aureus infections are also common in pigs and are isolated from approximately 40% of embolic lesions found in slaughter-pigs. Conclusions In patients who develop septic complications, changes in PCT occur earlier than changes in IL-6. Furthermore, BNP performs in the same fashion as PCT and correlates better than IL-6 with the clinical data of the infection status. In conclusion, monitoring PCT seems to be useful in early diagnosis of septic complications in patients undergoing cardiac surgery and more sensitive on the variations in IL-6. The combined study of variations in PCT and BNP could improve the diagnostic accuracy in these patients. S17 g p g Objective To establish a porcine model of severe sepsis. Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 70 mg/kg r-roscovitine or vehicle was injected intraperitoneally. Twenty-four hours later bronchoalveolar lavage (BAL) was performed and differential cell counts were determined. Bacterial pneumonia was induced by inoculation of 5 x 104 CFU S. pneumoniae. r-Roscovitine (70 mg/kg) or vehicle was administered 24 hours later in combination with antibiotic therapy (ceftriaxon; 20 mg/kg). Mice were sacrificed after 48 hours. In a second experiment, mice were infected and treated at 24 and 72 hours and sacrificed 96 hours post infection. 70 mg/kg r-roscovitine or vehicle was injected intraperitoneally. Twenty-four hours later bronchoalveolar lavage (BAL) was performed and differential cell counts were determined. Bacterial pneumonia was induced by inoculation of 5 x 104 CFU S. pneumoniae. Induction of severe Staphylococcus aureus sepsis in pigs The number of apoptotic cells in the splenic white pulp was increased at 48 hours. Conclusions With this study, we reproduced earlier findings that r-roscovitine treatment reduces CDK activity and induces apoptosis in neutrophils; we demonstrated that r-roscovitine diminishes inflammatory responses in sterile inflammation; and we found that r-roscovitine treatment in bacterial pneumonia is detrimental early in infection but beneficial at later time points. We believe that the negative effect of r-roscovitine reflects the importance of neutrophil antibacterial defense early in infection. Yet, during resolution of infection, apoptosis of neutrophils induced by r-roscovitine could present a way of damage control as opposed to unwanted side effects of neutrophil function. Conclusions All infected pigs developed sepsis with metastatic abscesses and at 48 hours severe sepsis was present with signs of dysfunction of the liver and the coagulation system. The splenic apoptotic response indicates reduced function and immuno- suppression. P39 M Kassim1, M Mansor1, M Achoui2, OS Yan1, S Devi3, KM Yusoff4 1Department of Anesthesiology, 2Department of Pharmacology, 3Department of Medical Microbiology, and 4Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Critical Care 2009, 13(Suppl 4):P40 (doi: 10.1186/cc8096) M Kassim1, M Mansor1, M Achoui2, OS Yan1, S Devi3, KM Yusoff4 Cyclin-dependent kinase inhibitor r-roscovitine reduces lipoteichoic acid lung inflammation and improves the resolution of antibiotic-treated Streptococcus pneumoniae pneumonia 1Department of Anesthesiology, 2Department of Pharmacology, 3Department of Medical Microbiology, and 4Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Critical Care 2009, 13(Suppl 4):P40 (doi: 10.1186/cc8096) P41 Are phenylcarboxylic acids really markers in severe sepsis? Are phenylcarboxylic acids really markers in severe sepsis? Objective A multidisciplinary public health campaign was developed to educate a local urban community about recognizing the signs and symptoms of sepsis, preventing infection, and seeking treatment early. Targeting education efforts at the community level engages consumers to become involved in the care of their health. NV Beloborodova, AS Khodakova, AJ Olenin Bakoulev Scientific Center of Cardiovascular Surgery, Moscow, Russian Federation Critical Care 2009, 13(Suppl 4):P41 (doi: 10.1186/cc8097) Introduction Laboratory diagnostics of sepsis need to be improved. There is no evidence in the literature whether microbial metabolites could be used as sepsis markers. As a result of large- scale screening of microbial compounds we showed that levels of some phenylcarboxylic acids (PCAs) were increased in blood of septic patients. The content of p-hydroxyphenyllactic acid (HPLA), phenyllactic acid (PLA) and p-hydroxyphenylacetic acid (HPAA) was significantly higher in blood of patients with severe sepsis compared with control groups. The aim of the present study is to evaluate the sensitivity and specificity of PLA, HPLA and HPAA as markers for severe sepsis diagnostics. Methods A multimedia approach including print, television broadcast, hospital website on the Internet, news article and advertisement in the newspaper were widely distributed to maximize the ability to reach citizens throughout the area. A live seminar presented by two physicians and a clinical nurse specialist was strategically used to enhance learning, and participants completed an evaluation upon completion of the seminar. Results Ninety-six percent of the participants who attended the live seminar rated the program as excellent. The participants shared that the topic was very educational, informative, and felt that their questions were answered. Because of the education media campaign that targeted 320,000 households and the hospital-wide implementation of the sepsis bundles, the mortality for severe sepsis decreased from 40% at baseline to 32.8%, which was a 18% relative risk reduction (Figure 1). Methods In total, 264 blood samples from 200 adults were included to research. All persons were divided into groups with infectious complications after cardiosurgery (35 severe sepsis, 35 local infection complication) and others (33 non-infection compli- cation after surgery, 30 smooth recovery after surgery, 42 before surgery and 25 healthy volunteers). Clinical characteristics and procalcitonin (PCT), a well established biomarker of sepsis, were assessed in all patients. Impact of community-based education on sepsis Impact of community-based education on sepsis K Choy, CA Agcaoili, K Halimi Washington Hospital Healthcare System, Fremont, CA, USA Critical Care 2009, 13(Suppl 4):P42 (doi: 10.1186/cc8098) Introduction Sepsis is an uncontrolled infection that can develop very quickly throughout the body. Sepsis can strike anyone at any age and people with pre-existing medical conditions may be at greater risk. Patients with sepsis often present to the emergency department from home. Community education programs focus on raising public awareness of sepsis, its signs and symptoms, and can positively impact outcomes. Introduction Sepsis is an uncontrolled infection that can develop very quickly throughout the body. Sepsis can strike anyone at any age and people with pre-existing medical conditions may be at greater risk. Patients with sepsis often present to the emergency department from home. Community education programs focus on raising public awareness of sepsis, its signs and symptoms, and can positively impact outcomes. pneumonia Obtained data indicate that quantitative measurement of PLA, HPLA and HPAA in blood could be used for sepsis diagnostics in clinical practice and also as a predictor of outcome in high-risk surgery. Conclusions The following levels of PCAs are appropriate for diagnostic of sepsis: HPAA 8 x 10–6 M (sensitivity 64.3%, speci- ficity 88.9%), HPLA 3 x 10–6 M (sensitivity 75%, specificity 66.4%), PLA 1 x 10–6 M (sensitivity 75%, specificity 71.6%). Obtained data indicate that quantitative measurement of PLA, HPLA and HPAA in blood could be used for sepsis diagnostics in clinical practice and also as a predictor of outcome in high-risk surgery. pneumonia AJ Hoogendijk1,2, JJTH Roelofs3, MHP van Lieshout1,2, DC Blok1,2, T van der Poll1,2, CW Wieland2,4 1Center for Infection and Immunity Amsterdam, 2Center for Experimental and Molecular Medicine, 3Department of Pathology, and 4Laboratory of Experimental Intensive Care and Anesthesiology, Academic Medical Center, Amsterdam, the Netherlands Critical Care 2009, 13(Suppl 4):P39 (doi: 10.1186/cc8095) Introduction Malaysian honey (Gelam) has antibacterial activity and it also has a high antioxidant capacity and free radical scavenger activities. Honey extracts showed potent activity against TNFα in L929 cell and NO in RAW 264.7 macrophage as well as inhibitory effects on the prostaglandin E2 and nitric oxide (NO) in inflammatory tissues of rat. Sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (for example, TNF, IL-1) cytokines and inducible enzymes such as inducible nitric oxide (iNOS) synthase and heme oxygenase 1 (HO-1) and late such as high-mobility group box 1 (HMGB1). Introduction Streptoccocus pneumoniae pneumonia remains associated with high morbidity and mortality. Antibiotic treatment frequently is insufficient in limiting lung damage due to inflam- mation. Therefore, additional treatment strategies are needed. The drug r-roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was demonstrated to reduce inflammation in several models of inflammation. Objective To investigate the role of honey as an immunomodulator in sepsis induced by LPS in rats. Methods Four groups (n = 6) of rats were used. The treatment group received honey with LPS, the positive control group were given LPS (5 mg/kg), the negative control group were given saline only, while the fourth group were only given honey; all doses were 1 ml by intravenous route. Blood samples were collected 4 hours later and all rats were sacrificed after 24 hours. TNFα, IL-1β, IL-6, IL-10, NO, HO-1 and HMGB1 were quantified using ELISA. The effect of honey on coagulation (PT and APPT) in whole blood ex vivo from healthy volunteers (n = 10) was measured. Objective We studied the potential of r-roscovitine to modulate host defense during sterile inflammation and bacterial infection of the lung. Methods Isolated neutrophils were treated with 20 μM r-roscovitine and CDK and caspase 3 activity were determined by western blot analysis. Sterile lung inflammation was induced by intranasal administration of 100 μg lipoteichoic acid (LTA), a prominent cell wall component of Gram-positive bacteria. Simultaneously S18 Available online http://ccforum.com/supplements/13/S4 Results After 4 hours of treatment, the cytokines, NO and HO-1 were measured in all groups. pneumonia Honey showed evidence of immuno- modulatory effects with reduced cytokines (TNFα (P < 0.001), IL-1β (P < 0.001), IL-10 (P < 0.001)) and NO (P < 0.037) in the treatment group, while the change in IL-6 was not significant between all groups, HO-1 (P < 0.001) was increased in the treatment group, but only slightly increased in the honey group. After 24 hours of treatment, HMGB1 (P <0.025) and IL-1β (P <0.001) were reduced in the treatment group as well. HO-1 (P <0.013) continuously increased in all groups. Curiously, honey alone induced TNFα (P <0.001) and IL-1β (P <0.03) at 4 hours, and HO-1 (P <0.028) at 24 hours compared with saline. Honey prolonged the time of PT and APPT in a dose-dependent manner. Results After 4 hours of treatment, the cytokines, NO and HO-1 were measured in all groups. Honey showed evidence of immuno- modulatory effects with reduced cytokines (TNFα (P < 0.001), IL-1β (P < 0.001), IL-10 (P < 0.001)) and NO (P < 0.037) in the treatment group, while the change in IL-6 was not significant between all groups, HO-1 (P < 0.001) was increased in the treatment group, but only slightly increased in the honey group. After 24 hours of treatment, HMGB1 (P <0.025) and IL-1β (P <0.001) were reduced in the treatment group as well. HO-1 (P <0.013) continuously increased in all groups. Curiously, honey alone induced TNFα (P <0.001) and IL-1β (P <0.03) at 4 hours, and HO-1 (P <0.028) at 24 hours compared with saline. Honey prolonged the time of PT and APPT in a dose-dependent manner. Conclusions Honey behaves as immunomodulator by acting in two ways, by inducing HO-1, TNFα, and IL-1β and at the same time inhibiting cytokines, NO and HMGB1 that is induced by LPS. However, the exact mechanism remain unclear, but our suggestion is that since honey induces HO-1, TNFα and IL-1β this may cause changes or inhibition in the signaling of cytokines and NF-κB. Honey could therefore be used as a pharmacological tool in sepsis in the future. 34.7) x 10–6 M vs. 2.8 (2.2 to 5.0) x 10–6 M correspondingly), but not for HPAA. Conclusions The following levels of PCAs are appropriate for diagnostic of sepsis: HPAA 8 x 10–6 M (sensitivity 64.3%, speci- ficity 88.9%), HPLA 3 x 10–6 M (sensitivity 75%, specificity 66.4%), PLA 1 x 10–6 M (sensitivity 75%, specificity 71.6%). P41 Severe sepsis was diagnosed according to consensus criteria, also a level of PCT ≥2 ng/ml was an additional criterion. Blood concentrations of PCAs were deter- mined by gas chromatography–mass spectrometry. IL-33 protects mice from sepsis by inhibiting TLR4 signaling aureus was attempted using the following techniques directly from the positive bottles: (1) morphology and organization of the cocci on Gram stain (10 minutes), (2) rapid tube coagulase (2 hours), (3) direct DNAse test using toluedine blue agar (4 hours), (4) species-specific multiplex PCR (4 hours), (5) direct inoculation of selective commercial chromogenic media (18 hours), and (6) direct inoculation of mannitol-supplemented Mueller–Hinton agar (MMHA) combined with disc diffusion susceptibility testing (18 hours). Identifications were compared with the standard laboratory identification at 36 hours. Objective To investigate the role of the IL-33/ST2 pathway in experimental sepsis. Methods and results We show that IL-33 treatment (1 μg/mouse, intravenously) markedly reduced mortality in WT mice (50%, P <0.01) undergoing experimental sepsis induced by the cecal ligation and puncture (CLP) model. We did not detect any differences between ST2 KO and WT mice in peritoneal bacterial load and mortality rate after CLP, suggesting that the endogenous IL-33 does not participate in the pathophysiology sepsis. However, while the exogenous injection of IL-33 markedly reduced the CLP- induced mortality in WT mice, IL-33 failed to do so in ST2–/– mice, indicating the critical role of ST2 on the protective effect of IL-33. Notably, we found that IL-33-treated mice developed significantly increased neutrophil infiltration in the peritoneal cavity (fourfold) and more efficient bacterial clearance than untreated mice (n = 10) after CLP. Moreover, IL-33 treatment leads to marked reduction (30 to 70%) of systemic proinflammatory cytokines (TNFα, IL-6 and CXCL2) but not a shift toward a Th2 immune response (IL-4 and IL-13). The chemokine receptor CXCR2 plays a central role in the recruitment of neutrophils into the site of infection. Flow cytometry analysis showed that direct activation of TLR4 in neutrophils downregulates the expression of CXCR2 and, consequently, impaired CXCL2-driven and CLP-driven neutrophil migration, in vitro and in vivo respectively. Notably, IL-33 prevented the downregulation of CXCR2 on circulating neutrophils during CLP in vivo or LPS-treated neutrophils in vitro. Finally, we demonstrated that IL-33 reversed the TLR4-induced reduction of CXCR2 via the inhibition of LPS-induced G-protein-coupled receptor kinase-2 (GRK2) expression, a potent negative regulator of CXCR2. Conclusions Altogether we provide here a novel mechanism of Results Definitive laboratory identification revealed S. epidermidis 87% (n = 98), S. aureus 11% (methicillin-sensitive S. aureus (MSSA) n = 9, methicillin-resistant S. aureus (MRSA) n = 3), Micrococcus spp. 2% (n = 2); one culture contained a mixture of S. IL-33 protects mice from sepsis by inhibiting TLR4 signaling Introduction The ability to rapidly identify and determine the antimicrobial susceptibility of bacterial pathogens is an undisputed requirement for strategies aimed at improving the management of sepsis. Staphylococci are amongst the most common organisms isolated from blood cultures but it is difficult to rapidly distinguish between those representing contamination of the blood culture with harmless skin commensals (Staphylococcus epidermidis) and those that contain pathogenic species (Staphylococcus aureus). IL-33 protects mice from sepsis by inhibiting TLR4 signaling JC Alves-Filho1,2, F Sonego1, FO Souto1, A Freitas1, WA Verri Jr1, D Xu2, FQ Cunha1, FY Liew2 1Department of Pharmacology, School of Medicine of Ribeirao Preto, University of São Paulo, Brazil; 2Division of Immunology, Infection and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, UK Critical Care 2009, 13(Suppl 4):P43 (doi: 10.1186/cc8099) p g p ( p y ) Objective A number of phenotypic and genotypic tests with a vast range of complexity, speed and cost have been proposed to help distinguish these organisms. We evaluated the sensitivity, specificity and speed of a range of these tests compared with the standard laboratory identification protocol which takes up to 48 hours. Introduction Sepsis is an acute systemic inflammation following infection, with a high mortality rate and limited therapeutic options. IL-33 is a recently identified member of the IL-1 family that binds to ST2 receptor, which is preferentially expressed on Th2 and mast cells. Accordingly, the IL-33/ST2 pathway is closely associated with the activation and production of type-II cytokines (IL-4, IL-5 and IL- 13). However, ST2 has been implicated in inhibiting macrophage- dependent inflammation in response to LPS by negatively regulating Toll-like receptor-4 (TLR4) activation. Although TLRs have been implicated as an important element of host defense against infections, evidence indicates that these receptors may also play a detrimental role in the pathophysiology of sepsis. y p p Methods Positive blood culture sets (BACT/Alert 3D) (n = 113) in which Gram-positive cocci in clusters were seen on the initial film were included. Further identification as S. epidermidis versus S. IL-33 protects mice from sepsis by inhibiting TLR4 signaling aureus and S. epidermidis. The sensitivity and specificity of each of the direct techniques was calculated as follows: (1) microscopy 53% and 98%, (2) rapid coagulase 92% and 100%, (3) direct DNAse 69% and 98%, (4) multiplex PCR 88% (no amplification in 12 samples) and 99%, (5) chromogenic S. aureus media 90% and 100%, and (6) MMHA 100% and 78%. Conclusions All the methods had specificity >95%, except for MMHA, although this has the added advantage of providing susceptibility results. Chromogenic agar had the highest sensitivity and specificity, although it provided a result only 12 hours quicker than the standard protocol. Rapid tube coagulase was highly specific and one of the most rapid tests. This may be the most useful, inexpensive technique providing preliminary results to guide empirical therapy, especially in resource-poor settings. P44 Conclusions This community-based education program on sepsis demonstrated that education programs offered to the community will improve overall outcomes and promote quality care of sepsis patients. In the pursuit of evaluating the effectiveness of the program, there will be ongoing monitoring of its impact. Future education programs will continue to sustain improvements. Comparison of microscopic, phenotypic and molecular techniques for the rapid identification and susceptibility testing of Staphylococci from positive blood culture bottles Comparison of microscopic, phenotypic and molecular techniques for the rapid identification and susceptibility testing of Staphylococci from positive blood culture bottles A Shah, NC Gordon, L Pheel, DW Wareham St Bartholomew’s and The London NHS Trust, London, UK Critical Care 2009, 13(Suppl 4):P44 (doi: 10.1186/cc8100) A Shah, NC Gordon, L Pheel, DW Wareham Figure 1 (abstract P42) Figure 1 (abstract P42) Figure 1 (abstract P42) Figure 1 (abstract P42) Severe sepsis mortality. Results The levels of PCA in two control groups (healthy people and patients before surgery) were HPAA 0.4 to 0.8 x 10–6 M, HPLA 1.2 to 1.5 x 10–6 M, PLA 0.3 to 0.4 x 10–6 M and were not significantly different. Otherwise the levels of HPAA, HPLA and PLA as 11.6 (3.3 to 33.6) x 10–6 M, 7.5 (3.0 to 14.4) x 10–6 M and 1.8 (1.1 to 4.9) x 10–6 M in all severe sepsis patients were significantly increased versus control groups and versus all other groups (P <0.0001). In addition the levels of PCAs from surviving and nonsurviving severe sepsis patients were compared with each other. Nonsurviving sepsis patients had a significantly higher content of PLA (3.6 (1.5 to 6.4) x 10–6 M vs. 1.2 (0.8 to 1.6) x 10–6 M for survivors); the same trend was observed for HPLA (12.5 (5.6 to Severe sepsis mortality. S19 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Acknowledgements Financial support from Wellcome Trust, MRC and FAPESP. Pre-treatment with simvastatin prevents LPS-induced hyporesponsiveness of porcine isolated coronary artery S Al-Shalmani1, S Chinniah2, R Mahajan2, V Wilson1 1School of Biomedical Sciences, and 2Academic Division of Anaesthesia and Intensive Care, University of Nottingham Medical School, Nottingham, UK Critical Care 2009, 13(Suppl 4):P46 (doi: 10.1186/cc8102) Introduction Several retrospective studies suggest that prior use of statins can reduce hospital mortality in patients diagnosed with either bacteraemia or sepsis. Simvastatin has been shown to modify the proinflammatory effect of lipopolysaccharide (LPS) on neutrophils and endothelial cells. However, it is not clear whether these effects are also manifest on vascular smooth muscle, which becomes hyporesponsive to vasoconstrictor agents due to the induction of nitric oxide synthase. We have investigated the effect of pre-treatment with simvastatin on LPS-induced changes in contractions of porcine isolated coronary artery (PCA). Introduction Several retrospective studies suggest that prior use of statins can reduce hospital mortality in patients diagnosed with either bacteraemia or sepsis. Simvastatin has been shown to modify the proinflammatory effect of lipopolysaccharide (LPS) on neutrophils and endothelial cells. However, it is not clear whether these effects are also manifest on vascular smooth muscle, which becomes hyporesponsive to vasoconstrictor agents due to the induction of nitric oxide synthase. We have investigated the effect of pre-treatment with simvastatin on LPS-induced changes in contractions of porcine isolated coronary artery (PCA). Methods Segments (5 mm) of the PCA were dissected from hearts and incubated in Krebs–Henseleit (K–H) solution at 37°C in the presence of an antibiotic mixture (60 μg/ml benzylpenicillin and 20 μg/ml streptomycin sulphate), with or without 1 μg/ml LPS, 3 μM simvastatin or a combination of the two (simvastatin added 60 minutes before LPS). After 16 to 18 hours, segments were prepared for isometric tension recording in K–H solution. The segments were then exposed to cumulatively increasing concen- trations of KCl and then U46619. In some experiments, some segments were exposed to 10 μM 1400W, a selective inhibitor of inducible nitric oxide synthase prior to the addition of the agonists. Responses are shown as gram weight or calculated as the concentration causing 50% of the maximum effect (–log EC50). Differences between mean values were assessed by ANOVA (post-hoc Dunnett test). Pre-treatment with simvastatin prevents LPS-induced hyporesponsiveness of porcine isolated coronary artery R lt KCl d U46619 d i d d Methods Segments of the PCA were dissected from hearts and incubated (2 x 5 mm) in DMEM at 37°C in the presence of an antibiotic mixture (60 μg/ml benzylpenicillin, and 20 μg/ml strepto- mycin sulphate), with or without 1 μg/ml LPS, 3 μM simvastatin or a combination of the two (simvastatin added 60 minutes before LPS). The medium also contained 1 mM L-arginine. After 24 hours, segments were removed and weighed (mg wet weight). The nitrite/nitrate content (nmol) of the bathing medium was deter- mined by spectrophotometry using the Griess reaction. In a separate experiment, segments were prepared for immunohisto- chemical determination of the presence of iNOS and CD31. Differences between mean values were assessed by ANOVA (post-hoc Dunnett test). Methods Segments (5 mm) of the PCA were dissected from hearts and incubated in Krebs–Henseleit (K–H) solution at 37°C in the presence of an antibiotic mixture (60 μg/ml benzylpenicillin and 20 μg/ml streptomycin sulphate), with or without 1 μg/ml LPS, 3 μM simvastatin or a combination of the two (simvastatin added 60 minutes before LPS). After 16 to 18 hours, segments were prepared for isometric tension recording in K–H solution. The segments were then exposed to cumulatively increasing concen- trations of KCl and then U46619. In some experiments, some segments were exposed to 10 μM 1400W, a selective inhibitor of inducible nitric oxide synthase prior to the addition of the agonists. Responses are shown as gram weight or calculated as the concentration causing 50% of the maximum effect (–log EC50). Differences between mean values were assessed by ANOVA (post-hoc Dunnett test). Results Under control conditions the coronary artery segments produced 25.9 ± 3.7 nmol/mg wet weight nitrite/nitrate (n = 8) over 24 hours. Exposure to 1 μg/ml LPS caused a sevenfold increase in nitrite/nitrate production (199.0 ± 40.6 nmol/mg wet weight, n = 8). Although 3 μM simvastatin did not affect basal nitrite/nitrate production, it inhibited the response to LPS (35.6 ± 7.4 nmol/mg wet weight nitrite/nitrate) by 94.7 ± 5.7% (n = 8). Immunohistochemical assessment of four arteries revealed the presence of CD31 on endothelial cells under control conditions. Exposure to 1 μg/ml LPS was associated with an increase in endothelial CD31 and the appearance of iNOS in the adventitia. P46 P46 either bacteraemia or sepsis. In an accompanying abstract we demonstrated that pre-treatment with simvastatin prevents LPS- induced hyporesponsiveness of the porcine isolated coronary artery (PCA); an observation consistent with the clinical data. Although this effect of simvastatin is qualitatively similar to that of a known inhibitor of inducible nitric oxide synthase (1400W), it unlikely that direct inhibition of the enzyme is implicated. We have investigated whether the beneficial effect of simvastatin on LPS- induced changes in the PCA involves alteration in the induction of nitric oxide synthase (iNOS). P46 Pre-treatment with simvastatin prevents LPS-induced hyporesponsiveness of porcine isolated coronary artery S Al-Shalmani1, S Chinniah2, R Mahajan2, V Wilson1 1School of Biomedical Sciences, and 2Academic Division of Anaesthesia and Intensive Care, University of Nottingham Medical School, Nottingham, UK Critical Care 2009, 13(Suppl 4):P46 (doi: 10.1186/cc8102) Evidence that simvastatin prevents induction of nitric oxide synthase by LPS in the porcine isolated coronary artery S Al-Shalmani1, S Chinnah2, R Mahajan2, V Wilson1 1School of Biomedical Sciences, and 2Academic Division of Anaesthesia and Intensive Care, University of Nottingham Medical School, Nottingham, UK Critical Care 2009, 13(Suppl 4):P45 (doi: 10.1186/cc8101) Conclusions Altogether we provide here a novel mechanism of action of IL-33 and establish a potential therapeutic role of this new cytokine in sepsis. Introduction Several retrospective studies suggest that prior use of statins can reduce hospital mortality in patients diagnosed with Acknowledgements Financial support from Wellcome Trust, MRC and FAPESP. S20 Available online http://ccforum.com/supplements/13/S4 Pre-treatment with simvastatin prevents LPS-induced hyporesponsiveness of porcine isolated coronary artery Co-incubation of segments with 3 μM simvastatin and LPS produced a profile similar to that of control segments (CD31- positive, iNOS-negative, n = 4). Results KCl and U46619 caused concentration-dependent contraction of the PCA. Table 1 shows that treatment with 1 μg/ml LPS overnight (and subsequent removal) significantly reduced the maximum response to KCl and U46619 in the PCA by 32.0 ± 4.5% (n = 12) and 28.9 ± 12.3% (n = 12), without changing the potency of either agent. These effects of LPS on vasoconstrictor responses were not observed when 10 μM 1400W was added to the organ bath, after removal of the endotoxin (Table 1). LPS also failed to impair constrictor responses if 3 μM simvastatin was present during the incubation period and was subsequently removed (Table 1). Conclusions Simvastatin suppressed the induction of nitric oxide caused by LPS and the associated increase in nitrite/nitrate production. This finding helps to explain our observation that simvastatin prevented LPS-induced hyporesponsiveness of the coronary artery, and is also consistent with clinical studies suggesting that prior use of statins may afford protection against bacterial sepsis. Table 1 (abstract P46) Effects of statins on postoperative sepsis, systemic inflammatory response syndrome and mortality after colorectal surgery Multiple organ dysfunction syndrome: the scapegoat? Assessment of organ dysfunction between surviving and dying mice in the acute phase of polymicrobial sepsis A Khan1, D Yeung1, B Wyatt1, T Rafai1, J Byant1, A Coates1, E Fitzgerald2, A Acheson2, V Wilson1 1School of Biomedical Sciences, and 2Division of Surgery, University of Nottingham Medical School, Nottingham, UK Critical Care 2009, 13(Suppl 4):P47 (doi: 10.1186/cc8103) Introduction Colorectal surgery carries significant risks of postoperative morbidity and mortality. One of the major hazards is an increased risk of sepsis; an important component of which is systemic inflammatory response syndrome (SIRS). Several recent studies suggest that the noncholesterol-related, pleiotropic effects of statins may limit the development of sepsis and associated inflammation. This study investigates the impact of prior statin therapy on the incidence and outcome of postoperative sepsis and SIRS in colorectal surgery patients. Introduction Multiple organ dysfunction syndrome (MODS) frequently complicates sepsis contributing to poor outcome. Yet the evolution of MODS in the early septic mortality (ESM) is unclear. To delineate the ESM–MODS relationship, we compared the development and magnitude of organ injury between dying and surviving mice in the acute phase (days 1 to 5) of polymicrobial sepsis. Methods Female OF-1 mice were subjected to cecal ligation and puncture (CLP). In the first mouse subset, 20 μl blood was collected daily for 5 days or until death (mice followed for 28 days). To define the pre-lethal changes in circulating parameters, mice were retrospectively divided into two groups based on outcome in the acute sepsis: DEAD (all died within 5 days, n = 39) and survivors (SUR; alive at day 28, n = 40). In the second subset, mice were sacrificed within 24 hours of projected death (based on the body T <28°C, 100% specificity, n = 7) and matched with SUR (body T >35°C, 100% sensitivity) from the same post-CLP day and controls. Methods A retrospective cohort analysis of 577 patients who underwent curative surgery for colorectal cancer was conducted to evaluate postoperative morbidity and mortality (within 30 days of surgery). The primary endpoints were: 30-day in-hospital mortality, admission to intensive care (ICU), and a positive diagnosis of SIRS or sepsis. Results Prior to admission, 21.7% of patients were taking either simvastatin, atorvastatin, fluvastatin, pravastatin or rosuvastatin. Patients on statins were significantly older than those not on statins (statin – 74.7 years (SD = 6.5) vs. Effects of statins on postoperative sepsis, systemic inflammatory response syndrome and mortality after colorectal surgery nonstatin – 69.2 years (SD = 13.4), P = 0.022), more likely to have pre-existing co- morbidities and in receipt of antidiabetic agents and other cardiovascular drugs. Table 1 shows there was no difference in mortality rate between the two groups. Furthermore, the incidence of nosocomial infection and sepsis did not differ between the statin and nonstatin groups. Despite being more likely to be admitted to the ICU, the statin group was significantly less likely to develop either SIRS (in or out of the ICU) or postoperative wound infection or be admitted to the ICU for infective/inflammatory sequelae. y Results In the first subset, significant difference was observed between SUR and DIED in the circulating urea, ALT, LDH and glucose during the 1-day to 5-day time course but the magnitude of these changes varied among post-CLP days. Therefore, we used the day of death as a reference point clustering all pre-lethal parameter values as the 72, 48 and 24 hours prior-to-death (on any 1 to 5 post-CLP day) time points for comparison with the SUR values. A significant separation between SUR and DIED occurred generally at 24 hours prior to death: pre-lethal urea increased to 78 (vs. 40 mg/dl in SUR), ALT to 173 (vs. 106 U/l) and LDH to 798 (vs. 445 U/l), while pre-lethal glucose declined to 41 (vs. 74 mg/dl). In the second subset (only 24 hours prior-to-death time point), acute deaths were not preceded by a significant rise in creatinine (DIED 6.8 vs. 4.3 μM/l in SUR) and troponin I (239 vs. 119 pg/ml). Similarly, respiratory function of mitochondria in the liver and kidney was not impaired in either DIED and SUR compared with controls. Injury scores in the liver, kidney, heart and lung showed no apparent morphological disparity between moribund, surviving or control mice. Incidence of cell apoptosis (TUNEL) in organs was not increased in either of the groups. Conclusions The increase of selected organ function/metabolic markers was manifested at least 24 hours prior to death. Despite statistical significance, the relatively small magnitude of these changes questions organ failure as a direct cause of death in the early phase of CLP sepsis. Table 1 (abstract P47) Table 1 (abstract P46) S21 ( ) Effect of LPS, 1400W and simvastatin on maximum response and potency of vasoconstrictor agent on the PCA KCl U46619 Incubation conditions Maximum (g wt) –log EC50 Maximum (g wt) –log EC50 Control (n = 12) 10.23 ± 0.71 1.56 ± 0.04 10.39 ± 0.71 7.52 ± 0.08 LPS 6.83 ± 0.48* 1.60 ± 0.04 6.77 ± 0.80** 7.64 ± 0.06 LPS then 1400W 9.35 ± 1.21 1.64 ± 0.06 9.41 ± 0.46 7.54 ± 0.04 Control (n = 18) 12.13 ± 0.58 1.55 ± 0.01 13.46 ± 0.51 7.83 ± 0.01 LPS 8.41 ± 0.58** 1.51 ± 0.02 9.90 ± 0.47** 7.75 ± 0.06 Simvastatin and LPS 11.82 ± 0.34 1.53 ± 0.03 13.92 ± 0.38 7.73 ± 0.04 Data presented as mean ± SEM (n = 12 to 18). *P <0.05, **P <0.01, denotes a statistically significant difference from control. W and simvastatin on maximum response and potency of vasoconstrictor agent on the PCA Effect of LPS, 1400W and simvastatin on maximum response and potency of vasoconstrictor agent on the PC S21 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Conclusions Prolonged exposure to LPS caused hyporesponsive- ness of the PCA by a mechanism that appears to involve the induction of nitric oxide synthase. Since pre-treatment of the PCA with simvastatin reduced LPS-induced changes in vasoconstrictor responses, it unlikely that the effect of the statin involves direct inhibition of NOS (compare 1400W). These findings are consis- tent with clinical studies suggesting that prior use of statins may afford protection against bacterial sepsis. Conclusions Statin patients were older than nonstatin patients, and had a greater burden of co-morbidities, yet the mortality rate did not differ between the two groups. The possibility that prior use of statins may influence inflammatory or infective events associated with this surgical procedure is supported by the significantly lower incidence of wound infection, SIRS and sepsis in statin-treated patients, illustrating the potential for statins to confer protection against these insults in the most critically ill patients. Conclusions Statin patients were older than nonstatin patients, and had a greater burden of co-morbidities, yet the mortality rate did not differ between the two groups. Table 1 (abstract P46) The possibility that prior use of statins may influence inflammatory or infective events associated with this surgical procedure is supported by the significantly lower incidence of wound infection, SIRS and sepsis in statin-treated patients, illustrating the potential for statins to confer protection against these insults in the most critically ill patients. P48 Multiple organ dysfunction syndrome: the scapegoat? Assessment of organ dysfunction between surviving and dying mice in the acute phase of polymicrobial sepsis M Osuchowski1, K Weixelbaumer1, P Raeven1, D Remick2, K Reise1, A Kozlov1, M van Griensven1, H Redl 1, S Bahrami1 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in the Trauma Research Center of AUVA, Vienna, Austria; 2University of Boston, School of Medicine, Boston, MA, USA Critical Care 2009, 13(Suppl 4):P48 (doi: 10.1186/cc8104) Effects of statins on postoperative sepsis, systemic inflammatory response syndrome and mortality after colorectal surgery Available online http://ccforum.com/supplements/13/S4 The model incorporates a virtual clinician, an automated system to examine simulated patients’ status at clinically relevant intervals and administer standard of care interventions as necessary, thereby altering the dynamics of the disease state. The model reproduces many characteristics of systemic response to an infection, including the time course of cytokines, coagulation factors, clinical markers, early and late organ failure, and early versus late deaths. Conclusions The ability of our model to reproduce a large variety of patients with a relatively small number of parameter changes illustrates the robustness of the underlying biological processes being modeled. The model may help identify real signals in immensely variable and noisy multidimensional sepsis patient data, and distinguish real patient responses from clinical study-site- related variability. This model is currently undergoing further validation. Future capabilities include assessment of risk and benefit of new drugs for sepsis or new treatment strategies (for example, early goal-directed therapy) in different patient cohorts. Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 P49 demographics and co-morbidities (for example, COPD, cardio- vascular disease), a handful of parameters were changed to fit the model to median time course data from 14 subgroups. These parameters included pathogen virulence, effectiveness of anti- biotics, baseline status of patients at study inclusion, and occurrence of secondary infection. By overlaying variations in the above parameters about the median model, the model can encompass the entire spectrum of patients observed in the GenIMS study. The virtual clinician administers individualized treatment for each simulated patient. The model simulations provide clear evidence of changes in disease progression as a function of differences in treatment. Mathematical modeling of community-acquired pneumonia patients Mathematical modeling of community-acquired pneumonia patients J Sarkar1, DD Marathe1, AM Inglis1, KW Hurst1, JA Kellum2, DC Angus2, Y Vodovotz3, S Chang1 1Immunetrics Inc., Pittsburgh, PA, USA; 2Department of Critical Care Medicine, University of Pittsburgh, PA, USA; 3Department of Surgery, University of Pittsburgh, PA, USA Critical Care 2009, 13(Suppl 4):P49 (doi: 10.1186/cc8105) Introduction Sepsis is defined by the systemic response to an infection, governed by dynamic interactions between the tissues, immune cells and inflammatory mediators. We used the Immunetrics platform to build a large-scale mathematical model that encompasses these biological components. The model incorporates a virtual clinician, an automated system to examine simulated patients’ status at clinically relevant intervals and administer standard of care interventions as necessary, thereby altering the dynamics of the disease state. The model reproduces many characteristics of systemic response to an infection, including the time course of cytokines, coagulation factors, clinical markers, early and late organ failure, and early versus late deaths. Methods The ordinary differential equation-based model was used to simulate the progression of sepsis over a 30-day hospital stay. This model was fit to published human endotoxemia data as well as data from severely septic community-acquired pneumonia (CAP) patients from the GenIMS study. The model was fit to 15 biomarkers and clinical markers, including mean arterial pressure, PaO2, creatinine, TNFα, IL-6, and PAI-1. Introduction Sepsis is defined by the systemic response to an infection, governed by dynamic interactions between the tissues, immune cells and inflammatory mediators. We used the Immunetrics platform to build a large-scale mathematical model that encompasses these biological components. P50 Heart rate variability in the early resuscitation of septic shock RC Arnold1, DJ Lundy1, L Glaspey1, G Green2, AJE Seely2 1Cooper University Hospital, UMDNJ-Robert Wood Johnson Medical Center, Camden, NJ, USA; 2The Ottawa Hospital and Research Institute, Ottawa, ON, Canada Critical Care 2009, 13(Suppl 4):P50 (doi: 10.1186/cc8106) 2 Results Figure 1 illustrates simulated output as compared with the median time course data for surviving CAP patients without co- morbidities and for CAP patients without co-morbidities who died between 4 and 8 days after admission. After incorporating changes in physiological and immune function due to patient Introduction The assessment of heart rate variability (HRV) has provided valuable insight in sepsis. Impaired HRV has been shown Table 1 (abstract P47) S22 Outcomes for statin and nonstatin patients following colorectal surgery Parameter Statin Nonstatin Sig. Overall mortality 9/125 (7.2%) 27/452 (6.4%) 0.77 Nosocomial infections 42/125 (33.6%) 132/452 (29.2%) 0.40 Sepsis 10/75 (13.3%) 42/203 (20.7%) 0.22 SIRS 16/125 (12.8%) 148/452 (32.7%) <0.001 Wound infection 11/125 (8.8%) 66/452 (14.6%) 0.04 Admitted to the ICU 34/125 (27.2%) 63/452 (13.9%) <0.001 Sig,. significance. Outcomes for statin and nonstatin patients following colorectal surgery S22 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P49) Time domain factors: SDNN = standard deviation of the normal R–R interval; RMSSD = root mean square of the difference of successive R–R intervals. Frequency domain factors of HRV spectrum: LF = low frequency (0.04 to 0.15 Hz); HF = high frequency (0.18 to 0.4 Hz). Introduction Critically ill patients often have abnormally low plasma thyroxine (T4) concentrations even in the absence of thyroidal illnesses, with the lowest T4 values being observed in patients (particularly older individuals) with sepsis. Staphylococcal infection of the lung is the most common presentation of sepsis in the medical ICU setting, and is associated with a much higher mortality in older individuals than in younger. In studies where the lungs of rats are challenged with staphylococcal components lipoteichoic acid and peptidoglycan, we have noted negative corre- lations between plasma MIF and T4. Objective To examine the dynamics of MIF and T4 in a clinically relevant, large animal model of sepsis. Methods Adult female sheep (30 to 40 kg) with age approximately equivalent to humans of 55 years were surgically prepared for chronic study. After 5 days recovery, approximately 2.5 x 1011 CFU methicillin-resistant Staphylococcus aureus (ATCC 4300, a clinical isolate) were instilled into the lung via a bronchoscope. The animals were ventilated, and hemodynamics were monitored continuously for 24 hours post instillation. Blood and lymph samples were collected at baseline and at 3, 6, 12, 18, and 24 hours. MIF was measured by semiquantitative western blot and free-T4 by ELISA. to be diagnostic of sepsis, heralding its onset, and prognostic of its impact, correlating with the development of sepsis-induced organ failure and death. Objective To analyze the ability of HRV to act as a prognostic aid in the early resuscitation of septic shock, and to study the effect that early fluid resuscitation will have on the direction and magnitude of subsequent measurements of HRV in patients with septic shock. Methods Subjects were prospectively identified within the emergency department of an urban-based tertiary-care medical center during their initial evaluation and treatment for septic shock, defined as a systemic infection with a systolic blood pressure <90 mmHg after intravenous fluid or a serum lactate >4.0 mmol/l. Continuous cardiac telemetry was obtained for the assessment of HRV using a standardized set of multiple parameters including variables of frequency and time domain analyses. Fluid administration was recorded during the initial resuscitation. Figure 1 (abstract P49) Figure 1 (abstract P49) Comparison of time course data from model simulation (solid line) and median of patients (filled circles) from (a) surviving CAP patients without co- morbidities and (b) CAP patients without co-morbidities dying between 4 and 8 days after admission. Comparison of time course data from model simulation (solid line) and median of patients (filled circles) from (a) surviving CAP patients without co- morbidities and (b) CAP patients without co-morbidities dying between 4 and 8 days after admission. S23 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Figure 1 (abstract P50) Heart rate variability parameters measured during the initial resuscitation of patients with septic shock. Nonsurvivors showed significantly impaired variability as measured by LF and the HF/LF ratio. Data presented as logarithmic transformation to control for scale and allow visual comparison. Differences between survivors and nonsurvivors were calculated using raw averages without logarithmic conversion. Time domain factors: SDNN = standard deviation of the normal R–R interval; RMSSD = root mean square of the difference of successive R–R intervals. Frequency domain factors of HRV spectrum: LF = low frequency (0.04 to 0.15 Hz); HF = high frequency (0.18 to 0.4 Hz). Figure 1 (abstract P50) Figure 1 (abstract P50) independent of organ severity measurement. While the use of fluid administration in the early resuscitation of shock did not correlate with changes in HRV measured at 2 hours, this relationship may exist when measured at longer time points allowing for the physiologic response to manifest in the HRV trend. The assessment of HRV in patients with infection can identify those at high risk for clinical deterioration and can potentially serve as an endpoint of resuscitation in patients with septic shock. P51 Changes in MIF and thyroxine, in a clinically relevant large animal model of sepsis E Miller1, S Rehberg2, H Linge1, P Enkhbaatar2, L Traber2, D Traber2, Y Al-Abed1 1The Feinstein Institute for Medical Research, Manhasset, NY, USA; 2The University of Texas Medical Branch, Galveston, TX, USA Critical Care 2009, 13(Suppl 4):P51 (doi: 10.1186/cc8107) Heart rate variability parameters measured during the initial resuscitation of patients with septic shock. Nonsurvivors showed significantly impaired variability as measured by LF and the HF/LF ratio. Data presented as logarithmic transformation to control for scale and allow visual comparison. Differences between survivors and nonsurvivors were calculated using raw averages without logarithmic conversion. Available online http://ccforum.com/supplements/13/S4 We examined several classes of endogenous small molecules and their metabolites and observed that MIFnl1 binds to, and inhibits, the hydrophobic cavity of MIF in a dose-dependent manner with an IC50 of 15.8 μM. Importantly, MIFnl1 was a more potent inhibitor of MIF than ISO-1 (IC50 = 25 μM), the gold standard synthetic inhibitor of MIF. In addition, in plasma from patients with sepsis, we found an inverse correlation between the increased level of MIF and the decreased concentration of MIFnl1. Therefore, we hypothesized that supplementation of this ligand during sepsis should compensate for its dramatic reduction and improve survival in our peritonitis model of sepsis in C57/Bl6 mice. Administration of MIFnl1 improved the 7-day survival rate to 60% compared with 20% observed for the vehicle-treated mice. Conclusions Our data identify for the first time the presence of a natural, ligand antagonist of MIF in plasma. This suggests that, during severe sepsis, increased production and release of MIF leads to an imbalance of the MIF:MIFnl1 regulatory mechanism resulting in the development of an overwhelming systemic inflammatory response leading to cardiovascular collapse and death. A better understanding of the kinetics of MIF/ligand Introduction Macrophage migration inhibitory factor (MIF) is a pro- inflammatory cytokine that plays a critical role in the pathogenesis of sepsis. Plasma MIF concentrations are significantly higher in nonsurvivors than survivors of severe sepsis, and administration of antibodies that neutralize MIF activity improves survival in an experimental model of sepsis. Three-dimensional X-ray crystallo- graphy shows MIF has a homotrimeric conformation and we have determined that the hydrophobic cavity formed between two adjacent subunits of the homotrimer is required for the pro- inflammatory activity of the molecule. We have designed several small molecules that fit into the site critical for the proinflammatory action of MIF, and confirmed the interaction by the crystal structure of the MIF complex. Binding of MIF in this way inhibits its proinflammatory activity, improves the clinical outcome in sepsis, and recapitulates immunotherapy and gene deletion. However, no natural soluble ligand of MIF has been reported previously. Methods Lipoteichoic acid (LTA) and peptidoglycan (PGN) are major inflammatory components of the staphylococcal cell wall known to induce shock. Male Fischer 344 rats either Young (6 months) or Old (>18 months), approximately equivalent to humans of 18 and 60 years respectively, were anesthetized and LTA and PGN (1.5/5 mg/kg in 200 μl) were administered intra- tracheally. P54 Conclusions Our data identify for the first time the presence of a natural, ligand antagonist of MIF in plasma. This suggests that, during severe sepsis, increased production and release of MIF leads to an imbalance of the MIF:MIFnl1 regulatory mechanism resulting in the development of an overwhelming systemic inflammatory response leading to cardiovascular collapse and death. A better understanding of the kinetics of MIF/ligand regulation in patients with sepsis may lead to improved outcome in this devastating disease. Neutrophil recruitment by macrophage migration inhibition factor and CXCL1 to the lung following staphylococcal stimulation is significantly elevated in advanced age Available online http://ccforum.com/supplements/13/S4 Six hours later, blood was collected from the heart, and post mortem, the lungs were lavaged for analysis of the epithelial lining fluid. Young, untreated cage control animals (Control) were also assessed. Results The plasma MIF concentration significantly increased with LTA/PGN challenge (Control, 24.9 ± 0.6 ng/ml; Young, 38.8 ± 5.9 ng/ml; Old, 64.2 ± 12.1 ng/ml) and the mean concentration in the old animals was significantly higher than in the young; P = 0.004. Conversely, plasma free-T4 significantly decreased with LTA/PGN challenge (Control, 2.3 ± 0.9 ng/dl; Young, 1.7 ± 0.7 ng/dl; Old, 1.0 ± 0.4 ng/dl) and the mean concentration in the old animals was significantly lower than in the young; P <0.05. Significant age-specific differences were also noted in the response with respect to neutrophils and IL-6 within the lavage fluid. Hypothesis MIFnl1 binds to the hydrophobic cavity of MIF. Increased concentrations of MIF in sepsis deplete plasma MIFnl1 and lead to a critical MIF:MIFnl1 imbalance. Results We have discovered a natural ligand, designated MIFnl1, that binds the proinflammatory site of MIF with high affinity, and effectively modulates its activity. We examined several classes of endogenous small molecules and their metabolites and observed that MIFnl1 binds to, and inhibits, the hydrophobic cavity of MIF in a dose-dependent manner with an IC50 of 15.8 μM. Importantly, MIFnl1 was a more potent inhibitor of MIF than ISO-1 (IC50 = 25 μM), the gold standard synthetic inhibitor of MIF. In addition, in plasma from patients with sepsis, we found an inverse correlation between the increased level of MIF and the decreased concentration of MIFnl1. Therefore, we hypothesized that supplementation of this ligand during sepsis should compensate for its dramatic reduction and improve survival in our peritonitis model of sepsis in C57/Bl6 mice. Administration of MIFnl1 improved the 7-day survival rate to 60% compared with 20% observed for the vehicle-treated mice. Conclusions Typically, critically ill patients have abnormally low plasma T4 concentrations even in the absence of thyroidal illnesses (the so-called euthyroid sick phenomenon) with the lowest values being observed in septic and/or elderly individuals, suggesting a strong negative correlation between prognosis and T4 concentration. The data suggest that an increased early inflammatory response in older compared with young animals results in an exaggerated imbalance between MIF and T4, possibly leading to development of an uncontrolled systemic response. Neutrophil recruitment by macrophage migration inhibition factor and CXCL1 to the lung following staphylococcal stimulation is significantly elevated in advanced age HM Linge, K Takahashi, E Miller Cardiopulmonary Research Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset, NY, USA Critical Care 2009, 13(Suppl 4):P54 (doi: 10.1186/cc8124) Introduction Mortality from sepsis is greater in the elderly than in the young although incidence only increases slightly. Pulmonary infections caused by Staphylococcus aureus, which progress into sepsis, are a major cause of death in elderly patients. Adiponectin, multifaceted adipokine with anti-inflammatory properties, is secreted primarily from adipose tissue. It is increasingly acknowledged that the tissue microenvironment changes with old age. With increasing age, ectopic fat accumulates, increasing the possibility of elevated levels of adipose-derived mediators in the older individual. Figure 1 (abstract P49) A composite endpoint of increasing organ failure and in-hospital mortality was measured. Results The plasma concentration of MIF increased from 24.9 ± 4.5 ng/ml at baseline to 30.1 ± 2.5 ng/ml in the first 6 hours post instillation of the bacteria. During this time period, free-T4 concentration decreased from 1.6 ± 0.5 ng/dl to 0.4 ± 0.3 ng/dl. The concentration of MIF in the pulmonary lymph was approxi- mately 10% of the plasma level, and showed no time-dependent decrease, although the lymph flow increased approximately fourfold over the course of the study. Conclusions We have found that intratracheal instillation of staphylococcal cell wall components in rats results in an imbalance of plasma MIF and free-T4, and that pulmonary-derived MIF in sepsis induces cardiocirculatory depression. Here we show that, in a clinically relevant, large animal model of sepsis induced by pulmonary infection with methicillin-resistant S. aureus, increased plasma concentration of MIF and decreased free-T4 occurred in a similar manner to our rat studies, and that the total amount of MIF in the lymph also increased. Since strong negative correlations exist between prognosis and levels of T4 or MIF in patients with sepsis, our findings underscore interest in possible interactions between the MIF and T4 molecules that may be critical for the outcome of the disease. Results Prospective analysis of 15 patients with septic shock was made. The in-hospital mortality rate was 67% (10/15). Non- survivors had a significantly impaired HRV compared with survivors when measured through multiple parameters (P < 0.01), as seen in Figure 1. There was no difference between fluid administration between survivors and nonsurvivors (4,475 vs. 5,220 ml, P = NS). There was no relationship seen between intravenous fluid administration and the change in 2-hour HRV seen in the early resuscitation of septic shock. Conclusions In the early resuscitation of septic shock, HRV assessment can differentiate survivors from nonsurvivors, S24 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 P52 older individuals. While most animal models of sepsis have studied polymicrobial or Gram-negative sepsis from an abdominal origin in young animals, the most common presentation in the medical ICU is in older individuals (median age 64 years), with Gram-positive bacteria (predominantly Staphylococcus aureus) infection in the lung. Our recent studies reveal a critical role for macrophage migration inhibitory factor (MIF) in the pathogenesis of sepsis, and thyroxine (T4), two molecules with major implications in sepsis. Therefore, we reasoned that the age-related severity of sepsis may be due to an exaggerated imbalance between MIF and its inhibitor. Objective To examine age-dependent differences in inflammatory responses following pulmonary staphylococcal challenge. Discovery of a natural antagonist of macrophage migration inhibitory factor Discovery of a natural antagonist of macrophage migration inhibitory factor Discovery of a natural antagonist of macrophage migration inhibitory factor Y Al-Abed, C Metz, KF Cheng, B Aljabari, H Linge, M Ochani, X Lin, V Pavlov, T Coleman, K Tracey, EJ Miller The Feinstein Institute for Medical Research, Manhasset, NY, USA Critical Care 2009, 13(Suppl 4):P52 (doi: 10.1186/cc8108) Introduction Macrophage migration inhibitory factor (MIF) is a pro- inflammatory cytokine that plays a critical role in the pathogenesis of sepsis. Plasma MIF concentrations are significantly higher in nonsurvivors than survivors of severe sepsis, and administration of antibodies that neutralize MIF activity improves survival in an experimental model of sepsis. Three-dimensional X-ray crystallo- graphy shows MIF has a homotrimeric conformation and we have determined that the hydrophobic cavity formed between two adjacent subunits of the homotrimer is required for the pro- inflammatory activity of the molecule. We have designed several small molecules that fit into the site critical for the proinflammatory action of MIF, and confirmed the interaction by the crystal structure of the MIF complex. Binding of MIF in this way inhibits its proinflammatory activity, improves the clinical outcome in sepsis, and recapitulates immunotherapy and gene deletion. However, no natural soluble ligand of MIF has been reported previously. Hypothesis MIFnl1 binds to the hydrophobic cavity of MIF. Increased concentrations of MIF in sepsis deplete plasma MIFnl1 and lead to a critical MIF:MIFnl1 imbalance. Results We have discovered a natural ligand, designated MIFnl1, that binds the proinflammatory site of MIF with high affinity, and effectively modulates its activity. Age-associated increased inflammatory response to pulmonary bacterial challenge Age-associated increased inflammatory response to pulmonary bacterial challenge HM Linge, K Lin, Y Al-Abed, EJ Miller Cardiopulmonary Research, Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset, NY, USA Critical Care 2009, 13(Suppl 4):P53 (doi: 10.1186/cc8109) Introduction Sepsis, the systemic inflammation following infectious insult, is a major cause of morbidity and mortality particularly in Objective To investigate age-dependent changes in the intra- pulmonary response to staphylococcal challenge. We hypothesized S25 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4 Sepsis 2009 Corporation, Sagamihara, Japan). Auto anti-HMGB1 antibodies were detected in serum by a homemade ELISA test. Corporation, Sagamihara, Japan). Auto anti-HMGB1 antibodies were detected in serum by a homemade ELISA test. Corporation, Sagamihara, Japan). Auto anti-HMGB1 antibodies were detected in serum by a homemade ELISA test. that older animals have higher levels of adiponectin due to adipose tissue accumulation and that these levels will neutralize the proinflammatory consequences of staphylococcal stimulation of the lung. Results Forty-two septic shock patients were included. Median age was 70 (59 to 78) years, SAPS 2 was 68 (51 to 83), and the mortality rate was 29%. HMGB1 was undetectable in the plasma of a control population. In contrast, high levels of HMGB1 were found in all septic patients (median = 5.71 ng/ml at D7). Thirteen patients (38%) presented a significant production of auto anti- HMGB1 IgG antibodies during the course of sepsis. The age, sex ratio, median HMGB1 level and mortality rate were similar in patients producing (PPAb) and not producing antibodies (PNPAb). However, as compared with the PNPAb patients, the PPAb group had significant higher APACHE II (P = 0.027) and SOFA scores (P = 0.02) at the onset of shock and had a more important but nonsignificant decrease of the cardiovascular SOFA score between day 1 and day 7 (P = 0.063). Methods Young (6 months) and old (>18 months) male Sprague– Dawley rats were challenged intratracheally with S. aureus cell wall components lipoteichoic acid (LTA, 0.15 mg) and peptidoglycan (PGN, 0.5 mg) or saline alone. After 24 hours, plasma was collected and lungs lavaged post mortem. Concentrations of total protein, chemokines and MIF were assessed. P55 Naturally acquired anti-high mobility group box 1 antibodies during septic shock Age-associated increased inflammatory response to pulmonary bacterial challenge In vitro studies examined the accumulation of adiponectin in the culture medium of the adipocyte cell line 3T3-L1 following direct challenge with LTA/PGN (3 μg/ml; 100 μg/ml) for 6 hours. Results The older age group, compared with the young group receiving the same stimulus, showed significantly elevated alveolar levels of MIF and CXCL1 (KC), both involved in neutrophil recruitment. Neutrophils (controls: 0.029 ± 0.033, young: 13.9 ± 7.8, old: 29.3 ± 10.5, x 106) and the total number of cells (controls: 0.72 ± 0.26, young: 16.4 ± 9.7, old: 32.9 ± 11.2, x 106) within the alveolar space were significantly and age-dependently increased, following pulmonary insult. Plasma adiponectin did not change significantly. However, instillation of LTA/PGN significantly elevated the levels of adiponectin found within the alveolar space (controls: 3.9 ± 2.4, young: 22.5 ± 8.8, old: 29 ± 10.8 ng/ml, P <0.01). Interestingly, following LTA/PGN challenge of adipocytes, there was a significant decrease in adiponectin concentration in the culture medium (control: 20.3 ± 0.78, LTA/PGN: 18.23 ± 1.14 pg/ml, P = 0.016). Conclusions Naturally acquired anti-HMGB1 antibodies are produced during septic shock. The presence of antibodies is associated with higher severity scores and might be associated with an improvement of the haemodynamic dysfunction. The neutralizing capacity of these autoantibodies and their physio- logical role remain to be investigated. Naturally acquired anti-high mobility group box 1 antibodies during septic shock B Sauneuf1, D Grimaldi1,2,3, C Rousseau1, J-D Chiche1,2,3, C Desgranges1, J-P Mira1,2,3 1Department of Cellular Biology, Host–Pathogen Interactions, Cochin Institute, University Paris Descartes, INSERM U567, CNRS (UMR 8104), Paris, France; 2Medical Intensive Care Unit, Cochin Hospital, Paris, France; 3University Paris Descartes, Paris, France Critical Care 2009, 13(Suppl 4):P55 (doi: 10.1186/cc8125) Objective To assess capacity of S100A9 mRNA in whole blood from SS patients to predict survival and the occurrence of HAI. Objective To assess capacity of S100A9 mRNA in whole blood from SS patients to predict survival and the occurrence of HAI. Methods The authors conducted a cohort study. This study was conducted in two ICUs in Lyon University Hospital. The study included 166 SS patients and 44 healthy volunteers. PAXgene blood samples were obtained regularly in the course of the syndrome for S100A9 gene expression analysis using qRT-PCR. Results The overall mortality was 38% and the mean SAPS II on shock onset was 52. Thirty-seven patients (23%) experienced at least one HAI after septic shock. We found that S100A9 mRNA levels at days 1 to 3 after the onset of shock were significantly higher in SS patients compared with healthy volunteers (median: 1,460 vs. 16,620; P <0.0001) but not significantly different in nonsurviving versus surviving patients (median: 18,070 vs. 16,310; P = 0.1278). In contrast, systemic S100A9 mRNA levels measured at days 7 to 10 were significantly higher in the group of Introduction High mobility group box 1 (HMGB1) is a pleiotropic cytokine, implicated in the pathophysiology of sepsis. This alarmin, usually located in the nucleus, is released after tissue injury and activates various innate immunity receptors, leading to sustained inflammatory response. Inhibition of HMGB1 by anti-HMGB1 antibodies has been reported to decrease mortality in experimental models of sepsis. In the present work, we analyse whether HMGB1 secretion during septic shock leads to the production of naturally acquired anti-HMGB1 antibodies during septic shock. Methods All patients with septic shock criteria and no immunosuppression were included during a 6-month period of time. After informed consent, blood samples (200 μl) were drawn on the day of shock and at D3, D7, and D14. Plasma HMGB1 levels were measured, using a commercial ELISA kit (Sino-Test Introduction High mobility group box 1 (HMGB1) is a pleiotropic cytokine, implicated in the pathophysiology of sepsis. P56 Delayed increased S100A9 mRNA predicts hospital-acquired infection after septic shock Delayed increased S100A9 mRNA predicts hospital-acquired infection after septic shock M Fontaine1, A Pachot2, A Larue2, B Mougin2, C Landelle1, C Allombert2, F Venet3, M-A Cazalis2, G Monneret3, A Lepape1 1Intensive Care Units, Hospices Civils de Lyon, CH Lyon-Sud, Lyon, France; 2Joint Unit Hospices Civils de Lyon ‘bioMerieux, Hôpital E. Herriot, Lyon, France; 3Immunology Laboratory, Hospices Civils de Lyon, Hôpital E. Herriot, Lyon, France Critical Care 2009, 13(Suppl 4):P56 (doi: 10.1186/cc8126) Conclusions The proinflammatory but not anti-inflammatory components of the immune response (assessed by neutrophil recruitment and adiponectin concentrations, respectively) differed significantly between the age groups. Since adiponectin decreased on direct stimulation of adipocytes, the increased adiponectin within the lungs may reflect increased lung perme- ability, and/or production by other cells within the lung. These findings are important in understanding the response to pulmonary infections in the older patient and may lead to the identification of novel targets for age-dependent therapeutic strategies. Introduction Septic shock (SS) remains a serious disease with high mortality and increased risk of hospital-acquired infection (HAI). Access to biomarkers assessing prognosis of these outcomes is of utmost importance in order to select patients for future therapeutic strategies. Alarmins are normal cell constituents that can be released into the extracellular milieu during states of cellular stress and subsequently activate the innate immune system. Several alarmins of the S100 family are released by phagocytes in response to cell stress, recognized by RAGE and/or TLR4 on monocytes and therefore highlighted as relevant mediators in sepsis pathophysiology. Among them, S100A8 and S100A9 exist mainly as a heterodimer called calprotectin. However, several studies suggest independent functioning of S100A9, making it an interesting candidate biomarker in septic syndromes. Conclusions Our results showed that S100A9 mRNA is overexpressed in blood from SS. We showed that its persistent overexpression over time is associated with the occurrence of secondary HAI. This biomarker may be of major interest in identi- fying patients at increased HAI risk who could benefit from either targeted therapy aimed at restoring immune functions (in case of associated immunosuppression) or reinforced antibiotherapy and measures against cross-transmission. patients that were going to develop HAI compared with patients that were not (median: 10,140 vs. 7,160; P = 0.009). Multivariate analysis showed that the S100A9 mRNA level at days 7 to 10 after the onset of septic shock significantly increases the probability of HAI with odd ratios of 1.12 per unit (P = 0.0054). Available online http://ccforum.com/supplements/13/S4 Naturally acquired anti-high mobility group box 1 antibodies during septic shock This alarmin, usually located in the nucleus, is released after tissue injury and activates various innate immunity receptors, leading to sustained inflammatory response. Inhibition of HMGB1 by anti-HMGB1 antibodies has been reported to decrease mortality in experimental models of sepsis. In the present work, we analyse whether HMGB1 secretion during septic shock leads to the production of naturally acquired anti-HMGB1 antibodies during septic shock. Methods All patients with septic shock criteria and no immunosuppression were included during a 6-month period of time. After informed consent, blood samples (200 μl) were drawn on the day of shock and at D3, D7, and D14. Plasma HMGB1 levels were measured, using a commercial ELISA kit (Sino-Test S26 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 S27
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Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016
Journal of illicit economies and development
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Collins, J and Alarcón, KT. 2021. Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016. Journal of Illicit Economies and Development, 3(2), pp. 190–205. DOI: https://doi.org/10.31389/jied.93 Collins, J and Alarcón, KT. 2021. Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016. Journal of Illicit Economies and Development, 3(2), pp. 190–205. DOI: https://doi.org/10.31389/jied.93 Collins, J and Alarcón, KT. 2021. Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016. Journal of Illicit Economies and Development, 3(2), pp. 190–205. DOI: https://doi.org/10.31389/jied.93 RESEARCH Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 John Collins1 and Karen Torres Alarcón2 1 Global Initiative against Transnational Organised Crime, AT 2 London School of Economics (LSE), UK Corresponding author: John Collins (john.collins@globalinitiative.net) This article breaks new conceptual ground by questioning orthodox interpretations of nation state agency in the global drug wars. Specifically, it challenges the David vs. Goliath conception of Colombia as a passive, client state simply abiding to the United States’ hegemonic war on drugs. It provides the first published analysis of Colombia’s leadership during the UN General Assembly Special Session on Drugs (UNGASS) in 2016. It argues that the UN served as a useful forum for Colombia’s displacement of state building dilemmas, including drug control, and that Bogota utilised the UN as a proxy negotiating mechanism with the US and other international donors. Keywords: Colombia; War on Drugs; United States of America; FARC; Plan Colombia; Prohibi­ tion; Cocaine; Coca; Marijuana; Cannabis; United Nations; UNODC; UNGASS; Drug Trafficking; Drug Policy Introduction 191 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 This paper links broader revisionist drug histories with ‘mutual negotiation’ and ‘Colombian-agency’ based approaches to PC and the UNGASS in 2016. In this rendering, Bogota appears far less a client state of grander US regional designs, and far more an active protagonist both in terms of its own destiny and in terms of the international drug policy consensus. The government of President Juan Manuel Santos undoubtedly played a key role in challenging prohibitionist drug policy precepts in the run up to UNGASS 2016, arguing that prohibitionist drug policies had failed to achieve their aims and were imposing unacceptable burdens on producer and transit states. However, to fully understand the drives to do so, one must look beyond tradi­ tional narratives of David versus Goliath and instead view his government’s approach as a manifestation of complex domestic, regional, and international political imperatives. Only then, it argues, can a more com­ prehensive account of the politics of policy displacement within the war on drugs become apparent. The paper thereby utilises revisionist accounts of Colombian-US relations, particularly those focused on PC. It links these bilateral accounts with increased revisionism in both drug policy historiography, one which highlights greater role and agency to individual countries and localities, as well as with revisionist accounts of the evolution of the UN drug treaty system. It continues in this trend of challenging orthodox interpreta­ tions which ascribe sole agency to the hegemon, the US, and portraying national and local actors as lacking agency or control of their drug agenda. It in many ways reverses this narrative, highlighting the frequent role that drugs played within Colombian diplomatic initiatives and state building exercises, from the 1970s, through Plan Colombia up to the 2012–2016 drug policy reform era. The paper suggests a further research agenda, one which unpacks the internal power relations that the war on drugs informs and the frequent utility the latter provides to national elites’ agendas. Moreover, it challenges the general portrayal of UNGASS 2016 as an earnest effort by ‘client’ Latin American states to radically challenge the drug policy status quo. It instead highlights a continuation of a complex process of international bargaining and negotiation around funding, influence and policy choices, underpinned by a continuity in domestic coalition politics and state formation. Introduction This is not to say that many Colombian and other Latin American political elites did not hope for policy improvements via the UNGASS process. It instead highlights the UN as a forum where Colombia could safely displace the policy dilemmas of drugs and thereby both challenge a status quo that was increasingly out of step with reform debates, while leveraging the possibility of some additional funding and political legitimacy from new policy linkages such as development, gender, and human rights. This also served to remove the issue, to some degree, from fraught domestic politics and enabled a discussion on policy reform that would likely be infeasible in a domestic context. Further it enabled Colombia and other Latin American states to triangulate their complex bilateral drug diplomacy with the US via the UN and thereby sidestep difficult issues in an era of peace processes, policy fragmentation, popular delegitimization of the war on drugs, and cannabis legalization in the US and Latin America. Introduction This paper analyses nation state agency in the global drug wars. Specifically, it looks at the case of Colombia, long seen as ground zero in the United States (US)-led war on drug production and trafficking in the Ameri­ cas. Plan Colombia (PC) remains a centre point of this bilateral relationship and is one of the most discussed and debated US policy interventions in the region. The popular and academic conception of PC as a US imposition is widespread (Pastor 2001; Stokes 2016, 2005). However, this paper challenges this conventional wisdom both on the supposed passiveness of Colombian elites and society in accepting US impositions. Further it challenges the idea of the war on drugs being a solely US hegemonic construct rather than a multi­ state negotiated process, underpinned by numerous social, political, and economic power determinants that eschew simple analysis based on power asymmetries between states. p y p y This paper is based on an extensive literature review analysis and is intended to chart a new analytic frame­ work in understanding the relationship between member states and the international drug control system more broadly. It suggests a further research agenda based on primary source research and interviews with policy makers. Attempting to undertake both here would have resulted in a paper that is too thin on both points. The paper’s significant contribution to the field is a linking of the siloed research on (a) Plan Colombia and (b) the UN drug control system, particularly the latter’s more recent history. It uses these key case stud­ ies as they represent arguably the most significant bilateral and multilateral drug policy engagements for Colombia and ones of major national and international significance for the US as well as governments under the UN drug control system more broadly. Plan Colombia represents one of the most extensive and insti­ tutionalised bilateral counter-narcotics efforts in global drug control history as well as an epoch shaping strategic framework in Colombia’s drug wars and its state strengthening efforts. Meanwhile, Colombia’s engagement with the UN drug control system under President Santos, from 2012 through the UN General Assembly Special Session (UNGASS) on Drugs in 2016 represented a pivotal moment for international drug policy reform discussions, and one in which Colombia played a key strategic leadership and normative role. Rethinking Drug Control and Policy Displacement g g y p The Lancet Commission on Drug Policy discussed the many ways in which drug policies have too often worked to counter health, security and human rights (Csete et al. 2016). The high financial and human bur­ den of drug war policies and their disproportionate displacement onto poor and marginalized communities is a point widely accepted within the existing literature (Collins 2014a). The question is how these policies are displaced, imported, exported, and/or developed. The notion of drug control as a policy displacement from the global north, specifically the US, remains widespread in the policy literature. For the purposes of this article, we consider the concept of policy dis­ placement as equating to the viewpoint outlined by Mejia and Restrepo in 2014: From the perspective of producer and transit countries, prohibitionist drug policies can be under­ stood as a transfer of the costs of the ‘drugs problem’ faced by consumer countries to producer and transit countries. On the one hand, under complete legalisation, consumer countries would end up bearing most of the costs associated with drug consumption (Mejia & Restrepo 2014: 27). In other words, the costs of the policy of prohibition are widely borne by producer and transit countries, while the apparent public health benefits of reduced consumption are received by consumer countries. This is a point that scholars sympathetic to prohibition, utilizing a cost-benefit analysis from consumer states, agree with. Caulkins accepts that it does not apply to source or transshipment countries. If there were a country whose people would have no interest in using a drug, and that country were beset by violence, corruption and other ills 192 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 from hosting production or international trafficking, then that country might benefit from legalisa­ tion, even if final market countries would not (Caulkins 2014: 25). Meanwhile, proponents of what Collins has termed the ‘orthodox school’ of drug regime analysis (2021), por­ tray a uniform and analytically parsimonious story on the displacement of these costs. The line of thought within the orthodox school suggests that the war on drugs, and all interventions resulting therefrom, are the product of US hegemonic ambitions to instil its moralising and interventionist drug policies at the inter­ national level. Rethinking Drug Control and Policy Displacement It continues, arguing that the US legally enshrined prohibition via the League of Nations and subsequently the United Nations and pursued aggressive imperialist policies to enshrine these in subservi­ ent nations, either by UN legislative fiat or intervention by military-economic means (Bewley-Taylor 1999). y g y y ( y y ) PC is a neat fit within this orthodox narrative, suggesting a military intervention funded and guided by the US with minimal legitimacy within Colombia. Further, President Santos’ 2012–2016 leadership of global drug policy reform discussions have often assumed a David versus Goliath narrative, one of a small supplicant state challenging the hegemonic status quo. Neither of these conceptions is correct. Both miss the deep complexity of politics and power dynamics within global, US and Colombian drug policies; the US-Colombian relationship; PC and the UNGASS process. p p This is not, of course, to negate the role or responsibility of the US in propagating a broader policy of repressive drug policies internationally and how that interacted with national political imperatives. But interact it did. As is often highlighted in many other areas of drug policy, the war on drugs has served as a mechanism for other socio-economic goals, such as enshrining unequal power relations, racial divides and/ or maintaining socioeconomic inequalities. These same factors applied to local political elites and constella­ tions. The US in many ways exerted a form of regional policy hegemony, but as the Colombia example dem­ onstrates, it was often only at the invitation of, and thereby influence of, local political elites and alliances. The same is true for Colombia’s leadership role in international drug policy over the 2012–2016 period. Without a recognition of the policy shifts and national-international dynamic underlying it, the changing international drug policy landscape over this period remains inexplicable. Far from a David versus Goliath story of reformist states versus the US-dominated drug regime (Jelsma & Bewley-Taylor 2016; Oakford 2016), UNGASS 2016 represented a continuation of the negotiated power processes of Colombian internal and regional politics and Colombian-US relations. The latter point extends beyond US interventions in Colombia, as the US was undergoing its own legal fragmentation on cannabis, impacting national-international treaty law and state-federal law. From La Violencia to Plan Colombia It remains unclear why Colombia so radically monopolised the cocaine mar­ ket at the time (Thoumi 2003, 1995). Gaviria and Mejia posit that coca base from Bolivia and Peru, which they processed into cocaine and exported to the United States and Europe (Suárez & Santos 2018). It remains unclear why Colombia so radically monopolised the cocaine mar­ ket at the time (Thoumi 2003, 1995). Gaviria and Mejia posit that The dominance of Colombia might have been due to fortuitous events, historical accidents that were then exploited and maintained for economic reasons, and the experience and specialization that resulted might have created a competitive advantage (Gaviria & Mejia 2017: 3). The scale of the emerging illicit market began to further disrupt Colombian political economy. The New York Times wrote as early as 1978 that ‘the drug traffickers have arisen not only as a new economic class, but also as a powerful political force’ (Quoted In: Gaviria & Mejia 2017). Bogota adopted a repressive strategy early on, issuing decree 1206 and creating the National Narcotics Council (CNE) in 1973 (Zorro-Sánchez 2017). Some suggest that conditionality attached to US development assistance drove this institutional develop­ ments (Guáqueta 2005). While the timing coincided with the earliest counter-narcotics policies, the relative unimportance of Colombia in the early Nixonian war on drugs suggests as much a home-grown decision as an imposition of the US or the UN drug control system. Indeed, rather than fixating on drugs, in many ways the US at the time was focused on maintaining close clientelist ties with the Colombian military through training cooperation to serve as a bulwark against communism in the region (Richani 2020). With the growing dominance of drug cartels, violent crime soon ‘reached epidemic proportions’ and tripled between the 1970s and 1990s, increasing tenfold in some regions, while leaving others broadly untouched (Gaviria 2000: 2). Homicides almost doubled from 57 per 100,0000 in 1985, to 95 in 1993. In the province of Antioquia, where Medellín is the capital, the estimated homicide rate reached 400 per 100,000 in the early 1990s (Rosen 2014). In this climate, cartels bid for political protection and sought to corrupt government institutions through bribery, judicial intimidation, and murder, earning Colombia its infamous title as a ‘narco-state’ (Mendez 2017). From La Violencia to Plan Colombia The seeds of Plan Colombia are generally traced to the emergence of the country’s ‘unholy trinity’: insurgent organisations, vigilante paramilitary groups and the illegal drug industry (Manwaring 2002). The modern configuration of these groups in turn can be traced to the era known simply as La Violencia – the Violence (Franz 2016). La Violencia exploded in April 1948 with the assassination of Jorge Eliécer Gaitán, a social reformer presi­ dential candidate. Nationwide clashes between liberals and conservatives deepened decades-old social divi­ sions (Méndez 2012). While the conservative elites remained ostensibly aloof, their client groups killed an estimated 250,000, largely in the rural areas. This only served to further solidify urban-rural divisions and embed frontier justice systems (Méndez 2012). Eventually a relatively static political alignment emerged in the 1960s, divided between ‘leftist guerrillas, rightist paramilitaries and blackmarketeering mafias’, again only further mobilising discontent in rural areas (Méndez 2012: 92). The leftist guerrilla Revolutionary Armed Forces of Colombia (FARC) was founded in 1964. In its early years, the group supported socialist ideology, independent peasant republics and specific policies such as land reform, minimum agricultural prices, expanded agrarian credit, health care, and education for peasants (Franz 2016). Following US-backed military campaigns against the FARC, the group underwent an ideologi­ cal hardening emerging as a Marxist-Leninist guerrilla movement (Mendez 2017). The National Liberation Army (ELN) was also founded in 1964 by young Marxists educated in Cuba (Rosen 2014). Meanwhile, right wing paramilitaries, supported and financed by elite political groups, quickly began to form in the 1960s to counter the momentum of Marxist movements (Franz 2016). The most prominent group would emerge as the United Self-Defence Forces of Colombia (Autodefensas Unidas de Colombia, AUC), an umbrella paramili­ tary organised in 1998 (Richani 2020). In parallel, the mid-1970s in Colombia saw the emergence and consolidation of power by the drug cartels. The resilience of the coffee and emerald mafias had long testified to Bogota’s weakness to impose legal control in many regions (Méndez 2012). Finding a market gap, the Cali and Medellín cartels began to import 193 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 coca base from Bolivia and Peru, which they processed into cocaine and exported to the United States and Europe (Suárez & Santos 2018). From La Violencia to Plan Colombia The most illustrative example of the control and penetration of drug cartels in the country’s institutions was Pablo Escobar’s election as an alternate to the National Chamber of Representatives in 1982 (Bushnell 1993). Eventually political leaders began to view drug trafficking groups as a preeminent threat to the country’s institutions, prompting the agreement of a new extradition treaty between Colombia and the United States (Gaviria & Mejia 2017). The threat of extradition sought to curb the violence caused by drug cartels, but instead achieved the opposite effect: The feral, implacable violence engulfed the nation in assassinations of journalists, judges, politi­ cians, police officers and thousands of innocents, beginning with the virtual annihilation of the Colombian Supreme Court ten months after the first extraditions (Mendez 2017: 61). On November 6, 1985, members of the M-19 a leftist revolutionary guerrilla group took over the Palace of Justice in Bogotá, assassinating 100 hostages, including half of the Supreme Court. Various key extradition documents were destroyed that day, strengthening claims that the Medellín cartel financed part of the siege (Méndez 2017). On November 6, 1985, members of the M-19 a leftist revolutionary guerrilla group took over the Palace of Justice in Bogotá, assassinating 100 hostages, including half of the Supreme Court. Various key extradition documents were destroyed that day, strengthening claims that the Medellín cartel financed part of the siege (Méndez 2017). The following years were characterised both by the government’s ‘kingpin strategy’ and the reign of terror of drug cartels (Rosen 2014). Targeting key members of drug cartels resulted in notorious backlashes such as the assassination of Presidential candidate Luis Carlos Galán in 1989 or the bombing of Avianca Flight 203 (Rosen 2014). Responding to these circumstances, President César Gaviria requested technical assistance from the United States, bringing about the capture and death of Pablo Escobar in December 1993 (Crandall 2008). With the partial dismantling of the Medellin cartel, the power vacuum left in the illegal drug business was sought to be filled by FARC and soon AUC (Franz 2016). Colombia-US relations and the genesis of Plan Colombia h d b h l d l b fl l f Britto continues, The marijuana region served as a laboratory for the US and Colombian governments to experiment with a novel approach to statecraft and international cooperation, an approach that assumed drug production and traffic to be security threats that warranted bilateral military intervention in periph­ eral areas where national sovereignty and US hemispheric hegemony were challenged (Britto 2020: 3). The conditions for this were not just an aggressive US export of its war on drugs on an unwilling neighbour. The overlap in agendas, interests and international cooperation were the by-product of forces within both countries and their hemispheric and global strategies. To some degree the increased US focus on Latin America was a natural outgrowth of its broader foreign policy and its more constrained geographic focus on international drug policies. Increasingly marginalized from drug policy debates in the Middle East and Asia in the 1960s and 1970s, and following its failed effort to strengthen the Single Convention, the US seemed naturally to gravitate towards greater bilateral drug engagement in Latin America (Collins 2015). The second phase, during the 1980s, was characterised by a ‘slow expansion’ of the war on drugs in both countries alongside the rapid expansion in cocaine production and trafficking. As insurgents’ and cartels’ power grew, so too did kidnappings, assassinations and a broad trend of insecurity (Gaviria & Mejia 2017). US President Ronald Reagan responded by authorising foreign aid for Colombian law enforcement programmes thereby expanding US participation in the design of Colombia’s drug policies (Guáqueta 2005). The third phase, ‘deep institutionalization’ ran from 1989 through 1998 and was characterised by a juxtaposition of strong institutional cooperation as well as periodic diplomatic tensions and hostility between the two coun­ tries (Guáqueta 2005: 34; Suárez & Santos 2018). In 1989, US President George H.W. Bush authorised the provision of $65 million in military supplies to the Colombian army and police and in 1990 an extraordinary regional summit produced the Cartagena Declaration, announcing a new phase in the Colombian-US anti- drug alliance (Guáqueta 2005). The election in 1994 of Ernesto Samper led to an overt diplomatic rupture, in what Crandall describes as a period of ‘hypernarcotization’ of Colombian-US relations, as the pervasiveness of drug cartel money in Colombian political institutions became evident (Crandall 2008: 45). In 1995, the US government revoked President Samper’s visa and decertified Colombia due to insufficient drug cooperation (Rosen 2014). Colombia-US relations and the genesis of Plan Colombia h d b h l d l b fl l f Colombia-US relations and the genesis of Plan Colombia Richani describes the prolonged Colombian conflict as ‘a war system’ resulting from ‘fragmented hegemony’ under which ‘violence becomes a conflict management mechanism…regulating multiple sets of contentious relationships including intra-elite relationships’ (Richani 2020: 326). Within this war system, the US was an important protagonist but only insofar as it could balance within these competing relationships and power dynamics. The US was indeed a key support structure for parts of the Colombian state, but its relationship to drugs and other issues were mediated by its situational role within Bogota’s fragmented hegemony. Alexan­ dra Guáqueta argues that US-Colombia bilateral drug relations up until PC can be divided into four phases. 194 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 The first phase, in the 1970s, began with Nixon’s declaration of the war on drugs alongside the 1971 UN drug convention (Guáqueta 2005). Financial support from the US Agency for International Development (USAID) included conditional­ ity around building a drug policy infrastructure, which at the time was heavily focused on cannabis pro­ duction in the Caribbean region (Guáqueta 2005). As the state drug control apparatus grew, the US Drug Enforcement Administration (DEA) was there to embed, train, and conduct investigations (Nadelmann 1993). It was unclear at the time but at the beginning of the 1970s Colombia was just embarking on its two decades transition ‘from a coffee republic to a narcotics nations’ (Britto 2020: 1). Britto attributes the curious rise of the Colombian cannabis industry to ‘a series of state interventions that the Colombian government carried out’ with the support of the US and private business interests ‘in pursuit of agrarian development and nation-state formation’ (Britto 2020: 2). The early attempts to ‘mod­ ernise’ and ‘develop’ these outlier regions came not from US external pressure but internal anthropological, and in many ways fundamentally liberal, initiatives to encourage progress. The United Nations Educational, Scientific, and Cultural Organization (UNESCO) along with a collection of philanthropic groups and the US federal government, sought to create a mixed growth economy along the coast, which would move the economic centre of gravity of the region away from Venezuela and towards Bogota. The initiative failed, but associated projects laid key tangible and intangible infrastructure that was to partly-enable the development of the cannabis export industry (Britto 2020). Colombia-US relations and the genesis of Plan Colombia h d b h l d l b fl l f This coincided with a more hawkish US drug policy where both parties pushed a more assertive approach to foreign governments, one which the US State Department enthusiastically embraced. In response President Samper sought to reassure US and domestic audiences of his drug war sincerity. However, as one author comments, the reality was continuity of institutional cooperation and Samper ‘was not forcing prohibition onto the state bureaucracy because it was already institutionalized’ (Guáqueta 2005: 44). The fourth phase centred on the genesis of PC (Guáqueta 2005). Colombia was one of the most unstable and dangerous countries in the region, where a three-front war between FARC, paramilitary groups and drug cartels had critically undermined the government’s monopoly of force and exacerbated capital flight (Suárez & Santos 2018). This unravelling had also fundamentally changed Colombian-US relations. As Guáqueta writes, 195 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 The old war against drugs had nothing to do with the Colombian conflict. In contrast, the new war was concerned with ending the 40-year-old Colombian conflict in which drugs pose a major secu­ rity threat because they provide money and local social support to illegal armed groups (Guáqueta 2005: 46). The old war against drugs had nothing to do with the Colombian conflict. In contrast, the new war was concerned with ending the 40-year-old Colombian conflict in which drugs pose a major secu­ rity threat because they provide money and local social support to illegal armed groups (Guáqueta 2005: 46). The blueprint for PC originated during the presidential campaign of Andrés Pastrana. A conservative run­ ning a ‘Candidacy for Peace’ platform he called for a ‘new Marshall Plan’ along with peace talks with insur­ gent groups to lead Colombia back from the brink of collapse (Mendez 2017: 87). When elected in 1998, Pastrana unveiled a broader drug policy that included enforcement-oriented supply centric measures as well as alternative development measures. Seeking international funding Pastrana looked to the US (Suárez & Santos 2018). Alternative development is ostensibly a model of drug policy intervention which seeks to address the root causes of illicit drug production by offering support and incentives for farmers to transition beyond their reliance on drug crops (See for example: Brombacher & Westerbarkei 2019). Colombian Agency and Plan Colombia As early as 2005 Alexandra Guáqueta wrote that [m]ost academic and policy-oriented writing on illegal drugs in Colombia and the United States invariably focuses on diplomatic disagreements between the two countries caused by clashing pol­ icy interests and an imbalance of power (Guáqueta 2005: 27). She continues, Colombia-US relations and the genesis of Plan Colombia h d b h l d l b fl l f y g p ( p ) In 2000, after strong lobbying by the Colombian government, the Republican-dominated congress allo­ cated $1.3 billion to Plan Colombia with the support of President Bill Clinton (Suárez & Santos 2018). In theory, the aid package was to support judicial reform, foster alternative economic development, reduce drug supply, assist the Colombian police, and expand counter-narcotics operations (Oehme 2010). In practice, the majority of PC’s funding was destined to the Colombian military and in many ways the pro­ gramme supported a counter-insurgency effort against FARC (Guáqueta 2005). A report by the Government Accountability Office of the United States outlines the objectives and budget distribution of PC, emphasis­ ing with equal importance counter-narcotics and counter-insurgency efforts. The first of three general objec­ tives was ‘to reduce the flow of illicit narcotics and improve security’ (GAO 2008: 11). Specific targets were illicit crop eradication, interdiction activities and reclaiming control of FARC-dominated territory ‘through a variety of means, including the growth and professionalism of the Colombian military and National Police, systematic military engagement with insurgent groups, and an expanded police presence throughout the country’ (GAO 2008: 1). Given the interwoven nature of these objectives, it is impossible to distinguish how much of the $7.5 billion that was destined to PC by 2005 was intended for counterinsurgency or counter- narcotics efforts, or if one was prioritised over the other. She continues, The picture offered, that of a coerced U.S. imposition on a reticent and powerless Colombia is inac­ curate (Guáqueta 2005: 27). As in the broader discussion of Colombian-US relations, the question of agency in PC has featured in a number of academic debates and disciplines. Stances can be categorised into three groups: proponents of Colombian passiveness, proponents of mutual negotiation, and proponents of Colombian agency. Propo­ nents of Colombian passiveness predominate US foreign policy analysis, human rights, and security studies (Buxton 2006; Callaway 2008; Franke & Reed 2005; Holloway 2012; Rochlin 2011; Stokes 2007; William L. Marcy 2010). These lenses view PC as unilaterally imposed, based on American geopolitical and economic concerns. These echo closely the ‘orthodox’ interpretive lens for international drug control discussed else­ where in this article whereby nation states were simply railroaded, duped, or blindly led into a system of global prohibition at the behest of the US (Collins 2020). In a discourse analysis on American constructions of Latin American identities, Holloway highlights how conceptions of Latin American inferiority (derived via perceptions of particular qualities about Latin Americans, such as volatility, infantilism, and barbarism) underpin Colombian-US relations (Holloway 2012). Plan Colombia thus exemplified a ‘natural hemispheric leadership’ by the US, which was constructed from the perception of Latin American countries as ‘incapable of self-control and effective governance (...) [and] as a breeding ground for regional instability’ (Holloway 2012: iii). In what Holloway posits as the dominant Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 196 academic discourse on PC, where Colombia is defined through a composite set of identities such as ‘drug producer/distributor, impotent, unstable, corrupt, indifferent, backward, subordinate’, the US locates itself as the victim of an external threat, an unchecked trade, in which it had no involvement (Holloway 2012: 199). The US is thus defined as ‘the drug victim, powerful, controlled, honorable, guardian’ (Holloway 2012: 199). According to Holloway, this framework explains the US’ approval of the multibillion-dollar package to improve the Colombian military. However, the author emphasises how this framing of PC discourse does not entirely write off Colombian agency. In a context of weak institutions and poor governance, Colombia’s only option to fight the drug war against insurgent groups was to resort to the intervention of a better-equipped ally, such as the US (Holloway 2012). She continues, y ( y ) Presenting another angle of the Colombian passiveness argument, Stokes characterises PC as a continu­ ation of Cold War counter-insurgency efforts hidden under the facade of humanitarian aid and drug con­ trol (Stokes 2007). Framing it as a programme designed to counter insurgency groups which undermine American interests in South America, the author emphasises the disproportionate focus on suppressing FARC insurgents and not paramilitaries, despite their heavy involvement in drug trafficking. The empty humanitarian claims of PC are also evident in the rampant human rights violations committed by the Colombian military, the lack of oversight controls, and the presence of a ‘presidential waiver’ (Stokes 2007). Stokes views the ostensible goals of PC, for example social development, as ‘propaganda devices employed to construct the US as a moral agent in world politics while hiding motives that have more to do with geo- strategic reasoning’ (Stokes 2007: 72). In particular this refers to the US’ economic interests of stable oil production, which was threatened by the FARC and ELN bombings of oil pipelines. p y g p p Similarly, Oehme posits that Washington’s intervention in Colombian politics derived from a simple cost- benefit analysis (Oehme 2010). PC enabled the US to tackle insurgency and drug trafficking, two problems which were very costly to US interests. Therefore, despite Pastrana’s desire to focus on the social develop­ ment potential of PC, ‘the United States used diplomatic and economic leverage to ensure drug interdiction and massive aerial eradication, conducted by both Colombian military and police units, were the focus of the plan’ (Oehme 2010: 225). Buxton echoes this point from a drug control perspective by emphasising the dominance of the US’ model of criminalisation and supply eradication in international drug control frame­ works, which, she argues, have been devised to ultimately aid American interests (Buxton 2006). ‘Mutual negotiation’ authors suggest that Colombians had some limited degree of agency in the develop­ ment of PC. Crandall and Tickner argue that PC was a result of pragmatic negotiations and aligned interests between Bogotá and Washington, who ‘shared the common goals of promoting political stability, economic growth, and most important, anticommunism’ (Crandall 2008: 3; A. Tickner 2007). Guáqueta Argues that ultimately the Colombian-US drug relationship was one of continuity and overarching overlap of interests (Guáqueta, 2005). She continues, As he writes, ‘astute observers knew the Colombian state was headed for failure, and it was clear that the political elite were powerless by themselves to halt it’ (Mendez 2017: 181). The Colombian political elite ‘had far more to gain from the success, and far more to lose from the failure of their efforts to interest the US in intervening, than did the US itself’ (Méndez 2012: 13). More specifically, he argues that the Colombian government’s agency was apparent through its ‘Janus-facedness’ in framing PC according to the audience: a revamped military funded by American aid (Mendez 2017). As he writes, ‘astute observers knew the Colombian state was headed for failure, and it was clear that the political elite were powerless by themselves to halt it’ (Mendez 2017: 181). The Colombian political elite ‘had far more to gain from the success, and far more to lose from the failure of their efforts to interest the US in intervening, than did the US itself’ (Méndez 2012: 13). More specifically, he argues that the Colombian government’s agency was apparent through its ‘Janus-facedness’ in framing PC according to the audience: The Plan was represented to the US Congress as a supply interdiction effort in the drugs war; it was represented to the Colombia people as an internal peace and development plan; to the Europeans a process of accommodating romantic revolutionaries. Amongst themselves, the Colombian elites knew it was a state-building and rehabilitation project. The Pastrana administration was endeavour­ ing to win the support of stakeholders everywhere whilst avoiding the fallout from their adversarial interests (Mendez 2017: 184). The author suggests that the dominant rhetoric of Colombian passiveness and American imposition is the result of scholars only seeing the ‘face’ that was presented to the US – that of a country in desperate need of foreign intervention to sustain the war on drugs (Mendez 2017). Sandra Borda continues this narrative with a focus on Colombian government efforts to ‘pivot’ towards key international issues as a way to solidify the relevance of the Colombian conflict to donor governments. In this sense, the narrative Borda portrays is very much a drug war constructed in Bogota and sold in various forums in a manner tailored to perceived audiences’ interests (Borda 2016). This is something substantiated by Colombia’s engagement with the UN drug control system. The International Drug Control System g y The origins of multilateral drug control system are generally dated to the Shanghai Opium Commission of 1909 and the Hague Opium Convention of 1912 (McAllister 2012). The system developed over numerous subsequent treaties building on the aspirational foundations enunciated in the 1912 treaty, namely limiting global drug consumption to ‘medical and scientific purposes’. Its current legal architecture is built on three international drug conventions, the 1961 Single Convention on Narcotic Drugs (as amended by the 1972 Protocol), the 1971 Convention on Psychotropic Substances of 1971 and the 1988 United Nations Convention against Illicit Traffic in Narcotic Drugs and Psychotropic Substances (UNODC 2008). The international drug control system is often examined as an archetype robust regime. Colgan et al. write, ‘robust regimes’ emerge where there are low conflicts of interest and high concentrations of power (Colgan et al. 2012). Orthodox ‘prohibition regime’ theories are predicated on such a conception. Namely, the US represented the hegemon, providing stability, leverage and the diplomatic initiatives necessary for the production of a coherent Global Drug Prohibition Regime (GDPR) (Bewley-Taylor 2012, 1999). Under this conception, smaller or weaker states emerge as passive rule-takers, beholden to US power and its ideo­ logical pursuit of an interventionist war on drugs. This approach fits closely with orthodox narrative about Colombia’s client state role within the US war on drugs. Historiographical developments, such as the ‘new drug history’ approach to Latin America (Gootenberg & Campos 2015) shed a more complicating light. First, US drug hegemony represented a relatively delayed structural development (Collins 2020). The US was not even a member of the League of Nations; European states were more activist participants than earlier US-focused historiography gave credit to (Mills 2014); modern drug prohibitions did not originate in the US, but in Asia (Windle 2013); decolonization agendas often buttressed and drove prohibitions rather than passively inculcating them (Collins 2017a); the Cold War and the realities of bipolarity acted as a fundamental constraint on US drug hegemony and thereby the UN’s ability to adhere solely to US interests (Collins 2015; Kinder 1981). The analytic parsimony of the US-led global drug prohibition regime narrative does not, as such, hold up to empirical evaluation. She continues, Oehme also suggests that, despite the different strategic approaches between Washington and Bogotá, there was ‘a clear consensus about the overarching security imperative’ (Oehme 2010: 225). The Colombian agency authors, suggest that PC was conceived in Bogotá and pursued a Colombian stra­ tegic agenda (DeShazo et al. 2009; Marcella 2001; Mendez 2017; Suárez & Santos 2018). DeShazo et al. highlight how PC was a ‘broad-based approach to security and development’ in light of the government’s sovereignty gap due to FARC and ELN dominance in crucial territories (DeShazo et al. 2009: VII). In this view, the shift from Pastrana’s social development and counter-narcotics agenda to a counter-insurgency campaign in later years was more reflective of the differing political priorities between Pastrana and his suc­ cessor Álvaro Uribe Velez, rather than primarily the US’ interests for the region (DeShazo et al. 2009). In a different vein, Suárez argues that PC’s shift to a counter-insurgency agenda was a direct result of Pastrana’s decision to involve the international community to avoid state failure. Indeed, by inviting international aid, Pastrana inevitably surrendered to the pressures of intermestic issues (such as the failed peace negotiations with FARC) in the development of PC (Suárez & Santos 2018). This approach thereby decentres the hegemon in the negotiation process, seeks to uncover and reiterate the agency of the smaller power in exerting influ­ ence over the superpower and in some cases overcoming the reluctance of the hegemon ‘to be drawn too far into its internal affairs of its victim’ (Méndez 2012). Arlene Tickner and Álvaro Méndez, argue that Plan Colombia represented a continuation of a broader international phenomenon of ‘empire by invitation’ (Lundestad 1999; Méndez 2012). In the case of Plan Colombia, both Tickner and Mendez label it ‘intervention by invitation’ (Mendez 2017; A. B. Tickner 2007). Méndez builds on Tickner’s earlier exposition that President Pastrana proactively invited US intervention for the purposes of state rebuilding after a confluence of drug, security and economic emergencies, as well as ‘chronic debility with deep historical roots’ (A. B. Tickner 2007; Mendez 2017: 182). In this view, Pastrana’s agenda was characterised by a desire to leverage the FARC and ELN towards peace negotiations through 197 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 a revamped military funded by American aid (Mendez 2017). Colombia and International Drug Control This called for a special conference to discuss a global plan of action and reiterated the call for drug trafficking to be treated as a crime against humanity. This time Argentina, Bolivia, Brazil, Ecuador, Peru, and Venezuela signed, with Colombia absent (United Nations 1998). The UN General Assembly accepted the Quito Declaration as demonstrating the need for a new conven­ tion. Latin American suggestions to expand the jurisdiction of the International Court of Justice (ICJ) and t t t d t ffi ki i i t h it d t f th l b l ff t d This was followed in quick succession by the New York Declaration against Drug Trafficking and the Illicit Use of Drugs, submitted to the UN in 1985. This called for a special conference to discuss a global plan of action and reiterated the call for drug trafficking to be treated as a crime against humanity. This time Argentina, Bolivia, Brazil, Ecuador, Peru, and Venezuela signed, with Colombia absent (United Nations 1998). The UN General Assembly accepted the Quito Declaration as demonstrating the need for a new conven­ tion. Latin American suggestions to expand the jurisdiction of the International Court of Justice (ICJ) and to treat drug trafficking as a crime against humanity were massaged out of the proposals by less affected and non-interventionist states. As with the UNGASS 2016 process (see below), Latin American governments floated open ended and more radical departures from status quo policies with the ultimate outcome of pro­ ducing a brokered and more limited consensus shift. Within a year the emerging Convention proposal had received ‘worldwide’ support from governments as well as international, regional, and non-governmental organizations (United Nations 1998). g ( ) Governments had broadly and implicitly expanded a principle of shared responsibility on the issue of drug trafficking through an enumeration of 14 elements to be included in the convention. These included a focus on asset seizure, extradition, control of precursor chemicals, intelligence sharing, and all activities related to trafficking, including ‘crop substitution’ (United Nations 1998: 7). Within a decade, Latin American govern­ ments had gone from secondary actors in the multilateral drug story to key proponents of transnational legal developments. European governments, ostensibly seeking common ground, supported this idea of a shared responsibility of consumers and producers to mitigate the effects of policy displacement. Colombia and International Drug Control Colombia and International Drug Control Colombia’s involvement with the UN and drug control has often, if not generally, been driven by an effort ‘to advance its internal political agenda’ (Borda 2016: 103). Colombia’s engagement in UN negotiations of the 1961 and 1971 Conventions were relatively limited, aside from overtly acting as a good citizen of global insti­ tutions. This began to change in the 1970s and accelerated in the 1980s with the rapid expansion of, and policy interest in, the illicit drug trade in all parts of the world. Expert groups within the UN Commission on Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 198 Narcotic Drugs (CND) began advocating new treaty measures. Law enforcement officials in the Asia Pacific region advocated ‘streamlining mutual judicial and other assistance…to facilitate the tracing, freezing and forfeiture of the proceeds of drug crimes at the international level’ (United Nations 1998: 2). Extradition sup­ port became a key focus for the UN drug secretariat as did drug precursor control, intelligence sharing, and air and sea interdiction efforts (United Nations 1998). These issues, widely exceptionalised in Colombian-US bilateral relations at the time, were but one thread of a broader multilateral momentum on these issues. The US is also widely ascribed as the propagator of the archetypically prohibitionist 1988 Convention. In fact, Latin American governments provided the key impetus. Struggling to muster national and mul­ tinational responses to the growing power of illicit drug actors, Latin American governments sought to in some way displace the burden to, and via, the United Nations. Latin American governments submitted the Quito Declaration against Traffic in Narcotic Drugs to the UN General Assembly in 1984. Signed by Bolivia, Colombia, Ecuador, Nicaragua, Peru, and Venezuela, it called for, among other measures, drug traf­ ficking to be treated as a crime against humanity (United Nations 1998). Facing domestic opposition and a weak state apparatus, displacing the problem to the UN appeared a logical choice for Colombia and other governments. Adherence to international obligations was an easy bypass for complex domestic legislative processes, while strength in numbers would raise enforcement capabilities across the region, ostensibly preventing safe havens. p g This was followed in quick succession by the New York Declaration against Drug Trafficking and the Illicit Use of Drugs, submitted to the UN in 1985. Colombia and International Drug Control Europe remained reluctant to support repressive eradication policies and often sought other proactive frameworks grounded in development interventions (Brombacher & Westerbarkei 2019; Laurent 2017). The resulting blurred strategic relationship between states focusing on drug enforcement goals and those targeting devel­ opment outcomes has led many to critique development approaches to drugs as lacking a true development orientation, and being instead bound to drug control, supply reduction goals (Buxton 2015). The early efforts at Colombian Alternative Development (AD) saw a shift of eradication strategy away from a sole focus on fumigation and some attempt to incorporate crop substitution as a ‘complementary strategy’. This was in line with President Betancur’s National Rehabilitation Plan (PNR) (Zorro-Sánchez 2017: 68). The UN undertook the first efforts, ultimately focusing in Cauca and Nariño under the administration of President Virgilio Barco (1986–1990). These were generally isolated and aimed at halting the spread of cul­ tivation. They produced ‘no sustainable results’ (Zorro-Sánchez 2017: 69). The 1988 Convention had opened new avenues of substitution funding which encouraged the Colombian state to become more directly involved. The administration of César Gaviria (1990–1994) maintained a mixed, ‘contradictory’, approach, expanding on crop spraying, while scaling-up substitution programmes with UN support (Zorro-Sánchez 2017). The years 1993–2002 saw the government further expand and mainstream these interventions. International institutions, such as the International Development Bank (IDB) and nongovernmental devel­ opment organizations increasingly sought to engage. USAID also significantly increased support for sub­ stitution programs (Zorro-Sánchez 2017). New programmes, driven by community mobilisation in the 199 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 mid-1990s in opposition to fumigation of small-scale producers, led to a firm policy shift towards alterna­ tive development as a core policy approach rather than one subservient to fumigation (Zorro-Sánchez 2017). Although external funding helped create institutional structures and programmes that would be internal­ ised by the Colombian state and civil society, it was the delicate negotiation of various domestic coalitions and political movements which ultimately drove Colombian policy towards a greater development orienta­ tion. With the adoption of Plan Colombia in 1999 under President Andrés Pastrana, AD became one of its ten strategies, but many would argue was subservient to eradication efforts. Colombia and International Drug Control The US was a co-financier of these efforts, which some authors view as enabling the US to ensure a strong focus on fumigation alongside development efforts (Zorro-Sánchez 2017). ‘Shared responsibility’ formally became part of the international drug control strategy at the 1998 UNGASS (UNGA 1998). Further the 1998 UNGASS Action Plan had specifically included AD, in what some com­ mentators have referred to as a ‘crucial moment’ for donor countries to engage with drug control beyond purely policing-oriented approaches (Brombacher & Westerbarkei 2019: 90). The reality of course was that Colombia was a country ahead of the international curve and the 1998 UNGASS merely served to elaborate what was already an evolving reality on the ground. Nevertheless, ‘the principle of shared responsibility turned out to be the life insurance for AD in the decades to come’ (Brombacher & Westerbarkei 2019: 90). The administration of Álvaro Uribe again maintained some continuity, particularly based on the mix of economic alternatives and eradication, albeit under changed or evolved domestic institutional structures (Zorro-Sánchez 2017). ( ) Through the early 2000s Colombia maintained this framework as a good citizens of the drug control system, while promoting interests around AD and enforcing anti-production and trafficking laws (Borda 2016). The initiation of the US ‘war on terror’ led to a deprioritisation of the war on drugs, but the Uribe administration proved adroit at linking its domestic drug agenda to terror, for example the President in a speech to the UN in 2002: There is concern about weapons of mass destruction in the United Nations forum, and we share this anguish. Please, we must understand that drugs have the capability for mass destruction equivalent to that of the most feared chemical weapon (Quoted in: Borda 2016: 108). The administration also expanded its active linkage between drugs, human rights and the environment, but to a far less effective degree. As Borda writes, ‘[t]he central idea is that illegal drug money interposes itself between the state and its objective of guaranteeing the security and human rights of its citizens’ (Borda 2016: 114). This ultimately warped the mainstream vision of human rights, including state complicity in human rights abuses and ‘the penetration of the same [drug] monies into the Colombian political class and the very apparatus of the state’ (Barrett 2012; Borda 2016: 114; Lines 2017). Colombia and International Drug Control It would take the administration of President Santos before Colombia found a tone on drugs and human rights that would resonate with international human rights groups, largely framing human rights problems as systemic to the war on drugs itself (Collins 2017b). Further, sitting world leaders, including in Colombia and Mexico adopted a more reformist tone. In 2012 President Santos of Colombia wrote in a London School of Economics report, He continued, The Colombian Government strongly believes that the time has come to take a fresh look and we invite world leaders, scientists and experts to start an open, serious and honest debate about this war. The time has come to think outside the box. Our invitation is to dutifully study new formulas and approaches screened through an academic, scientific and non-politicised lens, because this war has proven to be extremely challenging and oftentimes, highly frustrating. This is a global problem that demands a global solution, and therefore a new international consensus is needed (Santos 2012: 2). Ultimately his scope for action was limited by his complex domestic coalition as well as the delicacies of the peace process. These were only complicated by regional dynamics and growing anti-drug war sentiments. All of these factors augured for a removal of the drug issue from domestic politics in so far as possible, while also suggesting the need for some significant changes in Colombian and international drug policy. Displac­ ing the dilemma to international fora to mitigate, and potentially to solve, seemed to represent perhaps the best option for the administration and one which it embraced. These constraints set the tone for the Colombian and Latin American reformist governments leading into the UNGASS 2016 meeting. They offered a challenge to the status quo within a consensus institutional setting in the hope of finding an evolved or potentially alternative consensus. In parallel, Latin American governments tended to follow a broad continuity in domestic policies. The Colombian peace process saw a much greater focus placed on peacebuilding and development in drug crop settings (Meger & Sachseder 2020), but this tended to follow in the broader domestic institutional lineage of drugs and development pol­ icies. Meanwhile, the US government tacitly enabled this dialogue, often against the more hawkish elements within its own administration. In 2012 Vice-President Joe Biden declared the debate on drug war orthodoxy in the region ‘totally legitimate’, paving the way for a more substantial regional dialogue (Williams & Cattan 2012). ) The Organization of American State (OAS) embraced this opening, producing the report Scenarios for the Drug Problem in the Americas, 2013–2025 (Organization of American States, 2012). Alongside this, Uruguay, Colorado and Washington State moved ahead with cannabis legalization. Again, the Obama administrations’ drug war scepticism won out, producing an effectively accommodationist approach at the Federal level, led by the Department of Justice. The Fragmentation of International Control, 2008–2016 The Fragmentation of International Control, 2008–2016 One author dates the onset of global drug control fragmentation to 2008 (Collins 2017c). US President Barack Obama, both a vocal sceptic of the war on drugs and attempting to repair regional relations following the perceived unilateralism of the George W. Bush administration, adopted a greater multilateral tone. This coincided with domestic fiscal crises which forced local governments to reassess drug war policies as well as to begin frank assessments of cannabis legalization as a revenue measure (Collins 2017c). Latin American leaders also began to speak out. The Latin American Commission on Drugs and Democracy, comprising a high-profile list of former presidents and leaders, launched a report challenging the war on drugs and advocating an open discussion on drug policy (Latin American Commission on Drugs and Democracy 2009). This was followed by a 2011 Global Commission on Drug Policy report which called for ‘not just alternatives to incarceration and greater emphasis on public health…but also decriminalization and experiments in legal regulation’ (The Global Commission on Drug Policy 2011). By 2014, the Global Commission was explicitly advocating a more robust focus on the legal regulation of drugs (The Global Commission on Drug Policy 2014). Further, sitting world leaders, including in Colombia and Mexico adopted a more reformist tone. In 2012 President Santos of Colombia wrote in a London School of Economics report, 200 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 Colombia has experienced progress in this fight, and historic results have been achieved. We are no longer the world’s top cocaine producer…However, we are now witnessing, with profound concern, how this situation is drifting to neighbouring countries, along with all the pain, violence and cor­ ruption it entails, which we have endured in our country for too many years (Santos 2012: 2). He continued, This forced the US State Department to seek an uncomfortable accommoda­ tion with the UN drug conventions, suggesting treaty flexibility and the reality that ‘some countries will have very strict drug approaches; other countries will legalize entire categories of drugs’ (Brownfield 2014). To some degree, the controversial US response, what one commentator termed the ‘Brownfield doc­ trine’ (Collins 2014b) was not just about squaring US local regulatory changes with international law. It had opened the door for Latin American governments to walk through, perhaps recognising that few, if any, would. None did. Instead the pursuit of consensus continued via the UN and its expansive, but hardly radical – in the sense of really challenging the tenets of the drug control system – UNGASS 2016 Outcome Document (United Nations General Assembly, 2016). ( y ) Civil society had staked heavy hopes that the UNGASS era would lead to a collapse of the consensus and the onset of radical systemic drug policy reforms. Many were left reeling in the immediate aftermath (Jelsma & Bewley-Taylor 2016). Some came to recognize the incremental victories it contained (Lines & Barrett 2016), and this ultimately became a consensus view among civil society with time. One commentator suggested that civil society had ignored political realities and ‘the assumption that the status-quo arose from a con­ spiracy of silence rather than the intersection of very tangible interests, concerns and political alignments’ (Collins, 2017c, p. 11). Instead, Collins argues, ‘member states were always and inevitably set to subjugate reformist rhetoric to consensus’ (Collins 2017c: 11). The 2016 Outcome Document moved beyond a three-pillar approach of drug control based on supply reduction, demand reduction and international cooperation, towards a seven-chapter thematic focus. This directly addressed areas of human rights, gender, and development. The concept of ‘development-oriented 201 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 drug control’, adopted in the 2009 Political Declaration and Plan of Action (Commission on Narcotic Drugs 2009), reached a new level with the UNGASS in 2016. Chapter VII assigns a key role to AD and also concep­ tually expands the range of development interventions towards urban drug markets and drug trafficking (Brombacher & Westerbarkei 2019; United Nations General Assembly 2016). He continued, For Colombia, the apotheosis of UNGASS 2016 in drugs and development terms coincided perfectly with a peace process that required large injections of domestic and international funding into areas affected by drug crop production. As UN discussions moved further beyond the sole focus on policing and enforcement the areas of potential donor overlap only increased. The real outcome of UNGASS 2016 for Colombia and other Latin American governments was not so much about a radical change in policies or consensus, but in a shift in emphasis, priorities and routes for scaling domestic, and to a lesser degree foreign, funding. For Colombia, the UNGASS process had, in many ways, been an instrument in pursuit of a wider state building project, rather than pursuing an end of drug policy reform in and of itself. Again, the narrative of Colombian agency looms large. Colombia was unquestionably a leading and highly credible voice in the UNGASS process, supported by the particular normative vision of its leader. This occurred at an exact moment when the US lost credibility among conservative states due to cannabis legalisation and a softening of the drug war rhetoric at home, again driven in no small part by the anti-drug war beliefs of President Obama and his Attorney General Eric Holder. Colombia’s reformist rhetoric almost undoubtedly derived from a strong drug war scepticism within the Santos administration, originating with the President himself. However, the UNGASS process must be viewed within the larger history of Colombian policy formation around drugs, security and development. Further, it must be seen as part of a broader effort to secure financial aid and international legitimacy for the fledgling peace process. Lastly, it represented a useful venue to displace the difficulties and complexi­ ties of negotiating a shift in drug policy emphasis and priorities away from tense and entrenched national fora and interests and towards legitimating international bodies which could lead on problem-solving and reformulating the approach from the outside. g pp Unable to maintain a broad drug policy reform coalition at home, the Colombian government displaced the policy problem, first to the OAS, through drug policy scenario planning (OAS 2013), and then to the UN. Conclusion This paper straddles between the Colombian agency and mutual negotiation framework for PC and drug control more broadly. It suggests a far greater Colombian agency than has been traditionally assumed, while recognising the clear and important role the US has played. That the pursuit of prohibition and a war on drugs has imposed immense harm on Colombia is beyond question. That Colombia has served as ground zero in this global fight is further accepted as consensus. This article challenges the conception of the war on drugs as a one-way policy displacement on Colombia as a drug production country. It looks to the evi­ dence of Plan Colombia and Colombia’s involvement with the UN drug control system, particularly around UNGASS 2016, to highlight that Colombia was at least a participant is a negotiation of displacement, both to international fora and from the US and UN drug control apparatus. Indeed, it highlights that Colombia utilised drug control to secure desired outcomes from US and UN institutions. Finally, it suggests that the OAS and UN served as useful fora for Colombia’s displacement of statebuilding dilemmas, including drug control, and utilised these to serve as a proxy negotiating forum with the US and other international donors. Taken at face value, Colombia’s efforts to ‘challenge’ the global consensus on drug policy in the UNGASS 2016 period seems like a major departure with the past. Instead, this paper has argued that it fits well within a continued evolution of Colombian drug policy within various national, bilateral, and international align­ ments. Viewed in isolation Colombia’s newfound reticence to blindly pursue a war on drugs appeared a radical departure. Viewed in terms of the history of bargaining and elite politics underpinning its approach to Colombian-US relations, most exemplified by Plan Colombia, it seems less exceptional. Colombia was part of a growing wave of disenchantment with war on drugs policies, both within the US and within producer and transit countries. It was well positioned to utilize its credible position to both externally challenge the status quo and simultaneously negotiate new power relations within the drug control system. This paper straddles between the Colombian agency and mutual negotiation framework for PC and drug control more broadly. It suggests a far greater Colombian agency than has been traditionally assumed, while recognising the clear and important role the US has played. He continued, Should these organizations be able to coalesce a new vision for drug policy and explain the technical implementation of such a policy, Colombia would benefit from a repatriation of drug policy control and the possibility of a more effective ‘post-war on drugs’ approach’. Should these organisations fail to elaborate a new vision, the political costs of this failure would not directly accrue to the Colombian government, nor the domestic political alliances underpinning it. Conclusion That the pursuit of prohibition and a war on drugs has imposed immense harm on Colombia is beyond question. That Colombia has served as ground zero in this global fight is further accepted as consensus. This article challenges the conception of the war on drugs as a one-way policy displacement on Colombia as a drug production country. It looks to the evi­ dence of Plan Colombia and Colombia’s involvement with the UN drug control system, particularly around UNGASS 2016, to highlight that Colombia was at least a participant is a negotiation of displacement, both to international fora and from the US and UN drug control apparatus. Indeed, it highlights that Colombia utilised drug control to secure desired outcomes from US and UN institutions. Finally, it suggests that the OAS and UN served as useful fora for Colombia’s displacement of statebuilding dilemmas, including drug control, and utilised these to serve as a proxy negotiating forum with the US and other international donors. Taken at face value, Colombia’s efforts to ‘challenge’ the global consensus on drug policy in the UNGASS 2016 period seems like a major departure with the past. Instead, this paper has argued that it fits well within a continued evolution of Colombian drug policy within various national, bilateral, and international align­ ments. Viewed in isolation Colombia’s newfound reticence to blindly pursue a war on drugs appeared a radical departure. Viewed in terms of the history of bargaining and elite politics underpinning its approach to Colombian-US relations, most exemplified by Plan Colombia, it seems less exceptional. Colombia was part of a growing wave of disenchantment with war on drugs policies, both within the US and within producer and transit countries. It was well positioned to utilize its credible position to both externally challenge the status quo and simultaneously negotiate new power relations within the drug control system. 202 Collins and Alarcón: Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016 As Méndez writes, ‘[t]he US government intervened in Colombia’s internal affairs on a much grander scale than previously contemplated, largely because the Colombians resolved to invite it’ (Méndez 2012: 12). References Barrett, D. 2012. Refections on Human Rights and International Drug Control. In Collins, J (Ed.), Governing the Global Drug Wars. LSE, London. Bewley-Taylor, DR. 2012. International Drug Control: Consensus Fractured. Cambridge University Press. DOI: https://doi.org/10.1017/CBO9781139057424 Bewley-Taylor, DR. 1999. The United States and International Drug Control, 1909–1997. Continuum, ­London. Borda, S. 2016. Colombia’s Multilateral Drug Policy During the Two Uribe Administrations: Towards ­Prohibitionist Activism in an Era of Détente. In Gaviria, A and Mejia, D (Eds.), Anti-Drug Policies in Colombia: Successes, Failures, and Wrong Turns, Vanderbilt University Center for Latin American Studies. Nashville: Vanderbilt University Press. DOI: https://doi.org/10.2307/j.ctv167556h.11 Britto, L. 2020. Marijuana Boom: The Rise and Fall of Colombia’s First Drug Paradise. Univ of California Press. DOI: https://doi.org/10.1525/9780520974265 Brombacher, D and Westerbarkei, J. 2019. From Alternative Development to Sustainable Development: The Role of Development Within the Global Drug Control Regime. Journal of Illicit Economies and ­Development, 1: 89–98. DOI: https://doi.org/10.31389/jied.12i Brownfield, WR. 2014. Trends in Global Drug Policy. Bushnell, D. 1993. The Making of Modern Colombia: A Nation in Spite of Itself. University of California Pres Bushnell, D. 1993. 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Competing Interests p g One of the authors is editor-in-chief of the journal where this article is published. Editorial procedures were followed to ensure the author had no knowledge or involvement in the article’s peer review process. Conclusion PC was borne of this vision of ‘soft’ US intervention on the Colombian political and military elites’ terms and indeed the Colombian government succeeded in drawing the US into its internal affairs to a far greater degree than it would have generally countenanced, evidenced by visible reluctance within the US Congress (Méndez 2012). The recent UNGASS experience follows this trend. Absent a stable governing coalition to enable domestic policy reforms, President Santos sought instead to displace some of the impetus for policy changes to a regional and, eventually, global level via the UNGASS. 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Journal of Illicit Economies and Development, 3(2), pp. 190–205 DOI: https://doi.org/10.31389/jied.93 Submitted: 20 January 2021 Accepted: 01 November 2021 Published: 23 November 2021 Copyright: © 2021 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/ licenses/by/4.0/. Copyright: © 2021 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/ licenses/by/4.0/. Journal of Illicit Economies and Development is a peer-reviewed open access journal published by LSE Press. 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Oridonin Sensitizes Cisplatin-Induced Apoptosis via AMPK/Akt/mTOR-Dependent Autophagosome Accumulation in A549 Cells Huahong Yang 1, Yun Gao 1, Xiaoye Fan 2, Xingkai Liu 3, Liping Peng 1* and Xinxin Ci 2* 1 Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun, China, 2 Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, China, 3 Department of Hepatopancreatobiliary Surgery, The First Hospital of Jilin University, Changchun, China Huahong Yang 1, Yun Gao 1, Xiaoye Fan 2, Xingkai Liu 3, Liping Peng 1* and Xinxin Ci 2* 1 Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun, China, 2 Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, China, 3 Department of Hepatopancreatobiliary Surgery, The First Hospital of Jilin University, Changchun, China Edited by: Edited by: Simona Rapposelli, University of Pisa, Italy Reviewed by: Daniela Cesselli, University of Udine, Italy Grazia Chiellini, University of Pisa, Italy *Correspondence: Liping Peng plp640317@163.com Xinxin Ci cixinxin@jlu.edu.cn Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology Received: 10 May 2019 Accepted: 30 July 2019 Published: 14 August 2019 Keywords: oridonin, non-small cell lung cancer, cisplatin sensitivity, autophagy, apoptosis, AMPK/Akt/mTOR ORIGINAL RESEARCH published: 14 August 2019 doi: 10.3389/fonc.2019.00769 ORIGINAL RESEARCH published: 14 August 2019 doi: 10.3389/fonc.2019.00769 ORIGINAL RESEARCH published: 14 August 2019 doi: 10.3389/fonc.2019.00769 ORIGINAL RESEARCH published: 14 August 2019 doi: 10.3389/fonc.2019.00769 AMPK-mediated autophagy and Akt/mTOR pathways play important roles in current cancer treatments. Oridonin (Ori), an ent-kaurane diterpenoid isolated from Isodon rubescens, exerts extensive anti-tumor potential and controversial effects on autophagy. In this study, we investigated the effect of Ori on the autophagy, apoptosis, and AMPK/Akt/mTOR pathways and determined whether Ori was related to the increased cisplatin sensitivity observed in A549 cells. First, we found that Ori suppressed Akt/mTOR, Bcl2 and autophagy flux with enhanced levels of Atg3, P62, and LC3II, which was also shown as the accumulation of autophagosomes. AMPK and pro-apoptotic proteins (caspase3, Bax, and PARP) were activated in Ori-treated cells. With the pretreatment of compound c (AMPK inhibitor), the activation of autophagosomes, apoptosis and the inhibition of Akt/mTOR pathways induced by Ori were all reversed. The Ori-activated apoptosis-related markers mentioned previously and the cell-killing effect were restrained by 3-MA (inhibitor of autophagosomes) treatment. Therefore, we hypothesized that the Ori-induced pro-apoptotic effect was mediated by AMPK/Akt/mTOR-dependent accumulation of impaired autophagosomes. Furthermore, Ori could increase the sensitivity of cisplatin through its increased cell-killing, autophagy-suppressing and apoptosis-inducing activities. In addition to sensitizing cisplatin, Ori also alleviated cisplatin-induced acute renal injury in vivo, manifested as depleted BUN, CRE, kidney index, and weight loss compared to the cisplatin group. In summary, apart from its protective effect on cisplatin-induced nephrotoxicity, Ori enhanced cisplatin sensitivity via its pro-apoptotic activity mediated by AMPK/Akt/mTOR-dependent autophagosome activation, which may be a potential therapeutic target for non-small cell lung cancer. Edited by: Simona Rapposelli, University of Pisa, Italy Reviewed by: Daniela Cesselli, University of Udine, Italy Grazia Chiellini, University of Pisa, Italy *Correspondence: Liping Peng plp640317@163.com Xinxin Ci cixinxin@jlu.edu.cn INTRODUCTION human glioma cells (21). Unfolded protein response suppresses cisplatin-induced apoptosis via autophagy regulation in human hepatocellular carcinoma cells (22). Therefore, in our study, we mainly studied the role of above pathways in cisplatin resistance in NSCLC cells. With the highest morbidity and mortality, lung cancer is a serious disease affecting public health (1). In terms of biological characteristics, lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for 80–85% of all lung cancers with poor prognosis (2). Cisplatin remains the first-line chemotherapy drug for NSCLC, despite the occurrence of cisplatin resistance. Numerous studies show that some cytotoxic agents can reverse the cisplatin resistance of NSCLC patients, however, the survival rates are still low (3–5). Therefore, there is an urgent need to develop new effective adjuvant therapies against NSCLC. An ent-kaurane diterpenoid isolated from Isodon rubescens, Oridonin (Ori) and its analogs exert anti-tumor potential in cancer cells (23–25). Besides, the mechanisms involved are mainly concentrated on autophagy and apoptosis (26, 27). However, the effect of Ori on autophagy in cisplatin-induced lung cancer cells has not been thoroughly elucidated to date. It has been verified that Ori could induce the conversion of LC3II/I in A549 cells, but the fusion of autophagosome- lysosome and the expression of P62 (marker of autophagy degradation) have not been investigated (27). So in our study, we aimed to explore the role and correlation of autophagy, apoptosis and AMPK/Akt/mTOR induced by Ori in cisplatin- treated A549 cells, and provided a new therapeutic target against carcinogenesis and cisplatin resistance in lung cancer. j p g Autophagy and relevant pathways are closely related to the emergence of cisplatin resistance (6). Autophagy, also known as “self-eating,” is the process of transporting damaged, denatured or aged proteins and organelles to lysosomes for digestion and degradation. Autophagy consists of three stages: formation of autophagosomes, fusion of autophagosome-lysosome and degradation of autophagolysosomes. There is growing evidence that autophagy takes part in many human diseases, such as cancer, neurodegenerative disease, metabolic diseases and immune-related disorders (7–9). From the studies of autophagy and cancer, autophagy takes part in tumorigenesis, metastasis and cisplatin resistance (10). Cisplatin resistance can be induced by the activation of autophagy, and the autophagy inhibitor chloroquine (CQ) can improve the sensitivity of endometrial cancer cells to cisplatin (11, 12). Citation: Yang H, Gao Y, Fan X, Liu X, Peng L and Ci X (2019) Oridonin Sensitizes Cisplatin-Induced Apoptosis via AMPK/Akt/mTOR-Dependent Autophagosome Accumulation in A549 Cells. Front. Oncol. 9:769. doi: 10.3389/fonc.2019.00769 August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 1 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. INTRODUCTION CQ blocks the fusion of autophagosomes and lysosomes and causes the accumulation of autophagosomes, which highlights the relationship between autophagosomes and cisplatin resistance. Moreover, recent studies show that the activation of autophagosomes inhibits the proliferation of tumor cells and sensitizes cisplatin-induced resistance in cervical cancer cells (13, 14). Therefore, finding a compound that induces autophagosome accumulation may be a new measure to reverse chemotherapy resistance. Frontiers in Oncology | www.frontiersin.org Cell Culture A549 (human NSCLC cell) and B2b (human bronchial epithelium cell) were purchased from the China Cell Line Bank (Beijing, China). Cells were maintained in DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic and incubated at 37◦C in a 5% CO2 atmosphere. As a vital effector of autophagy regulation, apoptosis can be initiated to counteract the proliferation, metastasis, and cisplatin resistance of cancer cells, so that many activators of apoptosis are used in cancer treatment (20). Studies have demonstrated that 3-MA (autophagy inhibitor) increased cisplatin-induced apoptosis by increasing endoplasmic reticulum stress in U251 Reagents Oridonin was obtained from Chengdu Pufeide Biotechnology Company. All of the primary antibodies LC3, P62, Atg3, Bax, Bcl2, caspase3, PARP, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, and β-actin were from Cell Signaling Technology or Abcam. FITC-annexin V and propidium iodide (PI) were from BD or Invitrogen. Cell culture medium DMEM, antibiotic- antimycotic and trypsin–EDTA were from Corning, MBI and Biofil, respectively. Fetal bovine serum (FBS) and PBS were obtained from BI. Autophagy detection kit was obtained from Abcam. Cell Counting Kit-8 (CCK-8) was purchased from Bimake. BUN and CRE detection kits were obtained from Nanjing jiancheng Biotechnology Company. BCA protein assay kit was provided from Thermo. Many signaling pathways are involved in the regulation of autophagy, such as AMPK and Akt/mTOR (15, 16). The mammalian target of rapamycin (mTOR) acts as a negative regulator of autophagy and exists in two complexes (mTORC1 and mTORC2). Protein kinase B (Akt) and AMP-activated protein kinase (AMPK) pathways can regulate mTORC1 positively and negatively (17). Previous studies have shown that Akt inhibition significantly reduces the phosphorylation of mTOR and enhances cell autophagy in human oral cancer CAR cells (18). Additionally, AMPK inhibits mTORC1 through direct phosphorylation of the tumor suppressor TSC2 on Ser 1387 or subunit Raptor on two conserved serines and subsequently triggers autophagy flux (17). In addition, targeting AMPK and Akt/mTOR signaling can overcome cisplatin resistance in ovarian and oral cancer cells, respectively (18, 19). Animals C57BL/6 WT mice were obtained from Liaoning Changsheng Technology Industrial, Co., Ltd. (Certificate SCXK2010-0001; Liaoning, China). All mice were kept in SPF-grade animal room and fed with sterile water and standard rodent chow. All animal studies were reviewed and approved by the Animal Welfare and Research Ethics Committee of Jilin University. Flow Cytometry (FCM) Analysis of Apoptosis After 18 h of drug treatment (20 µM Ori and 20 µM cisplatin), the cells were detached from the culture plates, washed with PBS and suspended in 5 µl of Annexin V binding buffer for 20 min in the dark. Then, the cells were stained with 2 µl of PI and analyzed with a FACS. Cell Viability Assay A549 and B2b cells were seeded in 96-well plates (1.5 × 104 cells/well) and treated with different doses of Ori or cisplatin for 18 h. Then 10 µl CCK-8 was added to every well for 1 h and measured at 450 nm according to the instructions. August 2019 | Volume 9 | Article 769 2 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. Oridonin Sensitizes Cisplat Ori regulates autophagy, AMPK, Akt/mTOR signaling and apoptosis. (A) The structure of Oridonin (Ori). (B) A549 cells and B2b cells were t es (10, 20, and 30 µM) of Ori for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered as 10 cology | www frontiersin org 3 August 2019 | Volum FIGURE 1 | Ori regulates autophagy, AMPK, Akt/mTOR signaling and apoptosis. (A) The structure of Oridonin (Ori). (B) A549 cells and B2b cells were treated with different doses (10, 20, and 30 µM) of Ori for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered as 100%. (Continued) August 2019 | Volume 9 | Article 769 3 Frontiers in Oncology | www.frontiersin.org Yang et al. Oridonin Sensitizes Cisplatin in A549 Cells FIGURE 1 | (C–H) Cells were treated with different doses of Ori (5, 10, and 20 µM) for 18 h. Representative western blots and statistical results showed the protein levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, caspase3, Bax, Bcl2, and PARP. (I,J) Cells were treated with Ori (20 µM) in the absence or presence of CQ (50 µM) for 18 h. Acidic vesicular organelles (autophagosomes) were stained with acridine orange. Scale bars: 10 µM. (K–N) Cells were treated with Ori (20 µM) in the absence or presence of CQ (50 µM) for 18 h. LC3 was measured with immunoblot and immunofluorescence. Scale bars: 20 µM. The results showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. ##p < 0.01 vs. the CQ group. FIGURE 1 | (C–H) Cells were treated with different doses of Ori (5, 10, and 20 µM) for 18 h. Representative western blots and statistical results showed the protein levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, caspase3, Bax, Bcl2, and PARP. Western Blot Cells were homogenized in RIPA lysis buffer that contained protease and phosphatase inhibitors. Protein concentrations were determined using a BCA protein assay kit (Thermo 23227). According to the instructions, reagent A and B were mixed together in a ratio of 50 to 1 and added into every well for the measurement of OD. Then proteins were quantified and separated by SDS-PAGE (10–12.5%) and transferred to PVDF membrane. Then blocked with non- fat milk for 1 h and incubated with primary antibodies overnight. The next day, the membrane were washed and followed with secondary antibodies for 1 h and detected by ECL. Experimental Models of AKI and Biochemical Assay The mice were fasted for 12 h and randomly divided into 4 groups: the control group without any treatment (Con, n = 5); cisplatin-treated group (20 mg/kg, Cis, n = 5); Ori- treated group without cisplatin (20 mg/kg, Ori, n = 5); Ori- treated group with cisplatin treatment (Ori+Cis, n = 5). Cisplatin (20 mg/kg) was injected for 72 h to construct an acute kidney injury model, and 20 mg/kg Ori was given for 3 consecutive days. Mice were sacrificed at 24 h after the last Ori administration and body and kidney weights were recorded. Blood was collected to test BUN and CRE levels according to the manufacturer’s instructions. Cell Viability Assay (I,J) Cells were treated with Ori (20 µM) in the absence or presence of CQ (50 µM) for 18 h. Acidic vesicular organelles (autophagosomes) were stained with acridine orange. Scale bars: 10 µM. (K–N) Cells were treated with Ori (20 µM) in the absence or presence of CQ (50 µM) for 18 h. LC3 was measured with immunoblot and immunofluorescence. Scale bars: 20 µM. The results showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. ##p < 0.01 vs. the CQ group. Immunofluorescence Staining Cells were seeded in glassy plates and treated with Ori (20 µM), cisplatin (20 µM) or compound c (5 µM) for 18 h. After fixation in 4% paraformaldehyde for 30 min, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with goat serum. Then, cells were incubated with primary antibody (anti- LC3) overnight and the corresponding secondary antibody. The nuclei were stained with DAPI for 20 min and images were captured with a confocal microscope. LC3 puncta were green and localized in the cytoplasm and nuclei were stained blue. Then the intensity of LC3 puncta was examined by Image J. RESULTS Ori Regulates Autophagy, AMPK, Akt/mTOR Signaling, and Apoptosis First, we treated cells with different doses of Ori (10, 20, and 30 µM), and found that Ori could significantly inhibit the viability of A549 cells in a dose-dependent manner. Then the same doses were used in B2b cells, and we ultimately chose Ori (5, 10, and 20 µM) for the following vitro experiments (Figure 1B). As autophagy and related pathways play a vital role in cancer treatment, we investigated whether Ori had Statistical Analysis Data presented are representatives from at least three independent experiments. All results in this study were expressed as the means ± SEM and analyzed using SPSS19.0 (IBM). Statistical analysis was employed the unpaired Student’s t-test by using GraphPad 5 Software. Value of p < 0.05 was considered significant. Quantification of Autophagosomes This step was performed using autophagy detection kit (Abcam 139484). Cells were seeded in 96-well plates and treated with different reagents (20 µM Ori, 20 µM cisplatin and 5 µM compound c) for 18 h. Starvation and CQ (50 µM) were positive controls. Then, the cells were stained with dual detection reagent and incubated for 30 min at 37◦C. After washed with assay buffer, the cells were fixed with 4% formaldehyde. Finally, images were captured with a fluorescence microscope and the fluorescence intensity of autophagosomes (green) was examined by Image J. Determination of Combination Index orange solution for 15 min and images were collected by fluorescence microscope. The interaction between Ori and cisplatin was determined by the combination index (CI) according to Chou-Talalay’s median- effect plots and isobologram principles (28). Cells were treated with different doses of each single drug or their combinations at the set molar ratios (1:1). The equation for the isobologram was shown as CI = (D)1/(Dx)1 + (D)2/(Dx)2. (Dx)1 and (Dx)2 indicated the individual doses of Ori and cisplatin required to inhibit a given level of cell viability, and (D)1 and (D)2 were the doses of them necessary to produce the same effect in combination. The combination effects were indicated as follows: CI = 1, additive effect; CI > 1, antagonism; CI < 1, synergism. Acridine Orange Staining g g A549 cells (1 × 106 per well) were treated with Ori (20 µM) and CQ (50 µM) for 18 h and incubated with acridine August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 4 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. FIGURE 2 | Ori initiates autophagy, apoptosis and Akt/mTOR signaling by the regulation of AMPK. Cells were treated with Ori (20 µM) in the absence or presence of compound c (5 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by fluorescence microscope. The green fluorescence intensity was analyzed by Image J. Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images were captured with confocal microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–J) Western blots and statistical results showed the protein levels of P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. FIGURE 2 | Ori initiates autophagy, apoptosis and Akt/mTOR signaling by the regulation of AMPK. Cells were treated with Ori (20 µM) in the absence or presence of compound c (5 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by fluorescence microscope. The green fluorescence intensity was analyzed by Image J. Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images were captured with confocal microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–J) Western blots and statistical results showed the protein levels of P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. August 2019 | Volume 9 | Article 769 5 Frontiers in Oncology | www.frontiersin.org Yang et al. Oridonin Sensitizes Cisplatin in A549 Cells Ori Initiates Autophagy, Apoptosis, and Akt/mTOR Signaling by the Regulation of AMPK the corresponding regulatory effects in A549 cells. Autophagy- related proteins (P62, LC3, and Atg3) were activated in a dose- dependent manner (Figures 1C,F). We subsequently observed that Ori (10 and 20 µM) evidently activated AMPK, caspase3, Bax, and PARP, but decreased the protein levels of Akt, mTOR, and Bcl2 (Figures 1D,E,G,H). From our results, 20 µM Ori showed the most obvious regulator effects. These results indicate that Ori can regulate autophagy, AMPK, Akt/mTOR signaling and apoptosis in A549 cells. From the results that P62 and LC3 were all overexpressed in Ori-treated cells, we added CQ (50 µM) through acridine orange staining, LC3 staining and Western blotting to explore whether Ori inhibited autophagy flux. Results showed that Ori (20 µM) induced more acidic impaired autophagosomes and LC3 expression in CQ-treated cells (Figures 1I–N), which indicated that Ori, similar to CQ, could block the maturation and degradation of autophagosomes. the corresponding regulatory effects in A549 cells. Autophagy- related proteins (P62, LC3, and Atg3) were activated in a dose- dependent manner (Figures 1C,F). We subsequently observed that Ori (10 and 20 µM) evidently activated AMPK, caspase3, Bax, and PARP, but decreased the protein levels of Akt, mTOR, and Bcl2 (Figures 1D,E,G,H). From our results, 20 µM Ori showed the most obvious regulator effects. These results indicate that Ori can regulate autophagy, AMPK, Akt/mTOR signaling and apoptosis in A549 cells. From the results that P62 and LC3 were all overexpressed in Ori-treated cells, we added CQ (50 µM) through acridine orange staining, LC3 staining and Western blotting to explore whether Ori inhibited autophagy flux. Results showed that Ori (20 µM) induced more acidic impaired autophagosomes and LC3 expression in CQ-treated cells (Figures 1I–N), which indicated that Ori, similar to CQ, could block the maturation and degradation of autophagosomes. Ori Alleviates Cisplatin-Induced Nephrotoxicity in vivo According to the above results, we found the protective effect of Ori in cancer treatment and could enhance the sensitivity of cisplatin. While clinically recognized that with the use of chemotherapy drugs, nephrotoxicity induced by cisplatin also appears. So we chose a dose of 20 mg/kg cisplatin intraperitoneal injection for 3 days to induce acute kidney injury and Ori (20 mg/kg) injection simultaneously as a treatment (Figure 6A). We examined some indicators of kidney function and found that Ori could reduce cisplatin-induced high levels of BUN, CRE, kidney index, and weight loss (Figures 6B–E). Ori Induces Apoptosis Through Autophagy Initiation consistent with related protein expression levels (Figures 5C,D). In summary, these results indicate that Ori possesses anti- tumor and cisplatin sensitizer potential by the activation of apoptosis, which was mediated by AMPK/Akt/mTOR-dependent autophagy initiation. Pro-apoptotic molecules are widely used to enhance the regulation of autophagy in cancer treatment. So we added 3- MA (autophagy inhibitor) to examine the relationship between autophagy and apoptosis in Ori-treated cells. First, we examined the effects of 3-MA (1 mM) and CQ (50 µM) on Ori-induced cytotoxicity by CCK-8 assay (Figure 3A). The combination of CQ and Ori exerted better cytotoxic effects in A549 cells, while 3-MA antagonized the anti-tumor effects of Ori. These results indicated that autophagosomes induced by Ori possessed cell-killing effects in A549 cells. Then, Western blotting was used to detect the proteins related to apoptosis and autophagy (Figures 3B–E). Autophagy and apoptosis activated by Ori (20 µM) were restrained by 3-MA. Above all, Ori induced apoptosis by the AMPK/Akt/mTOR-dependent impaired autophagosomes accumulation. DISCUSSION Ori, an ent-kaurane diterpenoid isolated from Isodon rubescens, functions as an anti-tumor compound. It has been demonstrated that Ori could regulate the levels of LC3II/I and some Atg proteins in A549 cells. However, Ori-regulated the levels of P62 in tumor growth and cisplatin resistance of lung cancer have not been investigated. P62 is critical protein to decide the process of autophagy flux. Generally, P62 will be degraded and autophagy flux is complete and fluent. Once the autophagy flux is blocked and the degradation of P62 will be also inhibited. Therefore, the levels of P62 and autophagosomes are vital in cancer treatment (27). In our study, Ori induced AMPK/Akt/mTOR-dependent autophagosome accumulation, which further activated apoptosis to inhibit lung cancer cell growth and increase cisplatin sensitivity. Moreover, Ori alleviated cisplatin-induced renal damage indexes (BUN, CRE and body weight). Based on the results mentioned later (Figures 5A,B), we chose the corresponding doses and tested whether Ori sensitized cisplatin-induced cell death by the regulation of autophagy. Immunofluorescence and Western blotting were used to display impaired autophagosome accumulation. LC3 puncta and autophagosomes formation were also promoted in the group of Ori (20 µM) plus cisplatin (20 µM) (Figures 4A–D). P62, LC3, Atg3, and AMPK/Akt/mTOR expressions were more obvious in cotreatment with Ori and cisplatin (Figures 4E–H). Taken together, these results indicate that Ori increases cisplatin sensitivity through the role of autophagy and AMPK/Akt/mTOR. Akt/mT AMPK AMPK signaling acts as an upstream effector of autophagy and participates in oncotherapy. Based on Ori’s protective effect on the above pathways, we explored the interrelation between them by the addition of compound c (AMPK inhibitor). The number of autophagosomes was counted and LC3 puncta formation was analyzed by immunofluorescence. Ori-induced autophagosomes and representative protein LC3 accumulation were reduced by compound c (Figures 2A–D). Then Ori- activated P62, LC3, Atg3, and apoptotic proteins (Bax, caspase3, PARP) were reduced by compound c (5 µM), while the low levels of Akt/mTOR and Bcl2 in Ori-treated cells were elevated (Figures 2E–J). FIGURE 3 | Ori induces apoptosis through autophagy initiation. (A) Cells were exposed to different concentrations (10 and 20 µM) of Ori either alone or in the presence of autophagy inhibitors (50 µM CQ and 1 mM 3-MA) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered as 100%. (B–E) Cells were seeded in 6-well plates and treated with Ori (20 µM) in the absence or presence of 3-MA (1 mM) for 18 h. Western blots and statistical results showed the protein levels of P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. FIGURE 3 | Ori induces apoptosis through autophagy initiation. (A) Cells were exposed to different concentrations (10 and 20 µM) of Ori either alone or in the presence of autophagy inhibitors (50 µM CQ and 1 mM 3-MA) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered as 100%. (B–E) Cells were seeded in 6-well plates and treated with Ori (20 µM) in the absence or presence of 3-MA (1 mM) for 18 h. Western blots and statistical results showed the protein levels of P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. August 2019 | Volume 9 | Article 769 6 Frontiers in Oncology | www.frontiersin.org Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. Frontiers in Oncology | www.frontiersin.org Ori Enhances Chemotherapeutic Sensitivity Through the Activation of Apoptosis Chemotherapy resistance has always been an important factor in reducing the survival rate of cancer patients, so researchers investigate sensitizers to reverse the resistance. On the basis of anti-tumor potential of Ori, we further examined whether Ori could enhance cisplatin sensitivity by CCK-8 assay. Cisplatin (10, 20 µM) showed slight inhibition of tumor growth, while cell viability was evidently suppressed after the addition of Ori (10, 20 µM) (Figure 5A). Addtionally, 30 µM cisplatin showed cell- killing effects on B2b cells. Then, added these data into CalcuSyn and calculated the combination index according to the formula. CI < 1 indicates synergism, while CI > 1 indicates antagonism. From our results, the combination of Ori (20 µM) and Cis (20 µM) showed the best synergism and these two doses were used in our experiments (Figure 5B). We further investigated the role of apoptosis in cisplatin plus Ori group, and the effect of Ori (20 µM) on apoptotic proteins (caspase3, Bax, Bcl2, and PARP) was more remarkable in cisplatin-treated cells (Figures 5E,F). Then we detected apoptosis activity by FASC and the results were Under stress conditions, autophagy exerts a pro-survival activity in carcinogenesis and drug resistance (29, 30). When autophagy occurs, cytoplasmic components (organelles, proteins) are wrapped by double-layer membrane vesicles and form autophagosomes, which are then transferred to lysosomes for degradation. This procedure aims to provide nutrients and energy for cellular activity. In these reactions, Atg genes and LC3 regulate the formation of autophagosome double-layer membranes. Under the participation of Atg3, LC3I is transformed into LC3II and tightly binds to the surface of autophagic vesicles, taking part in the extension of autophagosomes (31). As a vital tumor suppressor, P62 interacts with mTORC1 and negatively regulates lysosome acidification and autophagosome-lysosome fusion (32). Increasing evidence indicates that cisplatin can induce autophagy, and suppression of autophagy by 3-methyladenine(3-MA) strongly enhanced cisplatin sensitivity (21). Our results showed that Ori evidently inhibited cell growth and induced the over-expressions of August 2019 | Volume 9 | Article 769 7 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. FIGURE 4 | Ori enhances chemotherapeutic sensitivity through the modulation of autophagy and AMPK/Akt/mTOR pathways. A549 cells were exposed to Ori (20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by fluorescence microscope. The green fluorescence intensity was analyzed by Image J. Frontiers in Oncology | www.frontiersin.org Ori Enhances Chemotherapeutic Sensitivity Through the Activation of Apoptosis Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images were captured with confocal microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–H) Western blots and statistical results showed the protein levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, and mTOR. The results showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p < 0.01 vs. the Cis group. FIGURE 4 | Ori enhances chemotherapeutic sensitivity through the modulation of autophagy and AMPK/Akt/mTOR pathways. A549 cells were exposed to Ori (20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by fluorescence microscope. The green fluorescence intensity was analyzed by Image J. Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images were captured with confocal microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–H) Western blots and statistical results showed the protein levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, and mTOR. The results showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p < 0.01 vs. the Cis group. LC3II, Atg3, P62 and the accumulation of autophagosomes under confocal and general fluorescence microscopy. The increased levels of LC3 and P62 tend to block autophagosomal maturation and degradation (suppression of autophagy at its late stage), therefore we combined Ori and CQ to examine the induced changes in autophagy flux. As expected, Ori induced increased expression of LC3II and acidic autophagosomes in cells treated with CQ. Thus, these results suggested that Ori inhibited autophagy flux and exerted an effect similar to that of CQ to cause the activation of autophagosomes (Figure 1). Then, the anti-tumor potential and autophagy initiation of Ori were weakened by the addition of 3-MA, so we considered that Ori-induced autophagosome accumulation caused lung cancer cell death (Figure 2). Based on the abovementioned August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 8 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. Ori Enhances Chemotherapeutic Sensitivity Through the Activation of Apoptosis Oridonin Sensitizes Cisplatin in A549 Cells | Ori enhances chemotherapeutic sensitivity through the activation of apoptosis. (A) A549 cells were treated with different doses (10, 20, and 30 µM) of Ori nce or presence of cisplatin (10, 20, and 30 µM) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered B) Data was added into CalcuSyn and combination index (CI) was analyzed in this experiment. CI < 1 indicates synergism, while CI>1 indicates . A549 cells were exposed to Ori (20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (C,D) The percentages of apoptotic and necrotic cells mined by flow cytometry. (E,F) Western blots and statistical results showed the protein levels of caspase3, Bax, Bcl2, and PARP. The results showed the three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p < e Cis group. FIGURE 5 | Ori enhances chemotherapeutic sensitivity through the activation of apoptosis. (A) A549 cells were treated with different doses (10, 20, and 30 µM) of Ori in the absence or presence of cisplatin (10, 20, and 30 µM) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered as 100%. (B) Data was added into CalcuSyn and combination index (CI) was analyzed in this experiment. CI < 1 indicates synergism, while CI>1 indicates antagonism. A549 cells were exposed to Ori (20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (C,D) The percentages of apoptotic and necrotic cells were determined by flow cytometry. (E,F) Western blots and statistical results showed the protein levels of caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p < 0.01 vs. the Cis group. cisplatin sensitivity by the inhibition of autophagy flux (Figure 4). results, we investigated the role of Ori in cisplatin-treated cells. Frontiers in Oncology | www.frontiersin.org Ori Enhances Chemotherapeutic Sensitivity Through the Activation of Apoptosis Additionally, it has been demonstrated that a prominent hallmark of platinum resistance is the evasion of apoptosis. Apoptotic signaling maintains a balance between cell death and cell survival, however, the dysfunction of pro- and anti-apoptotic proteins contributes to platinum resistance by reducing apoptosis. More evidence shows that agents activating apoptosis by targeting the Bcl-2 family, P53 and caspases can have a protective effect on sensitizing resistant cells (38–41). Therefore, we observed the effect of Ori on the above pathways and found that Ori enhanced the levels of AMPK, Bax, caspase3 and PARP and suppressed Akt, mTOR and Bcl2 expression in A549 cells (Figure 1). In cisplatin-treated cells, Ori had more obvious regulatory effects on apoptosis and Akt/mTOR (Figures 4, 5). Therefore, we considered that Ori increased the sensitivity of cisplatin by regulating apoptosis, AMPK and Akt/mTOR. AMPK is activated during situations in which the cellular level of ATP is decreased and therefore will inhibit tumor cell growth and enhance chemotherapy effect (17). For instance, the expression of AMPK-α was decreased in human breast cancer tissues and the AMPK activator metformin could have a positive impact on the effect of chemotherapy (34, 35). In addition, the Akt/mTOR pathway and apoptosis also participate in cisplatin resistance in various cancers. Recent studies indicate that inhibition of autophagy by andrographolide re-sensitizes cisplatin-resistant non-small cell lung carcinoma cells via activation of the Akt/mTOR pathways (36). While silencing long non-coding RNA ROR improves sensitivity of non-small-cell lung cancer to cisplatin resistance by inhibiting PI3K/Akt/mTOR signaling pathway (37). Additionally, it has been demonstrated that a prominent hallmark of platinum resistance is the evasion of apoptosis. Apoptotic signaling maintains a balance between cell death and cell survival, however, the dysfunction of pro- and anti-apoptotic proteins contributes to platinum resistance by reducing apoptosis. More evidence shows that agents activating apoptosis by targeting the Bcl-2 family, P53 and caspases can Research has shown that AMPK and autophagy frequently refer to the upstream regulators of apoptosis (17, 42). So we investigated the correlation among these pathways using compound c (AMPK inhibitor) and 3-MA (autophagy inhibitor). Results indicated that the overexpression of autophagy and apoptosis related proteins by Ori were reversed by 3-MA (Figure 3), and Ori-induced Akt/mTOR, autophagy and apoptosis-relevant changes were all impaired by compound c (Figure 2). In keeping with these results, LC3 puncta activated by Ori were reduced in the pretreatment of these two inhibitors. Ori Enhances Chemotherapeutic Sensitivity Through the Activation of Apoptosis Ori showed increased expression of LC3II, Atg3 and P62 and decreased cell viability after cotreatment with cisplatin, and we reached the conclusion that Ori increased In the relevant pathways of cisplatin resistance, AMPK, Akt/mTOR and apoptosis also play an important role (18, 19, 33). August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 9 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. FIGURE 6 | Ori alleviates cisplatin-induced nephrotoxicity in vivo. (A) Experimental design of Ori’s therapeutic effects on cisplatin-induced kidney injury. After fasting for 12 h, intraperitoneal injection of cisplatin (20 mg/kg) was performed to establish an acute kidney injury model. Ori (20 mg/kg) was administered to the enterocoelia for 3 consecutive days. (B,C) Body and kidney weights were calculated and the kidney index was shown as kidney weight divided by body weight. (D,E) Blood was collected to detect the levels of BUN and CRE. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. ##p < 0.01 vs. the Cis group. FIGURE 6 | Ori alleviates cisplatin-induced nephrotoxicity in vivo. (A) Experimental design of Ori’s therapeutic effects on cisplatin-induced kidney injury. After fasting for 12 h, intraperitoneal injection of cisplatin (20 mg/kg) was performed to establish an acute kidney injury model. Ori (20 mg/kg) was administered to the enterocoelia for 3 consecutive days. (B,C) Body and kidney weights were calculated and the kidney index was shown as kidney weight divided by body weight. (D,E) Blood was collected to detect the levels of BUN and CRE. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. ##p < 0.01 vs. the Cis group. AMPK is activated during situations in which the cellular level of ATP is decreased and therefore will inhibit tumor cell growth and enhance chemotherapy effect (17). For instance, the expression of AMPK-α was decreased in human breast cancer tissues and the AMPK activator metformin could have a positive impact on the effect of chemotherapy (34, 35). In addition, the Akt/mTOR pathway and apoptosis also participate in cisplatin resistance in various cancers. Recent studies indicate that inhibition of autophagy by andrographolide re-sensitizes cisplatin-resistant non-small cell lung carcinoma cells via activation of the Akt/mTOR pathways (36). While silencing long non-coding RNA ROR improves sensitivity of non-small-cell lung cancer to cisplatin resistance by inhibiting PI3K/Akt/mTOR signaling pathway (37). Frontiers in Oncology | www.frontiersin.org DATA AVAILABILITY that Ori induced apoptosis by AMPK/Akt/mTOR-dependent autophagosome accumulation and increased cisplatin resistance in NSCLC cells. In addition, with the use of chemotherapy drugs, cisplatin-induced nephrotoxicity is observed (43). However, the protective effect of Ori as anti-tumor agent should not be interfered by the side effect of cisplatin. So we constructed a renal injury mouse model induced by cisplatin and investigated whether Ori could have a therapeutic effect. As expected, Ori could alleviate cisplatin-induced high levels of BUN and CRE, weight loss and kidney index (Figure 6). The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. Ori Enhances Chemotherapeutic Sensitivity Through the Activation of Apoptosis In summary, we could made conclusions August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 10 Yang et al. Oridonin Sensitizes Cisplatin in A549 Cells FIGURE 7 | Schematic model of the increased cisplatin sensitivity by Ori in A549 cells. Ori induced apoptosis and enhanced the chemotherapeutic efficacy of cisplatin via AMPK/Akt/mTOR-dependent impaired autophagosome accumulation (black lines). And the same binding targets between Ori and Cis were also exhibited (green lines). FIGURE 7 | Schematic model of the increased cisplatin sensitivity by Ori in A549 cells. Ori induced apoptosis and enhanced the chemotherapeutic efficacy of cisplatin via AMPK/Akt/mTOR-dependent impaired autophagosome accumulation (black lines). And the same binding targets between Ori and Cis were also exhibited (green lines). ETHICS STATEMENT All animal studies were reviewed and approved by the Animal Welfare and Research Ethics Committee of Jilin University. AUTHOR CONTRIBUTIONS In summary, Ori displayed anti-tumor bioactivity and decreased cisplatin resistance by apoptotic signaling activation, which was modulated by AMPK/Akt/mTOR-dependent autophagy inhibition. Besides, Ori had a therapeutic effect on cisplatin-induced nephrotoxicity (Figure 7). Our results suggested that Ori, as a specific autophagy modulator, could be potentially developed as an adjuvant for further cancer treatment and chemotherapy resistance. 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Oridonin Sensitizes Cisplatin in A549 Cells Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2019 Yang, Gao, Fan, Liu, Peng and Ci. 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. 42. Zhang B, Cui Y, Wang L, Zhao L, Hou C, Zeng Q, et al. Autophagy regulates high concentrations of iodide-induced apoptosis in SH- SY5Y cells. Toxicol Lett. (2018) 284:129–35. doi: 10.1016/j.toxlet.2017. 12.007 43. Crona DJ, Faso A, Nishijima TF, McGraw KA, Galsky MD, Milowsky MI. A systematic review of strategies to prevent cisplatin-induced nephrotoxicity. Oncologist. (2017) 22:609–619. doi: 10.1634/theoncologist.2016–0319 August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 13
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АГРОЭКОЛОГИЧЕСКОЕ ОБОСНОВАНИЕ ВНЕСЕНИЯ УДОБРЕНИЙ ПОД ОЗИМУЮ ПШЕНИЦУ СОРТА «МИРОНОВСКАЯ 61»
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WWW.SCILOOK.EU Совместно с: Институт морехозяйства и предпринимательства При научной поддержке: Экономическая академия им.Д.А.Ценова (Болгария) Московский государственный университет путей сообщения (МИИТ) Украи инская госуд дарств вен нна ая ака адем мия я железзнод дорож жного тра ансп портта Нау учно о-иссле едова ате ельский й прое екттно о-констр рукто орски ий инс ститтут морс скогго флотта Луганский государственный медицинский университет Харьковская медицинская академия последипломного образования Бельцкий Го осударс стве енн ный Ун ниве ерси итетт «Алеку Руссо» Инс ститу ут во одны ых проб блем и мелио орац ции На ацион нальной й академии аггра арны ых наукк Одесский нау учно о-исслед дова ател льский й ин нсти итутт связзи Входит в международные наукометрические базы РИНЦ INDEXCOPERNICUS (ICI 88.47) Н Международное периодическое научное издание Взгляд в АУЧНЫЙ Будущее International periodic scientific journal SCIENTIFIC LOOK INTO THE FUTURE НАУКО ОВИЙ ПОГЛ ЛЯД У МА АЙБУТНЄ Є Выпуск №13, апрель 2019 Issue №13, April 2019 Том 2 Part 2 Одессаа Куприенко СВ 2019 УДК 08 ББК 94 Н 347 ISSN 2415-766Х (Print) ISSN 2415-7538 (Online) Главный редактор: Шибаев Александр Григорьевич, доктор технических наук, профессор, Академик Головний редактор: Шибаєв Олександр Григорович, доктор технічних наук, професор, Академік Chief Editor: Shibaev Alexander Grigoryevich, Doctor of Technical Sciences, Professor, Academician Заместитель Главного редактора: Яценко Александр Владимирович, кандидат технических наук, профессор, Ректор ИМП Заступник головного редактора: Яценко Олександр Володимирович, кандидат технічних наук, професор, Ректор ІМП Deputy Chief Editor: Yatsenko Alexander Vladimirovich, Candidate of Technical Sciences, Professor, Rector of ISE Редакционный Совет: Более 150 докторов наук. Полный список представлен на страницах 3-5. Редакційна Рада: Понад 150 докторів наук. Повний список представлений на сторінках 3-5. Editorial Board: More than 150 doctors of sciences. The full list is available on pages 3-5. Н 347 Научный взгляд в будущее. – Выпуск 13. Том 2. – Одесса: КУПРИЕНКО СВ, 2019 – 134 с. Журнал предназначается для научных работников, аспирантов, студентов старших курсов, преподавателей, предпринимателей. The journal is intended for researchers, graduate students, senior students, teachers and entrepreneurs. Published quarterly. УДК 08 ББК 94 DOI: 10.30888/2415-7538.2019-13-02 © Коллектив авторов, научные тексты 2019 © Куприенко С.В., оформление 2019 Выпуск 13 Том 2 Редакционный Совет Попов Виктор Сергеевич, доктор технических наук, профессор, Россия Рокочинский Анатолий Николаевич, доктор технических наук, профессор, Украина Ромащенко Михаил Иванович, доктор технических наук, профессор, Украина Семенцов Георгий Никифорович, доктор технических наук, профессор, Украина Сухенко Юрий Григорьевич, доктор технических наук, профессор, Украина Устенко Сергей Анатольевич, доктор технических наук, доцент, Украина Хабибуллин Рифат Габдулхакович, доктор технических наук, профессор, Россия Червоный Иван Федорович, доктор технических наук, профессор, Украина Шайко-Шайковский Александр Геннадьевич, доктор технических наук, профессор, Украина Щербань Игорь Васильевич, доктор технических наук, доцент, Россия Бушуева Инна Владимировна, доктор фармацевтических наук, профессор, Украина Волох Дмитрий Степанович, доктор фармацевтических наук, профессор, Украина Георгиевский Геннадий Викторович, доктор фармацевтических наук, старший научный сотрудник, Украина Гудзенко Александр Павлович, доктор фармацевтических наук, профессор, Украина Тихонов Александр Иванович, доктор фармацевтических наук, профессор, Украина Шаповалов Валерий Владимирович, доктор фармацевтических наук, профессор, Украина Шаповалова Виктория Алексеевна, доктор фармацевтических наук, профессор, Украина Блатов Игорь Анатольевич, доктор физико-математических наук, профессор, Россия Кондратов Дмитрий Вячеславович, доктор физико-математических наук, доцент, Россия Лялькина Галина Борисовна, доктор физико-математических наук, профессор, Россия Малахов А В , доктор физико-математических наук, профессор, Украина Ворожбитова Александра Анатольевна, доктор филологических наук, профессор, Россия Лыткина Лариса Владимировна, доктор филологических наук, доцент, Россия Попова Таисия Георгиевна, доктор филологических наук, профессор, Россия Коваленко Елена Михайловна, доктор философских наук, профессор, Россия Липич Тамара Ивановна, доктор философских наук, доцент, Россия Майданюк Ирина Зиновиевна, доктор философских наук, доцент, Украина Светлов Виктор Александрович, доктор философских наук, профессор, Россия Стовпец А В , доктор философских наук, доцент, Украина Антрапцева Надежда Михайловна, доктор химических наук, профессор, Украина Бажева Рима Чамаловна, доктор химических наук, профессор, Россия Гризодуб Александр Иванович, доктор химических наук, профессор, Украина Ермагамбет Болат Толеуханович, доктор химических наук, профессор, Казахстан Максин Виктор Иванович, доктор химических наук, профессор, Украина Ангелова Поля Георгиева, доктор экономических наук, профессор, Болгария Безденежных Татьяна Ивановна, доктор экономических наук, профессор, Россия Бурда Алексей Григорьевич, доктор экономических наук, профессор, Россия Грановская Людмила Николаевна, доктор экономических наук, профессор, Украина Дорохина Елена Юрьевна, доктор экономических наук, доцент, Россия Климова Наталья Владимировна, доктор экономических наук, профессор, Россия Кочинев Юрий Юрьевич, доктор экономических наук, доцент, Россия Курмаев Петр Юрьевич, доктор экономических наук, профессор, Украина Лапкина Инна Александровна, доктор экономических наук, профессор, Украина Мельник Алёна Алексеевна, доктор экономических наук, доцент, Украина Миляева Лариса Григорьевна, доктор экономических наук, профессор, Россия Пахомова Елена Анатольевна, доктор экономических наук, доцент, Россия Резников Андрей Валентинович, доктор экономических наук, доцент, Россия Савельева Нелли Александровна, доктор экономических наук, профессор, Россия Соколова Надежда Геннадьевна, доктор экономических наук, доцент, Россия Стрельцова Елена Дмитриевна, доктор экономических наук, доцент, Россия Батыргареева Владислава Станиславовона, доктор юридических наук, , Украина Гетьман Анатолий Павлович, доктор юридических наук, профессор, Украина Кафарский Владимир Иванович, доктор юридических наук, профессор, Украина Кириченко Александр Анатольевич, доктор юридических наук, профессор, Украина Степенко Валерий Ефремович, доктор юридических наук, доцент, Россия Тонков Евгений Евгеньевич, доктор юридических наук, профессор, Россия Шепитько Валерий Юрьевич, доктор юридических наук, профессор, Украина Шишка Роман Богданович, доктор юридических наук, профессор, Украина Яровенко Василий Васильевич, доктор юридических наук, профессор, Россия Кантарович Ю Л , кандидат искусствоведения, , Украина Волгирева Галина Павловна, кандидат исторических наук, доцент, Россия Токарева Наталья Геннадьевна, кандидат медицинских наук, доцент, Россия Демидова В Г , кандидат педагогических наук, доцент, Украина Могилевская И М , кандидат педагогических наук, профессор, Украина Лебедева Лариса Александровна, кандидат психологических наук, доцент, Россия Шаповалов Валентин Валерьевич, кандидат фармацевтических наук, доцент, Украина Стовпец В Г , кандидат филологических наук, доцент, Украина Зубков Руслан Сергеевич, доктор экономических наук, доцент, Украина Толбатов Андрей Владимирович, кандидат технических наук, доцент, Украина Шарагов Василий Андреевич, доктор химических наук, доцент, Молдова Бухарина Ирина Леонидовна, доктор биологических наук, профессор, Россия Гребнева Надежда Николаевна, доктор биологических наук, профессор, Россия Гриценко Светлана Анатольевна, доктор биологических наук, доцент, Россия Каленик Татьяна Кузьминична, доктор биологических наук, профессор, Россия Князева Ольга Александровна, доктор биологических наук, доцент, Россия Кухар Елена Владимировна, доктор биологических наук, доцент, Казахстан Моисейкина Людмила Гучаевна, доктор биологических наук, профессор, Россия Нефедьева Елена Эдуардовна, доктор биологических наук, доцент, Россия Сентябрев Николай Николаевич, доктор биологических наук, профессор, Россия Стародубцев Владимир Михайлович, доктор биологических наук, профессор, Украина Тестов Борис Викторович, доктор биологических наук, профессор, Россия Тунгушбаева Зина Байбагусовна, доктор биологических наук, , Казахстан Фатеева Надежда Михайловна, доктор биологических наук, профессор, Россия Ахмадиев Габдулахат Маликович, доктор ветеринарных наук, профессор, Россия Шевченко Лариса Васильевна, доктор ветеринарных наук, профессор, Украина Анимица Евгений Георгиевич, доктор географических наук, профессор, Россия Сухова Мария Геннадьевна, доктор географических наук, доцент, Россия Иржи Хлахула, доктор геолого-минералогических наук, профессор, Чехия Федоришин Дмитро Дмитрович, доктор геолого-минералогических наук, профессор, Украина Кокебаева Гульжаухар Какеновна, доктор исторических наук, профессор, Казахстан Отепова Гульфира Елубаевна, доктор исторических наук, профессор, Казахстан Тригуб Петр Никитович, доктор исторических наук, профессор, Украина Элезович М Далибор , доктор исторических наук, доцент, Сербия Визир Вадим Анатольевич, доктор медицинских наук, профессор, Украина Федянина Людмила Николаевна, доктор медицинских наук, профессор, Россия Орлов Николай Михайлович, доктор наук государственного управления, доцент, Украина Величко Степан Петрович, доктор педагогических наук, профессор, Украина Гавриленко Наталия Николаевна, доктор педагогических наук, доцент, Россия Гилев Геннадий Андреевич, доктор педагогических наук, профессор, Россия Дорофеев Андрей Викторович, доктор педагогических наук, доцент, Россия Карпова Наталия Константиновна, доктор педагогических наук, профессор, Россия Мишенина Татьяна Михайловна, доктор педагогических наук, профессор, Украина Николаева Алла Дмитриевна, доктор педагогических наук, профессор, Россия Растрыгина Алла Николаевна, доктор педагогических наук, профессор, Украина Сидорович Марина Михайловна, доктор педагогических наук, профессор, Украина Смирнов Евгений Иванович, доктор педагогических наук, профессор, Россия Фатыхова Алевтина Леонтьевна, доктор педагогических наук, доцент, Россия Федотова Галина Александровна, доктор педагогических наук, профессор, Россия Ходакова Нина Павловна, доктор педагогических наук, доцент, Россия Чигиринская Наталья Вячеславовна, доктор педагогических наук, профессор, Россия Чурекова Татьяна Михайловна, доктор педагогических наук, профессор, Россия Латыгина Наталья Анатольевна, доктор политологических наук, профессор, Украина Сирота Наум Михайлович, доктор политологических наук, профессор, Россия Хребина Светлана Владимировна, доктор психологических наук, профессор, Россия Вожегова Раиса Анатольевна, доктор сельскохозяйственных наук, профессор, Украина Денисов Сергей Александрович, доктор сельскохозяйственных наук, профессор, Россия Жовтоног Ольга Игоревна, доктор сельскохозяйственных наук, , Украина Костенко Василий Иванович, доктор сельскохозяйственных наук, профессор, Украина Котляров Владимир Владиславович, доктор сельскохозяйственных наук, профессор, Россия Морозов Алексей Владимирович, доктор сельскохозяйственных наук, профессор, Украина Патыка Николай Владимирович, доктор сельскохозяйственных наук, профессор, Украина Ребезов Максим Борисович, доктор сельскохозяйственных наук, профессор, Россия Тарарико Юрий Александрович, доктор сельскохозяйственных наук, профессор, Украина Мальцева Анна Васильевна, доктор социологических наук, доцент, Россия Стегний Василий Николаевич, доктор социологических наук, профессор, Россия Тарасенко Лариса Викторовна, доктор социологических наук, профессор, Россия Аверченков Владимир Иванович, доктор технических наук, профессор, Россия Антонов Валерий Николаевич, доктор технических наук, профессор, Украина Быков Юрий Александрович, доктор технических наук, профессор, Россия Гончарук Сергей Миронович, доктор технических наук, профессор, Россия Захаров Олег Владимирович, доктор технических наук, профессор, Россия Калайда Владимир Тимофеевич, доктор технических наук, профессор, Россия Капитанов Василий Павлович, доктор технических наук, профессор, Украина Кириллова Елена Викторовна, доктор технических наук, доцент, Украина Коваленко Петр Иванович, доктор технических наук, профессор, Украина Копей Богдан Владимирович, доктор технических наук, профессор, Украина Косенко Надежда Федоровна, доктор технических наук, доцент, Россия Круглов Валерий Михайлович, доктор технических наук, профессор, Россия Кудерин Марат Крыкбаевич, доктор технических наук, профессор, Казахстан Лебедев Анатолий Тимофеевич, доктор технических наук, профессор, Россия Ломотько Денис Викторович, доктор технических наук, профессор, Украина Макарова Ирина Викторовна, доктор технических наук, профессор, Россия Морозова Татьяна Юрьевна, доктор технических наук, профессор, Россия Павленко Анатолий Михайлович, доктор технических наук, профессор, Украина Парунакян Ваагн Эмильевич, доктор технических наук, профессор, Украина Пачурин Герман Васильевич, доктор технических наук, профессор, Россия Першин Владимир Федорович, доктор технических наук, профессор, Россия Пиганов Михаил Николаевич, доктор технических наук, профессор, Россия Поляков Андрей Павлович, доктор технических наук, профессор, Украина Научный взгляд в будущее 3 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 Редакційна Рада Першин Володимир Федорович, доктор технічних наук, професор, Росія Піганов Михайло Миколайович, доктор технічних наук, професор, Росія Поляков Андрій Павлович, доктор технічних наук, професор, Україна Попов Віктор Сергійович, доктор технічних наук, професор, Росія Рокочінскій Анатолій Миколайович, доктор технічних наук, професор, Україна Ромащенко Михайло Іванович, доктор технічних наук, професор, Україна Семенцов Георгій Никифорович, доктор технічних наук, професор, Україна Сухенко Юрій Григорович, доктор технічних наук, професор, Україна Устенко Сергій Анатолійович, доктор технічних наук, доцент, Україна Хабібуллін Рифат Габдулхакович, доктор технічних наук, професор, Росія Червоний Іван Федорович, доктор технічних наук, професор, Україна Шайко-Шайковскій Олександр Геннадійович, доктор технічних наук, професор, Україна Щербань Ігор Васильович, доктор технічних наук, доцент, Росія Бушуєва Інна Володимирівна, доктор фармацевтичних наук, професор, Україна Волох Дмитро Степанович, доктор фармацевтичних наук, професор, Україна Георгієвський Геннадій Вікторович, доктор фармацевтичних наук, старший науковий співробітник, Україна Гудзенко Олександр Павлович, доктор фармацевтичних наук, професор, Україна Тихонов Олександр Іванович, доктор фармацевтичних наук, професор, Україна Шаповалов Валерій Володимирович, доктор фармацевтичних наук, професор, Україна Шаповалова Вікторія Олексіївна, доктор фармацевтичних наук, професор, Україна Блатов Ігор Анатолійович, доктор фізико-математичних наук, професор, Росія Кондратов Дмитро В'ячеславович, доктор фізико-математичних наук, доцент, Росія Лялькіна Галина Борисівна, доктор фізико-математичних наук, професор, Росія Малахов А В , доктор фізико-математичних наук, професор, Україна Ворожбітова Олександра Анатоліївна, доктор філологічних наук, професор, Росія Литкіна Лариса Володимирівна, доктор філологічних наук, доцент, Росія Попова Таїсія Георгіївна, доктор філологічних наук, професор, Росія Коваленко Олена Михайлівна, доктор філософських наук, професор, Росія Липич Тамара Іванівна, доктор філософських наук, доцент, Росія Майданюк Ірина Зіновіївна, доктор філософських наук, доцент, Україна Светлов Віктор Олександрович, доктор філософських наук, професор, Росія Стовпец А В , доктор філософських наук, доцент, Україна Антрапцева Надія Михайлівна, доктор хімічних наук, професор, Україна Бажев Риму Чамаловна, доктор хімічних наук, професор, Росія Гризодуб Олександр Іванович, доктор хімічних наук, професор, Україна Ермагамбет Болат Толеухановіч, доктор хімічних наук, професор, Казахстан Максін Віктор Іванович, доктор хімічних наук, професор, Україна Ангелова Поля Георгієва, доктор економічних наук, професор, Болгарія Безденежних Тетяна Іванівна, доктор економічних наук, професор, Росія Бурда Олексій Григорович, доктор економічних наук, професор, Росія Грановська Людмила Миколаївна, доктор економічних наук, професор, Україна Дорохіна Олена Юріївна, доктор економічних наук, доцент, Росія Климова Наталія Володимирівна, доктор економічних наук, професор, Росія Кочинев Юрій Юрійович, доктор економічних наук, доцент, Росія Курман Петро Юрійович, доктор економічних наук, професор, Україна Лапкіна Інна Олександрівна, доктор економічних наук, професор, Україна Мельник Олена Олексіївна, доктор економічних наук, доцент, Україна Міляєва Лариса Григорівна, доктор економічних наук, професор, Росія Пахомова Олена Анатоліївна, доктор економічних наук, доцент, Росія Резніков Андрій Валентинович, доктор економічних наук, доцент, Росія Савельєва Неллі Олександрівна, доктор економічних наук, професор, Росія Соколова Надія Геннадіївна, доктор економічних наук, доцент, Росія Стрельцова Олена Дмитрівна, доктор економічних наук, доцент, Росія Батиргареева Владислава Станіславовона, доктор юридичних наук, , Україна Гетьман Анатолій Павлович, доктор юридичних наук, професор, Україна Кафарський Володимир Іванович, доктор юридичних наук, професор, Україна Кириченко Олександр Анатолійович, доктор юридичних наук, професор, Україна Степенко Валерій Єфремович, доктор юридичних наук, доцент, Росія Тонков Євген Євгенович, доктор юридичних наук, професор, Росія Шепітько Валерій Юрійович, доктор юридичних наук, професор, Україна Шишка Роман Богданович, доктор юридичних наук, професор, Україна Яровенко Василь Васильович, доктор юридичних наук, професор, Росія Кантаровіч Ю Л , кандидат мистецтвознавства, , Україна Волгірева Галина Павлівна, кандидат історичних наук, доцент, Росія Токарєва Наталія Геннадіївна, кандидат медичних наук, доцент, Росія Демидова В Г , кандидат педагогічних наук, доцент, Україна Могилевська І М , кандидат педагогічних наук, професор, Україна Лебедєва Лариса Олександрівна, кандидат психологічних наук, доцент, Росія Шаповалов Валентин Валерійович, кандидат фармацевтичних наук, доцент, Україна Стовпец В Г , кандидат філологічних наук, доцент, Україна Зубков Руслан Сергійович, доктор економічних наук, доцент, Україна Толбатов Андрій Володимирович, кандидат технічних наук, доцент, Україна Шарагов Василь Андрійович, доктор хімічних наук, доцент, Молдова Бухаріна Ірина Леонідівна, доктор біологічних наук, професор, Росія Гребньова Надія Миколаївна, доктор біологічних наук, професор, Росія Гриценко Світлана Анатоліївна, доктор біологічних наук, доцент, Росія Каленик Тетяна Кузьмівна, доктор біологічних наук, професор, Росія Князєва Ольга Олександрівна, доктор біологічних наук, доцент, Росія Кухар Олена Володимирівна, доктор біологічних наук, доцент, Казахстан Моісейкіна Людмила Гучаевна, доктор біологічних наук, професор, Росія Нефедьева Олена Едуардівна, доктор біологічних наук, доцент, Росія Сентябрьов Микола Миколайович, доктор біологічних наук, професор, Росія Стародубцев Володимир Михайлович, доктор біологічних наук, професор, Україна Тестів Борис Вікторович, доктор біологічних наук, професор, Росія Тунгушбаева Зіна Байбагусовна, доктор біологічних наук, , Казахстан Фатєєва Надія Михайлівна, доктор біологічних наук, професор, Росія Ахмадієв Габдулахат Маликович, доктор ветеринарних наук, професор, Росія Шевченко Лариса Василівна, доктор ветеринарних наук, професор, Україна Аніміца Євген Георгійович, доктор географічних наук, професор, Росія Сухова Марія Геннадіївна, доктор географічних наук, доцент, Росія Іржі Хлахула, доктор геолого-мінералогічних наук, професор, Чехія Федоришин Дмитро Дмитрович, доктор геолого-мінералогічних наук, професор, Україна Кокебаева Гульжаухар Какеновна, доктор історичних наук, професор, Казахстан Отепова Гульфира Елубаевна, доктор історичних наук, професор, Казахстан Тригуб Петро Микитович, доктор історичних наук, професор, Україна Елезовіч М Далібор, доктор історичних наук, доцент, Сербія Візир Вадим Анатолійович, доктор медичних наук, професор, Україна Федяніна Людмила Миколаївна, доктор медичних наук, професор, Росія Орлов Микола Михайлович, доктор наук з державного управління, доцент, Україна Величко Степан Петрович, доктор педагогічних наук, професор, Україна Гавриленко Наталія Миколаївна, доктор педагогічних наук, доцент, Росія Гилев Геннадій Андрійович, доктор педагогічних наук, професор, Росія Дорофєєв Андрій Вікторович, доктор педагогічних наук, доцент, Росія Карпова Наталія Костянтинівна, доктор педагогічних наук, професор, Росія Мішеніна Тетяна Михайлівна, доктор педагогічних наук, професор, Україна Миколаєва Алла Дмитрівна, доктор педагогічних наук, професор, Росія Растригіна Алла Миколаївна, доктор педагогічних наук, професор, Україна Сидорович Марина Михайлівна, доктор педагогічних наук, професор, Україна Смирнов Євген Іванович, доктор педагогічних наук, професор, Росія Фатихова Алевтина Леонтіївна, доктор педагогічних наук, доцент, Росія Федотова Галина Олександрівна, доктор педагогічних наук, професор, Росія Ходакова Ніна Павлівна, доктор педагогічних наук, доцент, Росія Чигиринська Наталія В'ячеславівна, доктор педагогічних наук, професор, Росія Чурекова Тетяна Михайлівна, доктор педагогічних наук, професор, Росія Латигіна Наталія Анатоліївна, доктор політологічних наук, професор, Україна Сирота Наум Михайлович, доктор політологічних наук, професор, Росія Хребіна Світлана Володимирівна, доктор психологічних наук, професор, Росія Вожегова Раїса Анатоліївна, доктор сільськогосподарських наук, професор, Україна Денисов Сергій Олександрович, доктор сільськогосподарських наук, професор, Росія Жовтоног Ольга Ігорівна, доктор сільськогосподарських наук, , Україна Костенко Василь Іванович, доктор сільськогосподарських наук, професор, Україна Котляров Володимир Владиславович, доктор сільськогосподарських наук, професор, Росія Морозов Олексій Володимирович, доктор сільськогосподарських наук, професор, Україна Патика Микола Володимирович, доктор сільськогосподарських наук, професор, Україна Ребезов Максим Борисович, доктор сільськогосподарських наук, професор, Росія Тараріко Юрій Олександрович, доктор сільськогосподарських наук, професор, Україна Мальцева Анна Василівна, доктор соціологічних наук, доцент, Росія Стегній Василь Миколайович, доктор соціологічних наук, професор, Росія Тарасенко Лариса Вікторівна, доктор соціологічних наук, професор, Росія Аверченко Володимир Іванович, доктор технічних наук, професор, Росія Антонов Валерій Миколайович, доктор технічних наук, професор, Україна Биков Юрій Олександрович, доктор технічних наук, професор, Росія Гончарук Сергій Миронович, доктор технічних наук, професор, Росія Захаров Олег Володимирович, доктор технічних наук, професор, Росія Калайда Володимир Тимофійович, доктор технічних наук, професор, Росія Капітанів Василь Павлович, доктор технічних наук, професор, Україна Кирилова Олена Вікторівна, доктор технічних наук, доцент, Україна Коваленко Петро Іванович, доктор технічних наук, професор, Україна Копей Богдан Володимирович, доктор технічних наук, професор, Україна Косенко Надія Федорівна, доктор технічних наук, доцент, Росія Круглов Валерій Михайлович, доктор технічних наук, професор, Росія Кудерін Марат Крикбаевіч, доктор технічних наук, професор, Казахстан Лебедєв Анатолій Тимофійович, доктор технічних наук, професор, Росія Ломотько Денис Вікторович, доктор технічних наук, професор, Україна Макарова Ірина Вікторівна, доктор технічних наук, професор, Росія Морозова Тетяна Юріївна, доктор технічних наук, професор, Росія Павленко Анатолій Михайлович, доктор технічних наук, професор, Україна Парунакян Ваагн Емільович, доктор технічних наук, професор, Україна Пачурін Герман Васильович, доктор технічних наук, професор, Росія Научный взгляд в будущее 4 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 Editorial board Ustenko Sergey Anatol'yevich, Doctor of Technical Sciences, assistant professor, Ukraine Khabibullin Rifat Gabdulkhakovich, Doctor of Technical Sciences, Professor, Russia Chervonyy Ivan Fedorovich, Doctor of Technical Sciences, Professor, Ukraine Shayko-Shaykovskiy Aleksandr Gennad'yevich, Doctor of Technical Sciences, Professor, Ukraine Shibayev Aleksandr Grigor'yevich, Doctor of Technical Sciences, Professor, Ukraine Shcherban' Igor' Vasil'yevich, Doctor of Technical Sciences, assistant professor, Russia Bushuyeva Inna Vladimirovna, Doctor of Pharmaceutical Sciences, Professor, Ukraine Volokh Dmitriy Stepanovich, Doctor of Pharmaceutical Sciences, Professor, Ukraine Georgiyevskiy Gennadiy Viktorovich, Doctor of Pharmaceutical Sciences, Senior Researcher, Ukraine Gudzenko Aleksandr Pavlovich, Doctor of Pharmaceutical Sciences, Professor, Ukraine Tikhonov Aleksandr Ivanovich, Doctor of Pharmaceutical Sciences, Professor, Ukraine Shapovalov Valeriy Vladimirovich, Doctor of Pharmaceutical Sciences, Professor, Ukraine Shapovalova Viktoriya Alekseyevna, Doctor of Pharmaceutical Sciences, Professor, Ukraine Blatov Igor' Anatol'yevich, Doctor of Physical and Mathematical Sciences, Professor, Russia Kondratov Dmitriy Vyacheslavovich, Doctor of Physical and Mathematical Sciences, assistant professor, Russia Lyal'kina Galina Borisovna, Doctor of Physical and Mathematical Sciences, Professor, Russia Malakhov A V , Doctor of Physical and Mathematical Sciences, Professor, Ukraine Vorozhbitova Aleksandra Anatol'yevna, doctor of philology, Professor, Russia Lytkina Larisa Vladimirovna, doctor of philology, assistant professor, Russia Popova Taisiya Georgiyevna, doctor of philology, Professor, Russia Kovalenko Yelena Mikhaylovna, doctor of philosophical science, Professor, Russia Lipich Tamara Ivanovna, doctor of philosophical science, assistant professor, Russia Maydanyuk Irina Zinoviyevna, doctor of philosophical science, assistant professor, Ukraine Svetlov Viktor Aleksandrovich, doctor of philosophical science, Professor, Russia Stovpets A V , doctor of philosophical science, assistant professor, Ukraine Antraptseva Nadezhda Mikhaylovna, Doctor of Chemical Sciences, Professor, Ukraine Bazheva Rima Chamalovna, Doctor of Chemical Sciences, Professor, Russia Grizodub Aleksandr Ivanovich, Doctor of Chemical Sciences, Professor, Ukraine Yermagambet Bolat Toleukhanovich, Doctor of Chemical Sciences, Professor, Kazakhstan Maksin Viktor Ivanovich, Doctor of Chemical Sciences, Professor, Ukraine Angelova Polya Georgiyeva, Doctor of Economic Sciences, Professor, Bulgaria Bezdenezhnykh Tat'yana Ivanovna, Doctor of Economic Sciences, Professor, Russia Burda Aleksey Grigor'yevich, Doctor of Economic Sciences, Professor, Russia Granovskaya Lyudmila Nikolayevna, Doctor of Economic Sciences, Professor, Ukraine Dorokhina Yelena Yur'yevna, Doctor of Economic Sciences, assistant professor, Russia Klimova Natal'ya Vladimirovna, Doctor of Economic Sciences, Professor, Russia Kochinev Yuriy Yur'yevich, Doctor of Economic Sciences, assistant professor, Russia Kurmayev Petr Yur'yevich, Doctor of Economic Sciences, Professor, Ukraine Lapkina Inna Aleksandrovna, Doctor of Economic Sciences, Professor, Ukraine Mel'nik Alona Alekseyevna, Doctor of Economic Sciences, assistant professor, Ukraine Milyayeva Larisa Grigor'yevna, Doctor of Economic Sciences, Professor, Russia Pakhomova Yelena Anatol'yevna, Doctor of Economic Sciences, assistant professor, Russia Reznikov Andrey Valentinovich, Doctor of Economic Sciences, assistant professor, Russia Savel'yeva Nelli Aleksandrovna, Doctor of Economic Sciences, Professor, Russia Sokolova Nadezhda Gennad'yevna, Doctor of Economic Sciences, assistant professor, Russia Strel'tsova Yelena Dmitriyevna, Doctor of Economic Sciences, assistant professor, Russia Batyrgareyeva Vladislava Stanislavovona, doctor of law, , Ukraine Get'man Anatoliy Pavlovich, doctor of law, Professor, Ukraine Kafarskiy Vladimir Ivanovich, doctor of law, Professor, Ukraine Kirichenko Aleksandr Anatol'yevich, doctor of law, Professor, Ukraine Stepenko Valeriy Yefremovich, doctor of law, assistant professor, Russia Tonkov Yevgeniy Yevgen'yevich, doctor of law, Professor, Russia Shepit'ko Valeriy Yur'yevich, doctor of law, Professor, Ukraine Shishka Roman Bogdanovich, doctor of law, Professor, Ukraine Yarovenko Vasiliy Vasil'yevich, doctor of law, Professor, Russia Kantarovich YU L , Ph D in History of Arts, , Ukraine Volgireva Galina Pavlovna, Candidate of Historical Sciences, assistant professor, Russia Tokareva Natal'ya Gennad'yevna, Candidate of Medical Sciences, assistant professor, Russia Demidova V G , Candidate of Pedagogical Sciences, assistant professor, Ukraine Mogilevskaya I M , Candidate of Pedagogical Sciences, Professor, Ukraine Lebedeva Larisa Aleksandrovna, Candidate of Psychological Sciences, assistant professor, Russia Yatsenko Oleksandr Volodymyrovych, Candidate of Technical Sciences, Professor, Ukraine Shapovalov Valentin Valer'yevich, Candidate of Pharmaceutical Sciences, assistant professor, Ukraine Stovpets V G , Candidate of Philology, assistant professor, Ukraine Ruslan Zubkov, Doctor of Economics, Associate Professor, Ukraine Tolbatov Andrey Vladimirovich, candidate of technical sciences, associate professor, Ukraine Sharagov Vasily Andreevich, Doctor of Chemistry, Associate Professor, Moldova Bukharina Irina Leonidovna, Doctor of Biological Sciences, Professor, Russia Grebneva Nadezhda Nikolayevna, Doctor of Biological Sciences, Professor, Russia Gritsenko Svetlana Anatol'yevna, Doctor of Biological Sciences, assistant professor, Russia Kalenik Tat'yana Kuz'minichna, Doctor of Biological Sciences, Professor, Russia Knyazeva Ol'ga Aleksandrovna, Doctor of Biological Sciences, assistant professor, Russia Kukhar Yelena Vladimirovna, Doctor of Biological Sciences, assistant professor, Kazakhstan Moiseykina Lyudmila Guchayevna, Doctor of Biological Sciences, Professor, Russia Nefed'yeva Yelena Eduardovna, Doctor of Biological Sciences, assistant professor, Russia Sentyabrev Nikolay Nikolayevich, Doctor of Biological Sciences, Professor, Russia Starodubtsev Vladimir Mikhaylovich, Doctor of Biological Sciences, Professor, Ukraine Testov Boris Viktorovich, Doctor of Biological Sciences, Professor, Russia Tungushbayeva Zina Baybagusovna, Doctor of Biological Sciences, , Kazakhstan Fateyeva Nadezhda Mikhaylovna, Doctor of Biological Sciences, Professor, Russia Akhmadiyev Gabdulakhat Malikovich, Doctor of Veterinary Science, Professor, Russia Shevchenko Larisa Vasil'yevna, Doctor of Veterinary Science, Professor, Ukraine Animitsa Yevgeniy Georgiyevich, Doctor of Geographical Sciences, Professor, Russia Sukhova Mariya Gennad'yevna, Doctor of Geographical Sciences, assistant professor, Russia Irzhi Khlakhula, Doctor of Geological and Mineralogical Sciences, Professor, Czech Republic Fedorishin Dmitro Dmitrovich, Doctor of Geological and Mineralogical Sciences, Professor, Ukraine Kokebayeva Gul'zhaukhar Kakenovna, Doctor of Historical Sciences, Professor, Kazakhstan Otepova Gul'fira Yelubayevna, Doctor of Historical Sciences, Professor, Kazakhstan Trigub Petr Nikitovich, Doctor of Historical Sciences, Professor, Ukraine Elezovich M Dalibor, Doctor of Historical Sciences, assistant professor, Serbia Vizir Vadim Anatol'yevich, Doctor of Medical Sciences, Professor, Ukraine Fedyanina Lyudmila Nikolayevna, Doctor of Medical Sciences, Professor, Russia Orlov Nikolay Mikhaylovich, Doctor of Science in Public Administration, assistant professor, Ukraine Velichko Stepan Petrovich, doctor of pedagogical sciences, Professor, Ukraine Gavrilenko Nataliya Nikolayevna, doctor of pedagogical sciences, assistant professor, Russia Gilev Gennadiy Andreyevich, doctor of pedagogical sciences, Professor, Russia Dorofeyev Andrey Viktorovich, doctor of pedagogical sciences, assistant professor, Russia Karpova Nataliya Konstantinovna, doctor of pedagogical sciences, Professor, Russia Mishenina Tat'yana Mikhaylovna, doctor of pedagogical sciences, Professor, Ukraine Nikolayeva Alla Dmitriyevna, doctor of pedagogical sciences, Professor, Russia Rastrygina Alla Nikolayevna, doctor of pedagogical sciences, Professor, Ukraine Sidorovich Marina Mikhaylovna, doctor of pedagogical sciences, Professor, Ukraine Smirnov Yevgeniy Ivanovich, doctor of pedagogical sciences, Professor, Russia Fatykhova Alevtina Leont'yevna, doctor of pedagogical sciences, assistant professor, Russia Fedotova Galina Aleksandrovna, doctor of pedagogical sciences, Professor, Russia Khodakova Nina Pavlovna, doctor of pedagogical sciences, assistant professor, Russia Chigirinskaya Natal'ya Vyacheslavovna, doctor of pedagogical sciences, Professor, Russia Churekova Tat'yana Mikhaylovna, doctor of pedagogical sciences, Professor, Russia Latygina Natal'ya Anatol'yevna, Doctor of Political Sciences, Professor, Ukraine Sirota Naum Mikhaylovich, Doctor of Political Sciences, Professor, Russia Khrebina Svetlana Vladimirovna, Doctor of Psychology, Professor, Russia Vozhegova Raisa Anatol'yevna, doctor of agricultural sciences, Professor, Ukraine Denisov Sergey Aleksandrovich, doctor of agricultural sciences, Professor, Russia Zhovtonog Ol'ga Igorevna, doctor of agricultural sciences, , Ukraine Kostenko Vasiliy Ivanovich, doctor of agricultural sciences, Professor, Ukraine Kotlyarov Vladimir Vladislavovich, doctor of agricultural sciences, Professor, Russia Morozov Aleksey Vladimirovich, doctor of agricultural sciences, Professor, Ukraine Patyka Nikolay Vladimirovich, doctor of agricultural sciences, Professor, Ukraine Rebezov Maksim Borisovich, doctor of agricultural sciences, Professor, Russia Tarariko Yuriy Aleksandrovich, doctor of agricultural sciences, Professor, Ukraine Mal'tseva Anna Vasil'yevna, Doctor of Sociology, assistant professor, Russia Stegniy Vasiliy Nikolayevich, Doctor of Sociology, Professor, Russia Tarasenko Larisa Viktorovna, Doctor of Sociology, Professor, Russia Averchenkov Vladimir Ivanovich, Doctor of Technical Sciences, Professor, Russia Antonov Valeriy Nikolayevich, Doctor of Technical Sciences, Professor, Ukraine Bykov Yuriy Aleksandrovich, Doctor of Technical Sciences, Professor, Russia Goncharuk Sergey Mironovich, Doctor of Technical Sciences, Professor, Russia Zakharov Oleg Vladimirovich, Doctor of Technical Sciences, Professor, Russia Kalayda Vladimir Timofeyevich, Doctor of Technical Sciences, Professor, Russia Kapitanov Vasiliy Pavlovich, Doctor of Technical Sciences, Professor, Ukraine Kirillova Yelena Viktorovna, Doctor of Technical Sciences, assistant professor, Ukraine Kovalenko Petr Ivanovich, Doctor of Technical Sciences, Professor, Ukraine Kopey Bogdan Vladimirovich, Doctor of Technical Sciences, Professor, Ukraine Kosenko Nadezhda Fedorovna, Doctor of Technical Sciences, assistant professor, Russia Kruglov Valeriy Mikhaylovich, Doctor of Technical Sciences, Professor, Russia Kuderin Marat Krykbayevich, Doctor of Technical Sciences, Professor, Kazakhstan Lebedev Anatoliy Timofeyevich, Doctor of Technical Sciences, Professor, Russia Lomot'ko Denis Viktorovich, Doctor of Technical Sciences, Professor, Ukraine Makarova Irina Viktorovna, Doctor of Technical Sciences, Professor, Russia Morozova Tat'yana Yur'yevna, Doctor of Technical Sciences, Professor, Russia Pavlenko Anatoliy Mikhaylovich, Doctor of Technical Sciences, Professor, Ukraine Parunakyan Vaagn Emil'yevich, Doctor of Technical Sciences, Professor, Ukraine Pachurin German Vasil'yevich, Doctor of Technical Sciences, Professor, Russia Pershin Vladimir Fedorovich, Doctor of Technical Sciences, Professor, Russia Piganov Mikhail Nikolayevich, Doctor of Technical Sciences, Professor, Russia Polyakov Andrey Pavlovich, Doctor of Technical Sciences, Professor, Ukraine Popov Viktor Sergeyevich, Doctor of Technical Sciences, Professor, Russia Rokochinskiy Anatoliy Nikolayevich, Doctor of Technical Sciences, Professor, Ukraine Romashchenko Mikhail Ivanovich, Doctor of Technical Sciences, Professor, Ukraine Sementsov Georgiy Nikiforovich, Doctor of Technical Sciences, Professor, Ukraine Sukhenko Yuriy Grigor'yevich, Doctor of Technical Sciences, Professor, Ukraine Научный взгляд в будущее 5 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 О журнале Международный научный периодический журнал "Научный взгляд в будущее" получил большое признание среди отечественных и зарубежных интеллектуалов. Сегодня в журнале публикуются авторы из России, Украины, Молдовы, Казахстана, Беларуси, Чехии, Болгарии, Литвы Польши и других государств. Учрежден в 2015 году. Периодичность выхода: ежеквартально. Основными целями журнала "Научный взгляд в будущее" являются: • cодействие обмену знаниями в научном сообществе; • помощь молодым ученым в информировании научной общественности об их научных достижениях; • cоздание основы для инноваций и новых научных подходов, а также открытий в неизвестных областях; • содействие объединению профессиональных научных сил и формирование нового поколения ученыхспециалистов в разных сферах. Журнал целенаправленно знакомит читателя с оригинальными исследованиями авторов в различных областях науки, лучшими образцами научной публицистики. Публикации журнала "Научный взгляд в будущее" предназначены для широкой читательской аудитории – всех тех, кто любит науку. Материалы, публикуемые в журнале, отражают актуальные проблемы и затрагивают интересы всей общественности. Каждая статья журнала включает обобщающую информацию на английском языке. Журнал зарегистрирован в РИНЦ SCIENCE INDEX и INDEXCOPERNICUS. Про журнал Міжнародний науковий періодичний журнал "Науковий погляд у майбутнє" отримав велике визнання серед вітчизняних і зарубіжних інтелектуалів. Сьогодні в журналі публікуються автори з Росії, України, Молдови, Казахстану, Білорусі, Чехії, Болгарії, Литви, Польщі та інших держав. Дата заснування в 2015 році. Періодичність виходу: щоквартально Основними цілями журналу є: • cприяння обміну знаннями в науковому співтоваристві; • допомога молодим вченим в інформуванні наукової громадськості про їх наукові досягнення; • створення основи для інновацій і нових наукових підходів, а також відкриттів в невідомих областях; • сприяння об'єднанню фахових наукових сил і формування нового покоління вчених-фахівців в різних сферах. Журнал цілеспрямовано знайомить читача з оригінальними дослідженнями авторів в різних областях науки, кращими зразками наукової публіцистики. Публікації журналу призначені для широкої читацької аудиторії - усіх тих, хто любить науку. Матеріали, що публікуються в журналі, відображають актуальні проблеми і зачіпають інтереси всієї громадськості. Кожна стаття журналу включає узагальнюючу інформацію англійською мовою. Журнал зареєстрований в РИНЦ SCIENCE INDEX і INDEXCOPERNICUS. About the journal The International Scientific Periodical Journal "Scientific look into the future" has gained considerable recognition among domestic and foreign researchers and scholars. Today, the journal publishes authors from Russia, Ukraine, Moldova, Kazakhstan, Belarus, Czech Republic, Bulgaria, Lithuania, Poland and other countries. Journal Established in 2015. Рeriodicity of publication: Quarterly The journal activity is driven by the following objectives: • Broadcasting young researchers and scholars outcomes to wide scientific audience • Fostering knowledge exchange in scientific community • Promotion of the unification in scientific approach • Creation of basis for innovation and new scientific approaches as well as discoveries in unknown domains The journal purposefully acquaints the reader with the original research of authors in various fields of science, the best examples of scientific journalism. Publications of the journal are intended for a wide readership - all those who love science. The materials published in the journal reflect current problems and affect the interests of the entire public. Each article in the journal includes general information in English. The journal is registered in the RISC SCIENCE INDEX and INDEXCOPERNICUS. Научный взгляд в будущее 6 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 Требования к статьям Статьи должны соответствовать тематическому профилю журнала, отвечать международным стандартам научных публикаций и быть оформленными в соответствии с установленными правилами. Они также должны представлять собой изложение результатов оригинального авторского научного исследования, быть вписанными в контекст отечественных и зарубежных исследований по этой тематике, отражать умение автора свободно ориентироваться в существующем библиографическом контексте по затрагиваемым проблемам и адекватно применять общепринятую методологию постановки и решения научных задач. Все тексты должны быть написаны литературным языком, отредактированы и соответствовать научному стилю речи. Некорректность подбора и недостоверность приводимых авторами фактов, цитат, статистических и социологических данных, имен собственных, географических названий и прочих сведений может стать причиной отклонения присланного материала (в том числе – на этапе регистрации). Все таблицы и рисунки в статье должны быть пронумерованы, иметь заголовки и ссылки в тексте. Если данные заимствованы из другого источника, на него должна быть дана библиографическая ссылка в виде примечания. Название статьи, ФИО авторов, учебные заведения (кроме основного языка текста) должны быть представлены и на английском языке. Статьи должны сопровождаться аннотацией и ключевыми словами на языке основного текста и обязательно на английском языке. Аннотация должна быть выполнена в форме краткого текста, который раскрывает цель и задачи работы, ее структуру и основные полученные выводы. Аннотация представляет собой самостоятельный аналитический текст и должна давать адекватное представление о проведенном исследовании без необходимости обращения к статье. Аннотация на английском (Abstract) должна быть написана грамотным академическим языком. Приветствуется наличие УДК, ББК, а также (для статей по Экономике) код JEL (https://www.aeaweb.org/jel/guide/jel.php) Принятие материала к рассмотрению не является гарантией его публикации. Зарегистрированные статьи рассматриваются редакцией и при формальном и содержательном соответствии требованиям журнала направляются на экспертное рецензирование, в том числе через открытое обсуждение с помощью веб-ресурса www.sworld.education. В журнале могут быть размещены только ранее неопубликованные материалы. Вимоги до статей Статті повинні відповідати тематичному профілю журналу, відповідати міжнародним стандартам наукових публікацій і бути оформленими відповідно до встановлених правил. Вони також повинні представляти собою виклад результатів оригінального авторського наукового дослідження, бути вписаними в контекст вітчизняних і зарубіжних досліджень з цієї тематики, відображати вміння автора вільно орієнтуватися в існуючому бібліографічному контексті по піднятим проблемам і адекватно застосовувати загальноприйняту методологію постановки і вирішення наукових завдань. Всі тексти повинні бути написані літературною мовою, відредаговані і відповідати науковому стилю мовлення. Некоректність підбору і недостовірність наведених авторами фактів, цитат, статистичних та соціологічних даних, власних імен, географічних назв та інших відомостей може стати причиною відхилення надісланого матеріалу (в тому числі - на етапі реєстрації). Всі таблиці і рисунки в статті повинні бути пронумеровані, мати заголовки і посилання в тексті. Якщо дані запозичені з іншого джерела, на нього повинні бути бібліографічні посилання у вигляді примітки. Назва статті, ПІБ авторів, навчальні заклади (крім основної мови тексту) повинні бути представлені і на англійській мові. Статті повинні супроводжуватися анотацією та ключовими словами на мові основного тексту і обов'язково англійською мовою. Анотація повинна бути виконана у формі короткого тексту, який розкриває мету і завдання роботи, її структуру та основні отримані висновки. Анотація представляє собою самостійний аналітичний текст і повинна давати адекватне уявлення про проведене дослідження без необхідності звернення до статті. Анотація англійською (Abstract) повинна бути написана грамотною академічною мовою. Заохочується наявність УДК, ББК, а також (для статей по Економіці) код JEL (https://www.aeaweb.org/jel/guide/jel.php) Ухвалення матеріалу до розгляду не є гарантією його публікації. Зареєстровані статті розглядаються редакцією і при формальному і змістовному відповідно до вимог журналу направляються на експертне рецензування, в тому числі через відкрите обговорення за допомогою веб-ресурсу www.sworld.education. У журналі можуть бути розміщені тільки раніше неопубліковані матеріали. Requirements for articles Articles should correspond to the thematic profile of the journal, meet international standards of scientific publications and be formalized in accordance with established rules. They should also be a presentation of the results of the original author's scientific research, be inscribed in the context of domestic and foreign research on this topic, reflect the author's ability to freely navigate in the existing bibliographic context on the problems involved and adequately apply the generally accepted methodology of setting and solving scientific problems. All texts should be written in literary language, edited and conform to the scientific style of speech. Incorrect selection and unreliability of the facts, quotations, statistical and sociological data, names of own, geographical names and other information cited by the authors can cause the rejection of the submitted material (including at the registration stage). All tables and figures in the article should be numbered, have headings and links in the text. If the data is borrowed from another source, a bibliographic reference should be given to it in the form of a note. The title of the article, the full names of authors, educational institutions (except the main text language) should be presented in English. Articles should be accompanied by an annotation and key words in the language of the main text and must be in English. The abstract should be made in the form of a short text that reveals the purpose and objectives of the work, its structure and main findings. The abstract is an independent analytical text and should give an adequate idea of the research conducted without the need to refer to the article. Abstract in English (Abstract) should be written in a competent academic language. The presence of UDC, BBK Acceptance of the material for consideration is not a guarantee of its publication. Registered articles are reviewed by the editorial staff and, when formally and in substance, the requirements of the journal are sent to peer review, including through an open discussion using the web resource www.sworld.education Only previously unpublished materials can be posted in the journal. Научный взгляд в будущее 7 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 Положение об этике публикации научных данных и ее нарушениях Редакция журнала осознает тот факт, что в академическом сообществе достаточно широко распространены случаи нарушения этики публикации научных исследований. В качестве наиболее заметных и вопиющих можно выделить плагиат, направление в журнал ранее опубликованных материалов, незаконное присвоение результатов чужих научных исследований, а также фальсификацию данных. Мы выступаем против подобных практик. Редакция убеждена в том, что нарушения авторских прав и моральных норм не только неприемлемы с этической точки зрения, но и служат преградой на пути развития научного знания. Потому мы полагаем, что борьба с этими явлениями должна стать целью и результатом совместных усилий наших авторов, редакторов, рецензентов, читателей и всего академического сообщества. Мы призываем всех заинтересованных лиц сотрудничать и участвовать в обмене информацией в целях борьбы с нарушением этики публикации научных исследований. Со своей стороны редакция готова приложить все усилия к выявлению и пресечению подобных неприемлемых практик. Мы обещаем принимать соответствующие меры, а также обращать пристальное внимание на любую предоставленную нам информацию, которая будет свидетельствовать о неэтичном поведении того или иного автора. Обнаружение нарушений этики влечет за собой отказ в публикации. Если будет выявлено, что статья содержит откровенную клевету, нарушает законодательство или нормы авторского права, то редакция считает себя обязанной удалить ее с веб-ресурса и из баз цитирования. Подобные крайние меры могут быть применены исключительно при соблюдении максимальной открытости и публичности. Положення про етику публікації наукових даних і її порушеннях Редакція журналу усвідомлює той факт, що в академічній спільноті досить широко поширені випадки порушення етики публікації наукових досліджень. В якості найбільш помітних можна виділити плагіат, відправлення в журнал раніше опублікованих матеріалів, незаконне привласнення результатів чужих наукових досліджень, а також фальсифікацію даних. Ми виступаємо проти подібних практик. Редакція переконана в тому, що порушення авторських прав і моральних норм не тільки неприйнятні з етичної точки зору, але і служать перешкодою на шляху розвитку наукового знання. Тому ми вважаємо, що боротьба з цими явищами повинна стати метою і результатом спільних зусиль наших авторів, редакторів, рецензентів, читачів і усієї академічної спільноти. Ми закликаємо всіх зацікавлених осіб співпрацювати і брати участь в обміні інформацією з метою боротьби з порушенням етики публікації наукових досліджень. Зі свого боку редакція готова докласти всіх зусиль до виявлення та припинення подібних неприйнятних практик. Ми обіцяємо вживати відповідних заходів, а також звертати пильну увагу на будь-яку надану нам інформацію, яка буде свідчити про неетичну поведінку того чи іншого автора. Виявлення порушень етики тягне за собою відмову в публікації. Якщо буде виявлено, що стаття містить відвертий наклеп, порушує законодавство або норми авторського права, то редакція вважає себе зобов'язаною видалити її з веб-ресурсу і з баз цитування. Подібні крайні заходи можуть бути застосовані виключно при дотриманні максимальної відкритості і публічності. Regulations on the ethics of publication of scientific data and its violations The editors of the journal are aware of the fact that in the academic community there are quite widespread cases of violation of the ethics of the publication of scientific research. As the most notable and egregious, one can single out plagiarism, the posting of previously published materials, the misappropriation of the results of foreign scientific research, and falsification of data. We oppose such practices. The editors are convinced that violations of copyrights and moral norms are not only ethically unacceptable, but also serve as a barrier to the development of scientific knowledge. Therefore, we believe that the fight against these phenomena should become the goal and the result of joint efforts of our authors, editors, reviewers, readers and the entire academic community. We encourage all stakeholders to cooperate and participate in the exchange of information in order to combat the violation of the ethics of publication of scientific research. For its part, the editors are ready to make every effort to identify and suppress such unacceptable practices. We promise to take appropriate measures, as well as pay close attention to any information provided to us, which will indicate unethical behavior of one or another author. Detection of ethical violations entails refusal to publish. If it is revealed that the article contains outright slander, violates the law or copyright rules, the editorial board considers itself obliged to remove it from the web resource and from the citation bases. Such extreme measures can be applied only with maximum openness and publicity. Научный взгляд в будущее 8 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 https://www.scilook.eu/index.php/slif/article/view/slif13-008 DOI: 10.30888/2415-7538.2019-13-02-008 УДК 631.81 AGROECOLOGICAL GROUND of TOP-DRESSING UNDER WINTER WHEAT Of SORT «MURONIVSKA 61» АГРОЕКОЛОГІЧНЕ ОБГРУНТУВАННЯ ВНЕСЕННЯ ДОБРИВ ПІД ОЗИМУ ПШЕНИЦЮ СОРТУ « МИРОНІВСЬКА 61» Kudriawytzka A.N./Кудрявицька А.М. c.a.s. ., as.prof./ к.с.-г.н., доц. SPIN: 7001-1956 Yanik К. O./Яник К.О. National University of Life and Environmental Sciences of Ukraine Kyiv,street of Heroes of defensive,17,03041 Національний університет біоресурсів і природокористування України. м. Київ, вул. Героїв оборони, 17, 03041 Анотація. Дослідженнями на лучно-чорноземному карбонатному ґрунті встановлено, що тривале застосування мінеральних добрив на фоні післядії органічних, забезпечує приріст урожаю зерна районованого сорту ярої пшениці Миронівська яра на 1,73 т/га. Урожайність і якість зерна ярої пшениці підвищуються при внесенні полуторної норми мінеральних добрив на фоні післядії органічних, з відповідно високими показниками якості : збору білку- 0,64 т/га та збору «сирої» клейковини-1,36 т/га. Ключові слова: пшениця, урожайність, добрива, доза, білок, «сира» клейковина, сорт, ґрунт, сівозміна. Питання про вплив умов вирощування, біологічних особливостей сортів, впливу різних доз мінеральних та органічних добрив, на врожай зерна озимої пшениці має теоретичне та практичне значення [1,2]. Питання про агроекологічне обґрунтування внесення добрив, застосування різних доз мінеральних та органічних добрив на врожай зерна озимої пшениці має теоретичне та практичне значення [2,3]. Об’єктом дослідження є характер зміни вмісту елементів живлення в рослинах озимої пшениці сорту Миронівська 61 та ярої пшениці сорту Миронівська яра, врожайність нових сортів озимої та ярої пшениці за викорисання традиційних видів добрив. Предметом дослідження є вміст макроелементів в рослинах пшениці, врожай та якість зерна районованого сорту пшениці озимої Миронівська 61 та ярої Миронівська яра. Об’єктом дослідження була озима пшениця сорту Миронівська 61, попередником якої є конюшина. Дослід має трьохкратну повторність. Площа облікової ділянки 100 м2, посівної 175 м2. Стаціонарний дослід має 12 варіантів. Робочими варіантами в в зернобуряковій сівозміні були наступні: Контроль Післядія гною - фон Фон + P80 Фон + P80K80 Фон + N60P80K80 Фон + N75P120K120 Научный взгляд в будущее 119 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 N60P80K80 В результаті досліджень на лучно-чорноземному грубопилувато легкосуглинковому ґрунті встановлено, що тривале застосування мінеральних добрив на фоні післядії гною, сприяє підвищенню врожаю зерна озимої пшениці сорту Миронівська 61 на 11,4-26,3 ц/га (табл.1), при урожаї на контролі відповідно 32,7 ц/га. Озима пшениця має чітко виражені закономірності в нагромадженні вмісту білку і клейковини (табл.2). Таблиця 1 Вплив добрив на врожайність зерна озимої пшениці, ц/га Приріст до контролю Варіант досліду Врожайність, ц/га ц/га % Без добрив 32,7 100 (контроль) Післядія 30 т/га 4,1 11,4 34,8 гною (фон) Фон+Р80 45,5 12,8 39,1 Фон+Р80К80 46,2 13,5 41,3 Фон+N80Р80К80 54,9 22,2 67,8 Фон+N110Р120К120 59,0 26,3 80,4 N80Р80К80 46,1 13,4 40,9 Таблиця 2 Вплив тривалого застосування добрив на якість зерна озимої пшениці Збір «сирої» Збір білка клейковини «Сира» Білок, приріст приріст клейкови Варіант досліду % до до на, % ц/га ц/га контролю, контролю, ц/га ц/га Без добрив 9,2 3,0 20,1 6,6 (контроль) Післядія 30 т/га 11,6 5,1 2,1 21,2 9,3 2,7 гною (фон) Фон+Р80 11,9 5,4 2,4 21,5 9,8 3,2 Фон+Р80К80 12,1 5,6 2,6 21,1 9,7 3,1 Фон+N80Р80К80 12,4 6,8 3,8 25,6 14,0 7,4 Фон+N110Р120К120 12,7 7,5 4,5 27,2 16,0 9,4 N80Р80К80 12,1 5,6 2,6 24,1 11,1 4,5 НІР05,% 0,64 1,25 Висновки 1. Внесення мінеральних добрив на фоні післядії органічних підвищувало Научный взгляд в будущее 120 ISSN 2415-766Х (P) / 2415-7538 (O) Выпуск 13 Том 2 урожай зерна озимої пшениці до 26,3 ц\га, при урожаї на контролі 2,7ц/га. Найвищі врожаї зерна озимої і пшениці отримали при внесенні полуторної норми мінеральних добрив на фоні післядії органічних, відповідно 59,0 ц/га. 2. Озима пшениця має чітко виражені закономірності в нагромадженні вмісту білку і клейковини. Мінеральні добрива внесені на фоні післядії органічних підвищували вміст білку в озимій пшениці на 2,4-3,5% і клейковини на 1,1-7,1% при вмісті їх на контролі 9,2% і 20,1%. Література: 1. Агрохімічний аналіз: підр. для студ вищих навч. закл. / М.М. Городній, А.П. Лісовал, А.В. Бикін та ін.; – К.: Арістей. 2005. – 468с. 2. Антонова А.А. Відтворення родючості чорноземів. / Антонова А.А., Головінов А.А. // Агрохімічний вісник. - 2001. - №4. - С. 40–52. 3. Габібов М.А. Післядія мінеральних добрив при вирощуванні озимої пшениці / Габібов М.А. // Зернові культури. - 2001. - №1. - С. 11–19. References: 1. Agrohіmіchnij analіz: pіdr. dlya stud vishchih navch. zakl. / M.M. Gorodnіj, A.P. Lіsoval, A.V. Bikіn ta іn.; – K.: Arіstej. 2005. – 468s. 2. Antonova A.A. Vіdtvorennya rodyuchostі chornozemіv. / Antonova A.A., Golovіnov A.A. // Agrohіmіchnij vіsnik. - 2001. - №4. - S. 40–52. 3. Gabіbov M.A. Pіslyadіya mіneral'nih dobriv pri viroshchuvannі ozimoї pshenicі / Gabіbov M.A. // Zernovі kul'turi. - 2001. - №1. - S. 11–19. Abstract. On black carbonate soil it is set researches, that the protracted application of mineral fertilizers is on a background the afteraction of organic, the Миронівська furious provides the increase of harvest of grain of the districted sort of furious wheat on 1,73 t/he. The productivity and quality of grain of furious wheat rise at bringing of one-and-a-half norm of mineral fertilizers on a background the afteraction of organic, from accordingly by the high indexes of quality : of collection of albumen of 0,64 т/and and to collection of "raw" gluten of 1,36 t/he. Key words: wheat, productivity, fertilizers, dose, albumen, "raw" gluten, sort, soil, crop rotation. Стаття відправлена 02.04.2019 г. © Кудрявицька А.М. Научный взгляд в будущее 121 ISSN 2415-766Х (P) / 2415-7538 (O) Экспертно-рецензионный Совет журнала Expert-Peer Review Board of the journal Абдулвелеева Рауза Рашитовна, кандидат педагогических наук, доцент, Оренбургский государственный университет, Россия Артюхина Марина Владимировна, кандидат экономических наук, доцент, Славянский государственный педагогический университет, Украина Афинская Зоя Николаевна, кандидат филологических наук, доцент, Московский государственный университет имени М.В. Ломоносова, Россия Башлай Сергей Викторович, кандидат экономических наук, доцент, Украинская академия банковского дела, Украина Белоус Татьяна Михайловна, кандидат медицинских наук, доцент, Буковинская государственная медицинская академия, Украина Бондаренко Юлия Сергеевна, кандидат педагогических наук, доцент, ПГУ им. Т.Г. Шевчекнко кафедра психологии, Казахстан Бутырский Александр Геннадьевич, кандидат медицинских наук, доцент, Медицинская академия имени С.И. Георгиевского ФГАОУ ВО КФУ имени В.И. Вернадского, Россия Василишин Виталий Ярославович, кандидат технических наук, доцент, Ивано-Франковский национальный технический университет нефти и газа, Украина Войцеховский Владимир Иванович, кандидат сельскохозяйственных наук, доцент, Национальный университет биоресурсов и природопользования Украины, Украина Гаврилова Ирина Викторовна, кандидат педагогических наук, доцент, Магнитогорский государственный технический университет имени Г.И.Носова, Россия Гинис Лариса Александровна, кандидат педагогических наук, доцент, Южный федеральный университет, Россия Гутова Светлана Георгиевна, кандидат философских наук, доцент, Нижневартовский государственный университет, Россия Зубков Руслан Сергеевич, кандидат экономических наук, ГП НПКГ "Зоря"-"Машпроект" г. Николаев, Украина Ивлев Антон Васильевич, кандидат экономических наук, доцент, Магнитогорский государственный технический университет имени Г.И.Носова, Россия Идрисова Земфира Назиповна, кандидат экономических наук, доцент, Уфимский государственный авиационный технический университет, Россия Илиев Веселин, кандидат технических наук, доцент, Болгария Кириллова Татьяна Климентьевна, кандидат экономических наук, доцент, Иркутский государственный университет путей сообщения, Россия Коваленко Татьяна Антольевна, кандидат технических наук, Поволжский государственный университет телекоммуникаций и информатики, Россия Котова Светлана Сергеевна, кандидат педагогических наук, доцент, Российский государственный профессиональнопедагогический университет, Россия Кухтенко Галина Павловна, кандидат фармацевтических наук, доцент, Национальный фармацевтический университет Украины, Украина Лобачева Ольга Леонидовна, кандидат химических наук, доцент, Горный университет, Россия Ляшенко Дмитрий Алексеевич, кандидат географических наук, доцент, Национальный транспортный университет, Украина Макаренко Андрей Викторович, кандидат педагогических наук, доцент, Донбасский государственный педагогический университет, Украина Мельников Александр Юрьевич, кандидат технических наук, доцент, Донбасская государственная машиностроительная академия, Украина Мороз Людмила Ивановна, кандидат экономических наук, доцент, Национальный университет "Львовская политехника", Украина Музылёв Дмитрий Александрович, кандидат технических наук, доцент, Харьковский национальный технический университет сельского хозяйства имени Петра Василенко, Украина Надопта Татьяна Анатолиевна, кандидат технических наук, ст. преп, Хмельницкий национальный университет, Украина Напалков Сергей Васильевич, кандидат педагогических наук, Нижегородский государственный университет имени Н.И. Лобачевского, Арзамасский филиал ННГУ, Россия Никулина Евгения Викторовна, кандидат экономических наук, доцент, Белгородский государственный национальный исследовательский университет, Россия Орлова Анна Викторовна, кандидат экономических наук, доцент, Белгородский государственный национальный исследовательский университет, Россия Осипов Виктор Авенирович, доктор географических наук, профессор, Тюменский государственный университет, Россия Привалов Евгений Евграфович, кандидат технических наук, доцент, Ставропольский государственный аграрный университет, Россия Пыжьянова Наталия Владимировна, кандидат искусствоведения, доцент, Украина Сегин Любомир Васильевич, кандидат филологических наук, доцент, Славянский государственный педагогический университет, Украина Сергиенко Александр Алексеевич, доктор медицинских наук, профессор, Львовский национальный медицинский университет им. Даниила Галицкого, Украина Сочинская-Сибирцева Ирина Николаевна, кандидат экономических наук, доцент, Кировоградский государственный технический университет, Украина Сысоева Вера Александровна, кандидат архитектурных наук, доцент, Белорусский национальный технический университет, Беларусь Тлеуов Асхат Халилович, доктор технических наук, профессор, Казахский агротехнический университет, Казахстан Ходжаева Гюльназ Казым кызы, кандидат географических наук, Россия Чигиринский Юлий Львович, кандидат технических наук, доцент, Волгоградский государственный технический университет, Россия Шехмирзова Анджела Мухарбиевна, кандидат педагогических наук, доцент, Адыгейский государственный университет, Россия Шпинковский Александр Анатольевич, кандидат технических наук, доцент, Одесский национальный политехнический университет, Украина Научный взгляд в будущее 129 ISSN 2415-766Х (P) / 2415-7538 (O) СОДЕРЖАНИЕ / CONTENTS Экономика и торговля Economy and trade Економіка і торгівля https://www.scilook.eu/index.php/slif/article/view/slif13-020 9 ESSENCE, FUNCTIONS AND MAIN TASKS OF RETAIL TRADE ENTERPRISES СУТНІСТЬ, ФУНКЦІЇ ТА ОСНОВНІ ЗАВДАННЯ ДІЯЛЬНОСТІ ПІДПРИЄМСТВ РОЗДРІБНОЇ ТОРГІВЛІ Chechui A.V./ Чечуй А. В. https://www.scilook.eu/index.php/slif/article/view/slif13-023 16 POTENTIAL OF THE TRADE ENTERPRISE: GENERAL APPROACHES TO FORMATION AND REALIZATION ПОТЕНЦІАЛ ТОРГОВЕЛЬНОГО ПІДПРИЄМСТВА: ЗАГАЛЬНОНАУКОВІ ПІДХОДИ ДО ФОРМУВАННЯ ТА РЕАЛІЗАЦІЇ Zhuvahina I.O. / Жувагіна І.О., Zamaraikina T.S. / Замарайкіна Т.С. https://www.scilook.eu/index.php/slif/article/view/slif13-033 22 FRAGMENTATION IN MANUFACTURE OF AUTOMOBILE COMPONENTS IN UKRAINE: EXPIRIENCE FOR BELARUS ФРАГМЕНТАЦІЯ У ВИРОБНИЦТВІ АВТОКОМПОНЕНТІВ В УКРАЇНІ: ДОСВІД ДЛЯ БІЛОРУСІ Kostenko N.V. / Костенко Н.В. https://www.scilook.eu/index.php/slif/article/view/slif13-043 27 FUTURE AND PROSPECTS OF GLOBALIZATION AND ENVIRONMENTAL INTEGRATION PROCESS СУПЕРЕЧНОСТІ ТА ПЕРСПЕКТИВИ ПРОЦЕСУ ГЛОБАЛІЗАЦІЇ ТА ПРИРОДООХОРОННОЇ ІНТЕГРАЦІЇ Rogach S.M. / Рогач С.М. Менеджмент и маркетинг Management and marketing Менеджмент і маркетинг https://www.scilook.eu/index.php/slif/article/view/slif13-031 33 MANAGEMENT COMMUNICATION IN SCIENCE AND THE INFORMATION ENVIRONMENT УПРАВЛІНСЬКА КОМУНІКАЦІЯ В НАУЦІ ТА ІНФОРМАЦІЙНОМУ СЕРЕДОВИЩІ Makovetska I. M. / Маковецька І.М. https://www.scilook.eu/index.php/slif/article/view/slif13-038 38 POLITICAL AND ADMINISTRATIVE DECHOTOMY - MYTH OR REALITY ПОЛИТИКО-АДМИНИСТРАТИВНАЯ ДИХОТОМИЯ – МИФ ИЛИ РЕАЛЬНОСТЬ Fetescu C. P. / Фетеску Ч. П. Научный взгляд в будущее 130 ISSN 2415-766Х (P) / 2415-7538 (O) https://www.scilook.eu/index.php/slif/article/view/slif13-041 43 FEATURES OF DEVELOPMENT OF THE RURAL SETTLEMENT OF MORDOVO-ISHUTKINA OF M. R. ISAKLINSKIY SAMARA REGION ОСОБЕННОСТИ РАЗВИТИЯ СЕЛЬСКОГО ПОСЕЛЕНИЯ МОРДОВО-ИШУТКИНО В СОСТАВЕ М.Р. ИСАКЛИНСКИЙ САМАРСКОЙ ОБЛАСТИ Kuznetsov M.V. / Кузнецов М.В., Bulankina E.V. / Буланкина Е. В. Образование и педагогика Education and pedagogy Освіта і педагогіка https://www.scilook.eu/index.php/slif/article/view/slif13-005 50 TOLERANCE AS THE BASE PRINCIPLE OF THE ORGANIZATION OF ACTIVITY OF INTERNATIONAL STUDENT'S ASSOCIATIONS ТОЛЕРАНТНОСТЬ КАК БАЗОВЫЙ ПРИНЦИП ОРГАНИЗАЦИИ ДЕЯТЕЛЬНОСТИ ИНТЕРНАЦИОНАЛЬНЫХ СТУДЕНЧЕСКИХ ОБЪЕДИНЕНИЙ Vorobjeva G.V./ Воробьёва Г.В., Baturina L.A. / Батурина Л.А., Lepikhov N.V./ Лепихов Н.В. https://www.scilook.eu/index.php/slif/article/view/slif13-006 54 SOME PROBLEMS OF MATHEMATICAL TRAINING OF ECONOMISTS НЕКОТОРЫЕ ВОПРОСЫ МАТЕМАТИЧЕСКОЙ ПОДГОТОВКИ ЭКОНОМИСТОВ Belova T.B. / Белова Т.Б., Mikhin M.N. / Михин М.Н. https://www.scilook.eu/index.php/slif/article/view/slif13-017 58 THE ROLE OF STUDENTS INDEPENDENT WORK IN STUDYING INFORMATICS AT UNIVERSITY РОЛЬ САМОСТОЯТЕЛЬНОЙ РАБОТЫ СТУДЕНТОВ ПРИ ИЗУЧЕНИИ ИНФОРМАТИКИ В ВУЗЕ Eroshenko A.V. / Ерошенко А.В. https://www.scilook.eu/index.php/slif/article/view/slif13-024 ТЕХНОЛОГІЯ ФОРМУВАННЯ НАВИЧОК ГРАМОТИ У ДІТЕЙ 62 З РОЗЛАДАМИ АУТИСТИЧНОГО СПЕКТРА Dmitriyeva I.V. / Дмитрієва І.В., Burlachenko A.G. / Бурлаченко А.Г. https://www.scilook.eu/index.php/slif/article/view/slif13-032 66 THE EXPEDIENCY OF USING AUTHENTIC TEXTS IN TEACHING FOREIGN LANGUAGE ЦЕЛЕСООБРАЗНОСТЬ ИСПОЛЬЗОВАНИЯ АУТЕНТИЧНЫХ ТЕКСТОВ ПРИ ОБУЧЕНИИ ИНОСТРАННОМУ ЯЗЫКУ Zaitseva S.E. / Зайцева С.Е. https://www.scilook.eu/index.php/slif/article/view/slif13-035 72 THE QUESTION OF EFFICIENCY OF TEACHING LECTURE К ВОПРОСУ ЭФФЕКТИВНОСТИ ЛЕКЦИОННОГО ОБУЧЕНИЯ Emchenko E.A. / Емченко Е.А. Научный взгляд в будущее 131 ISSN 2415-766Х (P) / 2415-7538 (O) https://www.scilook.eu/index.php/slif/article/view/slif13-037 78 PROFESSIONALLY ORIENTED TRAINING IN THE FRAMEWORK OF THE COMPETENCE APPROACH ПРОФЕССИОНАЛЬНО ОРИЕНТИРОВАННОЕ ОБУЧЕНИЕ В РАМКАХ КОМПЕТЕНТНОСТНОГО ПОДХОДА Belova N.M. / Белова Н.М., Mikhin M.N. / Михин М.Н. Физическое воспитание и спорт Physical education and sport Фізичне виховання і спорт https://www.scilook.eu/index.php/slif/article/view/slif13-007 83 FORMATION OF A HEALTHY LIFESTYLE OF YOUNGER STUDENTS IN THE CONDITIONS OF EXTRACURRICULAR ACTIVITIES ФОРМИРОВАНИЕ ЗДОРОВОГО ОБРАЗА ЖИЗНИ МЛАДШИХ ШКОЛЬНИКОВ В УСЛОВИЯХ ВНЕКЛАССНОЙ РАБОТЫ Vilgelm K.I. / Вильгельм К.И., Sevenuk S.A. / Севенюк С.А. https://www.scilook.eu/index.php/slif/article/view/slif13-039 87 PHYSICAL EDUCATION OF SCHOOLCHILDREN IN RURAL KAZAKH SCHOOL ФИЗИЧЕСКОЕ ВОСПИТАНИЕ ШКОЛЬНИКОВ В КАЗАХСКОЙ СЕЛЬСКОЙ ШКОЛЕ Dzhusupov U.S./ Джусупов У.С., Issayeva A.U. / Исаева А.У. Психология и социология Psychology and sociology Психологія і соціологія https://www.scilook.eu/index.php/slif/article/view/slif13-044 93 YOGOTSENTRISM YOUTH SCHOOL LEVEL IN THE TEXT OF THE DECLARATION УРОВЕНЬ ЭГОЦЕНТРИЗМА МЛАДШЕГО ШКОЛЬНИКА В ПРОЦЕССЕ ПОНИМАНИЯ ТЕКСТА PerepeliykT.D./ Перепелюк Т.Д. Философия Philosophy Філософія https://www.scilook.eu/index.php/slif/article/view/slif13-046 98 SPECIFICATION OF «FROZEN» CONFLICTS: SOCIAL AND PHILOSOPHICAL ASPECTS СПЕЦИФІКА «ЗАМОРОЖЕНИХ» КОНФЛІКТІВ: СОЦІАЛЬНО-ФІЛОСОФСЬКІ АСПЕКТИ Авер’янова Н.М. / Averianova N.M., Воропаєва Т.С. / Voropayeva T.S. Научный взгляд в будущее 132 ISSN 2415-766Х (P) / 2415-7538 (O) Юридические и политические науки Legal and political sciences Юридичні і політичні науки https://www.scilook.eu/index.php/slif/article/view/slif13-013 105 INTERNATIONAL COMMUNICATIONS OF UKRAINE WITH THE REPUBLIC OF POLAND: HISTORICAL ASPECTS AND DEVELOPMENT PERSPECTIVES МІЖНАРОДНІ ЗВ’ЯЗКИ УКРАЇНИ З РЕСПУБЛІКОЮ ПОЛЬЩА: ІСТОРИЧНИЙ АСПЕКТ ТА ПЕРСПЕКТИВИ РОЗВИТКУ Vanina O.Y. / Ваніна О.Ю. https://www.scilook.eu/index.php/slif/article/view/slif13-019 110 INTERNATIONAL RELATIONS OF UKRAINE WITH GREECE МІЖНАРОДНІ ВІДНОСИНИ УКРАЇНИ З ГРЕЦІЄЮ Sekunova Y.V. / Сєкунова Ю.В., Shevchuck I.Y./ Шевчук І.Ю. https://www.scilook.eu/index.php/slif/article/view/slif13-045 114 THE CRISIS OF INTERNATIONAL RELATIONS OF UKRAINE AND POLAND МІЖНАРОДНІ ВІДНОСИНИ УКРАЇНИ ТА РЕСПУБЛІКИ ПОЛЬЩА: ПРОБЛЕМНІ СТОРІНКИ ІСТОРІЇ ТА ПЕРСПЕКТИВИ РОЗВИТКУ Дзуг М.С./ Dzuh M. S., Сєкунова Ю.В./ Sekunova Y.V. Сельское, лесное, рыбное и водное хозяйство Agriculture, forestry, fishery and water management Сільське, лісове, рибне та водне господарство https://www.scilook.eu/index.php/slif/article/view/slif13-008 119 AGROECOLOGICAL GROUND of TOP-DRESSING UNDER WINTER WHEAT Of SORT «MURONIVSKA 61» АГРОЕКОЛОГІЧНЕ ОБГРУНТУВАННЯ ВНЕСЕННЯ ДОБРИВ ПІД ОЗИМУ ПШЕНИЦЮ СОРТУ « МИРОНІВСЬКА 61» Kudriawytzka A.N./Кудрявицька А.М., Yanik К. O./Яник К.О. https://www.scilook.eu/index.php/slif/article/view/slif13-028 122 USE OF GROWTH RATE INDICATORS FOR PUREBRED LARGE WHITE BREEDS AND THEIR HYBRIDS WITH LANDRACE, DUROC AND PIETRAIN BREEDS FOR COMPARATIVE ANALYSIS OF THE GROWTH OF ANIMALS ВИКОРИСТАННЯ ПОКАЗНИКІВ ІНТЕНСИВНОСТІ РОСТУ ЧИСТОПОРОДНИХ СВИНЕЙ ВЕЛИКОЇ БІЛОЇ ПОРОДИ ТА ЇХ ПОМІСЕЙ ІЗ ПОРОДАМИ ЛАНДРАС, ДЮРОК ТА П’ЄТРЕН ДЛЯ ПОРІВНЯЛЬНОГО АНАЛІЗУ РОСТУ ТВАРИН Barkar Ye.V. / Баркарь Є.В., Dekhtiar Yu.F. / Дехтяр Ю.Ф., Kot S.P. / Кот С.П. Научный взгляд в будущее 133 ISSN 2415-766Х (P) / 2415-7538 (O) Научное издание Международный периодический рецензируемый научный журнал НАУЧНЫЙ ВЗГЛЯД В БУДУЩЕЕ Выпуск 13 Том 2 На украинском, русском и английском языках Входит в международные наукометрические базы (высокий импактфактор): РИНЦ, INDEXCOPERNICUS (ICI 88.47) Научные достижения Авторов были также представлены для открытого обсуждения на международной научной конференции «Практическое значение современных научных исследований ‘2019» (с 8 по 9 апреля 2019 г.) Решением международной научной конференции работы, которые получили положительную оценку, были рекомендованы к изданию в журнале. www.sworld.education Разработка оригинал-макета – КУПРИЕНКО СВ Подписано в печать: 07.05.2019 Формат 60х84/16. Печать цифровая. Усл.печ.л. 7,79 Тираж 500. Заказ №ua13-1. 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The Trouble with “Female Sexuality”
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The Trouble with “Female Sexuality” Sarah Salih • King’s College London Recommended citation: Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. Using medieval western art to speak of female sexuality is difficult. Karma Lochrie argues that medieval women’s sexuality was organised in ways so alien to current categories that it requires careful excavation: Medieval hybrids that are incomprehensible today, such as ‘chaste marriage’ or even a kind of ‘willful virginity,’ were not only practised during the Middle Ages, but they suggest a much more diffused and complex interaction of categories than we are used to. Armed only with the heterosexual/homosexual divide and a presumption of heteronormativity, we cannot even begin to sort out such categories as Amazons, female masculinity, or even virginity.[1] Lochrie’s book, like most studies of medieval sexuality, is primarily concerned with textual sources. Can the visual arts contribute to this work of categorisation? This brief overview will suggest that such a focus tends if anything to find more uncertainties of various kinds; to indicate that “Female sexuality [in the visual arts] … wasn’t.” The encounters of women, the visual arts and eros, that is, are so heterogenous and their boundaries so unclear as to make the category elusive. Of course women in the Middle Ages had sexual experiences, desires, fantasies, pleasures and pains; and of course we cannot have direct access to the experiences of the long dead, though we can converse about them. But the very nature of artistic representation, whether visual or textual, means that such desires and pleasures become shared property, which cannot be said to belong to women more than to men – or indeed to the medieval rather than to the modern. Hans Belting argues that “The human being is the natural locus of images, a living organ for images”; thus a contemporary viewer, assessing the sexual content or impact of a medieval image, must put their own bodies and sensibilities forward as substitutes for those of medieval viewers.[2] men – or indeed to the medieval rather than to the modern. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). /10 61302/IRRS2726 The Trouble with “Female Sexuality” Hans Belting argues that “The human being is the natural locus of images, a living organ for images”; thus a contemporary viewer, assessing the sexual content or impact of a medieval image, must put their own bodies and sensibilities forward as substitutes for those of medieval viewers.[2] “Sexuality” itself, of course, is a post-medieval term, which is nevertheless, with appropriate caveats, regularly used. I do not propose not using it, but would note to begin with that it seems unlikely that its range quite matches any medieval domain of knowledge. Medieval textual sources tell us a number of quite different things about female sexuality: that a woman’s desire may be directed to men, women, herself, or lifeless things; that women have an insatiable desire to be penetrated; that they are naturally inclined towards chastity; that their reluctance can be overcome by violence or seduction.[3] Visual sources are no more consistent. I might identify both a painting of St. Catherine of Siena’s stigmatisation by a crucifix (fig. 1) and a female exhibitionist figure (fig. 2) as illustrating aspects of female sexuality, but there is no evidence that these images spoke to one another in any known medieval context: their conjunction is a product of my framing category. The split between sacred and profane desires is so pronounced that the category does not cohere. 2 2 Giovanni di Paolo, Stigmatization of St Catherine of Siena, tempera and gold on wood, 1460–1465. New York, Metropolitan Museum of Art, inv. no. 1997.117.3. (Met open use policy via ArtStor) Fig. 1. Giovanni di Paolo, Stigmatization of St Catherine of Siena, tempera and gold on wood, 1460–1465. New York, Metropolitan Museum of Art, inv. no. 1997.117.3. (Met open use policy via ArtStor) 3 Fig. 2. Female exhibitionist figure, stone, date unknown, Old Parish Church, Llandrindod Wells. Llandrindod Wells, Radnorshire Museum (Photo: John Harding) Fig. 2. Female exhibitionist figure, stone, date unknown, Old Parish Church, Llandrindod Wells. Llandrindod Wells, Radnorshire Museum (Photo: John Harding) The problem of sexual identities is not quite, as it is sometimes put, that the Middle Ages preceded them. Lochrie argues persuasively that the absence of a homosexual identity means that the presumption of a heteronormative framework must be questioned.[4] But the contemporary division of homo and heterosexualities is not the only way to map desires. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” Medieval women did not lack sexual identities (although men’s are harder to pin down): as maidens, wives, professed virgins and whores their presumed sexual activity was the foundational category that Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 4 determined their dress, demeanour, social position and material location. Thanks to the work of social historians on women’s lifecycles, we know quite a lot about these identities, their local variations and their slippages. These sexual identity categories, however, do not necessarily tell us about women’s sexual pleasures, pains and desires, or the extent to which they felt at one with or alienated from such identity categories. The Book of Margery Kempe shows its protagonist at various periods of her life as a sexually satisfied young wife, who had “ful many delectabyl thowtys, fleschly lustys, and inordinate lovys” of her husband’s body, as a wife later repulsed from marital sex as from “the mukke in the chanel,” and as the Bride of a Christ who claims the right of a husband to “be homly wyth the and lyn in thi bed wyth the.”[5] But Kempe’s book, despite its seeming frankness, is not a transparent witness but a crafted tale of a penitent sinner; meanwhile other desires, such as female homoeroticism, hardly break discursive cover at all.[6] The topic of “female sexuality in medieval art” includes two overlapping fields of activity: women as sexualised addressees and consumers of art, and the sexualised representation of women in art. Both raise so many problems of identification and interpretation that further examination shows them to be almost nothing but problems. It might seem more promising to begin with female sexual subjects; that is, female spectators of sexualised art, since, as Sherry Lindquist shows in this volume, it is well-known that devout women had intense affective responses to artefacts such as crucifixes. The phenomenon of ‘sex without sex’ between chaste people might be mediated through visual art.[7] The image of St. Catherine mentioned above depicts one of the most famous of such events, showing the saint’s ardor as she opens her arms to receive the stigmata from the crucifix. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” However, I would caution that art cannot be isolated from other triggers, that sexuality in such responses cannot be isolated from other drives and affects, and that women’s responses cannot be isolated from men’s; indeed, that the shared ground of devotional eros tends to break down gender difference. For the anchoritic reader of “The Wooing of Our Lord,” the crucifix is the site of union. The text directs her to speak to Christ in these words: My body hangs with your body nailed to the cross, enclosed on all sides within four walls, and I will hang with you and never again come off my cross until I die. For then I shall leap from the cross to rest, from sorrow to joy and to eternal bliss. Oh Jesus, it is so sweet to hang with you, for when I see you hanging beside me, your great sweetness totally frees me from pain.[8] Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 5 While she spoke, she would surely fix her eyes on the crucifix in her cell and incorporate that into this fulfilment of her union with Christ: the artefact would be inextricably linked with the height of her pleasure. It is not, however, the sole cause of it: this is a multi-media experience, combining words, the artefact, visionary capacity, and probably movement, all of which are necessary constituents of the pleasure. If it is complexly visual, it is also complexly erotic. To describe such practices either as channelling sexual response into devotional purposes, or as smuggling sexuality under cover of devotion, is to overclarify. The practice is designed to produce sexual and devotional emotions together, and which was experienced as the primary response would have varied from viewer to viewer, from moment to moment. Gender, however, was not a significant factor in this variability; all the available evidence suggests that women’s response to devotional art was not markedly different from men’s. Men were if anything more often interpellated as passionate spectators of crucifixes; Rupert of Deutz’s often-cited account of the passionate kiss of a crucifix figure is one of many such encounters.[9] The illustrations of the Rothschild Canticles are some of the most joyous images of mystical eros, as the figure of the Bride cavorts with a unicorn (fol. 51r; fig. 3), or reaches up to her Heavenly Bridegroom (fol. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). /10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” 66r; fig. 4). Jeffrey Hamburger’s extensive study of the manuscript sites it in the context of feminine spirituality, arguing “[t]hat the Rothschild Canticles was intended for a woman is virtually certain.”[10] Nevertheless, as Sarah Bromberg points out, the manuscript also includes images of men as lovers of Christ, and male readers would not have been excluded from identification with the Bride: “[a] male viewer of the Rothschild Canticles might be . . . accustomed to using the visually feminized symbol of the Sponsa in order to act out his own supplication in seeking divine union.”[11] If the position of the Bride is that of female sexuality, then it is not restricted to women. 6 6 Fig. 3. Bride and unicorn, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book and Manuscript Library, MS 404, fol. 51r (Beinecke Rare Book & Manuscript Library: fair use policy) Fig. 3. Bride and unicorn, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book and Manuscript Library, MS 404, fol. 51r (Beinecke Rare Book & Manuscript Library: fair use policy) 7 7 Fig. 4. Spiritual marriage, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book and Manuscript Library, MS 404, fol. 66r (Beinecke Rare Book & Manuscript Library: fair use policy) Fig. 4. Spiritual marriage, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book and Manuscript Library, MS 404, fol. 66r (Beinecke Rare Book & Manuscript Library: fair use policy) 8 8 Such positions are formed in networks of persons, places and material objects.[12] The anchoress contemplating the crucifix in “The Wooing of our Lord” speaks a script written for her by her spiritual director, gazes at a crucifix crafted by an artisan to a model lent in a workshop, perhaps donated by a patron or relative.[13] Her cell, the site of what Michelle Sauer calls “erotic enclosure,” was a social and material space marked out by lay and clerical practices.[14] The anchoress may be the point at which art meets eros, but this meeting is enabled by the network that provides and vivifies the crucifix figure and gives the anchoress the space to contemplate it. Women’s art was never exclusively so. Books made for specific elite patrons would also have been accessible to their families and household intimates. Imagery in nunnery churches would frequently have been visible to priests and visitors. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” And some images made for women, as Madeline Caviness famously argued, might be better regarded as having been made against women: she read the sexual aspects of the illustrative programme of the Hours of Jeanne d’Evreux as a coercive interpellation of the young queen into wifely subordination, arguing that “the aggressive phallic forms … may well have frightened the adolescent Jeanne.”[15] The book produces its reader as sexual subject, but offers her no pleasures. As Caviness goes on to say, however, the intent does not fix the meaning, and we must surmise a variety of responses: “I do not propose to substitute a monolithic personal reading but to allow the work a multiplicity of affective possibilities that might be accounted for by differences of mood, life stage, and gender in the readers of this particular book; it not only may have looked different to Jeanne in her celibate widowhood, but certainly must have done so to Charles V when he inherited it.”[16] Paula Gerson confirms that women might take an active interest in sexual imagery, arguing that a woman “not unlike Chaucer’s Wife of Bath” most likely commissioned similar bawdy imagery in her Book of Hours because she liked it.[17] Gerson’s identification of female agency in this manuscript is plausible, but it is harder to be sure of the importance of specifically sexual response; the emphasis on “visual attacks on male genitalia and buttocks” suggests that aggressive impulses were mixed in with sexual ones.[18] All we can say is that sexualised imagery was addressed to women, but how it affected their fears, pleasures and desires would depend on a multitude of factors. As these examples show, images that have sexual content cannot necessarily be assumed to have stimulated sexual responses. Indeed, this seems more typically to be a negative relation. As Martha Easton has argued, “the most sexually explicit images are found in religious spaces like churches, cathedrals, and devotional manuscripts, and depending on the context, can be read as censorious rather than celebratory of eroticism and love”; and my own previous study found that “[s]exual Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 9 form, sexual content and sexual affect so frequently diverge in medieval art that they must be assessed separately.”[19] Visual images of sexuality are often more connotative than explicit. The Trouble with “Female Sexuality” The presence of a rabbit may instruct the viewer to read erotic import into an otherwise innocuous conversation scene, because the animal’s French name, “con,” puns on the word for female genitals.[20] Any sexual pleasure from contemplating the organ at this double remove would be inextricably bound up with the pleasure of successful decoding of the signifying chain.[21] Meanwhile, apparently sexual images might mean something completely different; a late fifteenth-century drawing of a couple embracing (fig. 5) is to be understood as encoding an alchemical process. Sexually explicit images such as the exhibitionist figures on churches are usually more grotesque than alluring. Many were probably intended to be anti-erotic, a warning against sexual sin; others have been argued to be fertility images or apotropaic charms.[22] form, sexual content and sexual affect so frequently diverge in medieval art that they must be assessed separately.”[19] Visual images of sexuality are often more connotative than explicit. The presence of a rabbit may instruct the viewer to read erotic import into an otherwise innocuous conversation scene, because the animal’s French name, “con,” puns on the word for female genitals.[20] Any sexual pleasure from contemplating the organ at this double remove would be inextricably bound up with the pleasure of successful decoding of the signifying chain.[21] Meanwhile, apparently sexual images might mean something completely different; a late fifteenth-century drawing of a couple embracing (fig. 5) is to be understood as encoding an alchemical process. Sexually explicit images such as the exhibitionist figures on churches are usually more grotesque than alluring. Many were probably intended to be anti-erotic, a warning against sexual sin; others have been argued to be fertility images or apotropaic charms.[22] Fig. 5. Alchemical process, Miscellaneous Treatises on Alchemy, England, 2nd half of the 15th century. London, British Library, MS Harley 2407, fol. 57v, detail (© British Library Board) Fig. 5. Alchemical process, Miscellaneous Treatises on Alchemy, England, 2nd half of the 15th century. London, British Library, MS Harley 2407, fol. 57v, detail (© British Library Board) Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 10 Towards the end of the period it becomes easier to identify images that better fit modern expectations of erotic art. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). org/10 61302/IRRS2726 The Trouble with “Female Sexuality” Paula Nuttall establishes that non-narrative images of nude women, for the viewing pleasure either of men or mixed audiences, were produced in northern art of the late fifteenth century. The major themes seem to have been voyeuristic gazing on women as they bathe, and male enthrallment to female sexual allure; they may be combined, as in the picture known as “The Love Charm” (fig. 6).[23] But this of course says nothing very directly about female sexuality itself, though it does show what kinds of representations of women’s bodies were desirable to some viewers. Viewing pleasure, however, is rarely pure. Van Eyck’s lost picture of bathing women, for example, is known only from this description of its content as: women of uncommon beauty emerging from the bath, the more intimate parts of the body being with excellent modesty veiled in fine linen, and of one of them he has shown only the face and breast but has then represented the hind parts of her body in a mirror painted on the wall opposite, so you may see her back as well as her breast. In the same picture there is a lantern in the bath chamber, just like one lit, and an old woman seemingly sweating, a puppy lapping up water, and also horses, minute figures of men, mountains, groves, hamlets and castles, carried out with such skill you would believe one was fifty miles distant from another.[24] women of uncommon beauty emerging from the bath, the more intimate parts of the body being with excellent modesty veiled in fine linen, and of one of them he has shown only the face and breast but has then represented the hind parts of her body in a mirror painted on the wall opposite, so you may see her back as well as her breast. In the same picture there is a lantern in the bath chamber, just like one lit, and an old woman seemingly sweating, a puppy lapping up water, and also horses, minute figures of men, mountains, groves, hamlets and castles, carried out with such skill you would believe one was fifty miles distant from another.[24] 11 11 Fig. 6. The Love Charm, oil on panel, Germany or Lower Rhine, 1470–1480. Leipzig, Museum der Bildenden Künste (Public domain image) Fig. 6. The Love Charm, oil on panel, Germany or Lower Rhine, 1470–1480. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” Leipzig, Museum der Bildenden Künste (Public domain image) 12 Fig. 7. Vulva figure depicted as a pilgrim with pilgrim’s staff and rosary, lead tin, Arnemuiden, the Netherlands, 1425–1475. Family Van Beuningen, Langbroek, Inv.nr. 4462 [cat. HP3, afb. 3026] (photo: Medieval Badges Foundation) Fig. 7. Vulva figure depicted as a pilgrim with pilgrim’s staff and rosary, lead tin, Arnemuiden, the Netherlands, 1425–1475. Family Van Beuningen, Langbroek, Inv.nr. 4462 [cat. HP3, afb. 3026] (photo: Medieval Badges Foundation) This account suggests that enjoyment of this picture’s painterly playfulness and verisimilitude was inseparable from enjoyment of its images of female flesh. It rehearses a process of viewing that registers bodies, textures, surfaces, light and shade as problems of representation to which an elegant solution has been found. A patron’s pleasure in such art would surely also include an element of pride in the ownership of such a prestigious and innovative piece. Sexuality is inextricably bound up with other pleasures. I would argue that sexuality is not absent from medieval art, but that there are reasons why it is hard to find. In a variety of genres, media and motifs, sexuality is shown to be impersonal: disembodied, dispersed, alienated and alienating. It is not securely attached to gendered bodies or gendered subjectivities; it continually exceeds, attacks or distorts the entity that experiences it. One example of such a Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 13 representation is the corpus of sexual badges, featuring male and/or female sexual organs, usually in motion. The meanings of the badges are obscure, though they are plainly genital in form, and most critics agree that they are not erotically enticing. One of the best-known, the pilgrim-vulva (fig. 7), has been interpreted as a misogynist satire on women pilgrims.[25] Drawing on the deep-rooted division between the good woman in her household and the bad woman wandering the world, these badges say that a female pilgrim, identifiable here by her staff, hat and rosary, is nothing but a vulva on legs. To depict a person in this reductive fashion, as only a gaping organ, is to register desire as a force that unbalances the self, makes one nothing but sexuality. The same goes for the exhibitionist images on churches, also depicting both male and female organs, which share some forms with the badges. The Trouble with “Female Sexuality” Such an image, then, might convey the subjective experience of living in a sexed body; it is an image of the perceptual distortions of desire, working on similar principles to the modern image of the sensory homunculus (fig. 8), which maps the body according to the sensitivity of each part, producing a representation more truthful to the subjective experience of being in the body than to its outward appearance. To recognise oneself depicted in such an image, repellent and reductive as it is, would be a profoundly alienating experience in which identification would alternate with disavowal. representation is the corpus of sexual badges, featuring male and/or female sexual organs, usually in motion. The meanings of the badges are obscure, though they are plainly genital in form, and most critics agree that they are not erotically enticing. One of the best-known, the pilgrim-vulva (fig. 7), has been interpreted as a misogynist satire on women pilgrims.[25] Drawing on the deep-rooted division between the good woman in her household and the bad woman wandering the world, these badges say that a female pilgrim, identifiable here by her staff, hat and rosary, is nothing but a vulva on legs. To depict a person in this reductive fashion, as only a gaping organ, is to register desire as a force that unbalances the self, makes one nothing but sexuality. The same goes for the exhibitionist images on churches, also depicting both male and female organs, which share some forms with the badges. Such an image, then, might convey the subjective experience of living in a sexed body; it is an image of the perceptual distortions of desire, working on similar principles to the modern image of the sensory homunculus (fig. 8), which maps the body according to the sensitivity of each part, producing a representation more truthful to the subjective experience of being in the body than to its outward appearance. To recognise oneself depicted in such an image, repellent and reductive as it is, would be a profoundly alienating experience in which identification would alternate with disavowal. Fig. 8. Sensory Homunculus. This is all over the web with no obvious ownership. Fig. 8. Sensory Homunculus. This is all over the web with no obvious ownership. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 14 Fig. 9. Adam and Eve, Cædmon Manuscript, England, c. 1000. Bodleian Library, Oxford, MS Junius 11, p. 11 (© Oxford, Bodleian Library) Fig. 9. Adam and Eve, Cædmon Manuscript, England, c. 1000. Bodleian Library, Oxford, MS Junius 11, p. 11 (© Oxford, Bodleian Library) Anne Marie Rasmussen points out how often the genital badges are images of mobility: they walk, they ride, they fly, and some even crew ships. She connects them with comic stories about genitalia which leave their owners to wander the world: a penis attempts to hide in a nunnery, or a stray vagina is mistaken for a toad.[26] The Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 15 sexual organ, that is, is detachable from the person and from gender: it is imagined as a force which wanders the world under its own steam. And the badges themselves, of course, are mobile, in that, pinned to people’s clothing, they moved around the world, their meanings no doubt shifting as they appeared in different material and social spaces. This means that they may well not be firmly gendered: we have no information at all about who wore them and when, but there is no reason to suppose that they had to be gender-appropriate to their wearers. If “female sexuality” is located in a badge of a vagina-pilgrim, then female sexuality is a quality which is mobile, detachable, not owned by female persons. A motif such as the phallus tree, shared by badges and other media, has a similar concept of sexuality as something which is out there in the world, only temporarily appropriated by individuals, something which one might pick off a tree. Such images show coherent personhood and sexuality to be mutually exclusive, and desire as a disruptive force, a perception that is quite consistent with the Christian theorisation of the condition of post-lapsarian humanity. As St Augustine explained the effects of the Fall: “They experienced an unprecedented movement of their own disobedient flesh as punishment in kind, as it were, for their own disobedience. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” The soul, in fact, delighting now in its own freedom to do wickedness and scorning to serve God, was stripped of the former subjection of the body, and because it had wilfully deserted its own higher master, no longer kept its lower servant subject to its will.”[27] The experience of desire is an experience of splitting: body and soul, God and humanity, are recognised as distinct through the operation of desire. A human subject experiencing desire is registering its fallenness, its distance from its prelapsarian perfection, its non-self-identity. One does not have a sexuality because one is not one in these conditions, but a disordered collection of jostling entities. The prelapsarian Adam and Eve in the Anglo-Saxon Junius MS (fig. 9) anticipate this moment at which sexual awakening is registered at the cost of personal disintegration. They stand fully naked and barely distinguishable, with smooth groins, but their sexual parts are externalised to the genital-like forms of the plants that each grasps firmly. In the visual arts, this idea is not, however, limited to the theological or moral register, but is expressed also in popular and comic art and performance. Images of sexuality informed by such perspectives are likely to be uncomfortably placed in the category of “female sexuality,” both because this Augustinian model imagines female desire to be exactly analogous to male desire, and because sexuality is so disruptive of the subject that no-one can lay claim to one of their own. In such images and texts anything we might want to call “female sexuality” is already fraught, complicated and disrupted; there is no coherent package of desires, behaviours and relations that belongs to female subjects. 16 References References 1 Karma Lochrie, Heterosyncracies: Female Sexuality when Normal Wasn’t (Minneapolis: University of Minnesota Press, 2005), xv. 2 Hans Belting, An Anthropology of Images: Picture, Medium, Body, trans. Thomas Dunlap (Princeton: Princeton University Press, 2011), 37. https://doi.org/10.1515/9781400839780 3 Ancrene Wisse: A Corrected Edition of the Text in Cambridge, Corpus Christi College, MS 402, ed. Bella Millett, Early English Text Society, Original Series 325 (Oxford: Oxford University Press, 2005), 121; Ruth Mazo Karras, Sexuality in Medieval Europe: Doing unto Others (New York: Routledge, 2005), 79; Christine de Pizan, The Book of the City of Ladies, trans. Rosalind Brown-Grant (Harmondsworth: Penguin, 1999), 144; Kathryn Gravdal, Ravishing Maidens: Writing Rape in Old French Literature and Law (Philadelphia: University of Pennsylvania Press, 1991), 111. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” https://doi.org/10.9783/9780812200331 4 Lochrie, Heterosyncracies, xiv-xv. 5 The Book of Margery Kempe, ed. Barry Windeatt (Cambridge: D.S. Brewer, 2000), 332, 62, 196. 6 Jacqueline Murray, “Twice Marginal and Twice Invisible: Lesbians in the Middle Ages,” in Handbook of Medieval Sexuality, ed. Vern L. Bullough and James A. Brundage (New York: Garland, 2000), 191-222. 7 “Sex without sex” is the theme of a special issue of the Journal of the History of Sexuality 19, no. 1 (2010), ed. Sally N. Vaughn and Christina Christoforatou. 8 Þe Wohunge of ure Lauerd, ed. W. Meredith Thompson, EETS Original Series 241 (London: Oxford University Press, 1958), ll. 590-602; my translation. 9 Sara Lipton, “‘The Sweet Lean of his Head’: Writing about Looking at the Crucifix in the High Middle Ages,” Speculum 80, no. 4 (2005): 1172-1208. https://doi.org/10.1017/S003871340000138X 10 Jeffrey F. Hamburger, The Rothschild Canticles: Art and Mysticism in Flanders and the Rhineland circa 1300 (New Haven: Yale University Press, 1990), 3. 11 Sarah Bromberg, “Gendered and Ungendered Readings of the Rothschild Canticles,” Different Visions: A Journal of New Perspectives on Medieval Art 1 (2008), https://doi.org/10.61302/PMSR4045 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 17 12 The concept of network here is indebted to Bruno Latour, Reassembling the Social: An Introduction to Actor-Network Theory (Oxford: Oxford University Press, 2007). 13 I have previously discussed the scripting of the meditation in Sarah Salih, “Transvestism in the Anchorhold,” in The Milieu and Context of the Wohunge Group, ed. Susannah M. Chewning (Cardiff: University of Wales Press, 2009), 148-64. 14 Michelle Sauer, “Uncovering Difference: Encoded Homoerotic Anxiety within the Christian Eremetic Tradition in Medieval England,” Journal of the History of Sexuality 19, no. 1 (2010), 133-52, at 143. https://doi.org/10.1353/sex.0.0075 15 Madeline H. Caviness, “Patron or Matron? A Capetian Bride and a Vade Mecum for Her Marriage Bed,”Speculum 68, no. 2 (1993), 333-62, at 355. https://doi.org/10.2307/2864556 16 Caviness, “Patron or Matron,” 356. 17 Paula Gerson, “Margins for Eros,” Romance Languages Annual 5 (1993): 47-53, at 50. 18 Gerson, “Margins for Eros,” 52. 19 Martha Easton, “‘Was it Good for You Too?’ Medieval Erotic Art and it Audiences,” Different Visions: A Journal of New Perspectives on Medieval Art 1 (2008), https://doi.org/10.61302/BUIO3522, 14; Sarah Salih, “Erotica,” in A Cultural History of Sexuality in the Middle Ages,ed. Ruth Evans(Oxford: Berg, 2010), 181-212, 186. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” https://doi.org/10.5040/9781350049673-ch-009 20 Michael Camille, The Medieval Art of Love: Objects and Subjects of Desire (New York: Harry N. Abrams, 1998), 96. 21 A point previously made in Salih, “Erotica,” 189. 22 Anthony Weir and James Jerman, Images of Lust: Sexual Carvings on Medieval Churches (London: Routledge, 1999); Marian Bleeke, “Sheelas, Sex, and Significance in Romanesque Sculpture: The Kilpeck Corbel Series,” Studies in Iconography 26 (2005): 1-26; Eamonn Kelly, “Irish Sheela-na-gigs and Related Figures with Reference to the Collections of the National Museums of Ireland,” in Medieval Obscenities, ed. Nicola McDonald (Woodbridge: York Medieval Press, 2006), 124-37. 18 23 Paula Nuttall, “Reconsidering the Nude: Northern Tradition and Venetian Invention,” The Meanings of Nudity in Medieval Art, ed. Sherry C.M. Lindquist (Farnham, UK; Burlington, VT: Ashgate, 2012), 299-318. https://doi.org/10.4324/9781315085975-12 24 Quoted in Keith Christiansen, “The View from Italy,” in From Van Eyck to Bruegel: Early Netherlandish Painting in the Metropolitan Museum of Art, ed. Maryan Wynn Ainsworth and Keith Christiansen (New York: Metropolitan Museum of Art, 1998), 39-62, 39. 25 Malcolm Jones, The Secret Middle Ages: Discovering the Real Medieval World (Stroud: Sutton, 2002), 256. 26 Anne Marie Rasmussen, Wandering Genitalia: Sexuality and the Body in German Culture between the Late Middle Ages and Early Modernity (London: King’s College London Medieval Studies, 2009), 8-9. 27 Augustine of Hippo, The City of God Against the Pagans, 7 vols, trans. George E. McCracken, Philip Levine, William M. Green, Loeb Classical Library (London: Heinemann; Cambridge Massachusetts: Harvard University Press, 1966), Book 13, ch. 13. 23 19
https://openalex.org/W1992808911
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Toxicological Study of<i>Ocimum sanctum</i>Linn Leaves: Hematological, Biochemical, and Histopathological Studies
Journal of toxicology
2,014
cc-by
5,807
Hindawi Publishing Corporation Journal of Toxicology Volume 2014, Article ID 135654, 9 pages http://dx.doi.org/10.1155/2014/135654 Hindawi Publishing Corporation Journal of Toxicology Volume 2014, Article ID 135654, 9 pages http://dx.doi.org/10.1155/2014/135654 Hindawi Publishing Corporation Journal of Toxicology Volume 2014, Article ID 135654, 9 pages http://dx.doi.org/10.1155/2014/135654 Hindawi Publishing Corporation Journal of Toxicology Volume 2014, Article ID 135654, 9 pages http://dx.doi.org/10.1155/2014/135654 M. K. Gautam and R. K. Goel Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India Correspondence should be addressed to R. K. Goel; rkgoelbhu50@gmail.com Received 31 July 2013; Revised 28 November 2013; Accepted 28 November 2013; Published 29 January 2014 Academic Editor: Lucio Guido Costa Copyright © 2014 M. K. Gautam and R. K. Goel. This is an open access article distributed under the Creative Commons Attr License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properl Copyright © 2014 M. K. Gautam and R. K. Goel. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The present study was aimed to study the acute and subacute toxicity studies with orally administered 50% ethanolic leaves extract of Ocimum sanctum Linn (OSE). In acute toxicity tests, four groups of mice (𝑛= 6/group/sex) were orally treated with doses of 200, 600, and 2000 mg/kg, and general behavior, adverse effects, and mortality were recorded for up to 14 days. In subacute toxicity study, rats received OSE by gavage at the doses of 200, 400, and 800 mg/kg/day (𝑛= 6/group/sex) for 28 days, and biochemical, hematological, and histopathological changes in tissues (liver, kidney, spleen, heart, and testis/ovary) were determined. OSE did not produce any hazardous symptoms or death and CNS and ANS toxicities in the acute toxicity test. Subacute treatment with OSE did not show any change in body weight, food and water consumption, and hematological and biochemical profiles. In addition, no change was observed both in macroscopic and microscopic aspects of vital organs in rats. Our result showed that Ocimum sanctum extract could be safe for human use. 1. Introduction Ocimum sanctum Linn (Labiatae), known as holy basil, is a commonly used home remedy and has been advocated for various ailments like cold, fever, dysentery, hemorrhage and dyspepsia, glucoma, cataract, chronic conjunctivitis, and other painful eye diseases, as well as gastric and hepatic disorders in indigenous system of medicine [5]. The plant is endowed with a variety of pharmacological properties including antistress, antifertility, immunoregu- latory, hypoglycemic, antibacterial, antifungal, antiinflam- matory, anti-carcinogenic, antioxidant, and cyclooxygenase inhibitory [6]. OS has been reported to show gastroduo- denal ulcer protective, antisecretory, and gastric mucosal defense enhancing activities [7]. The leaves of O. sanctum (OS) contain a volatile oil composed of limonene, bor- neol, copaene, caryophyllene, and elemol, phenolic com- pounds (rosmarinic acid, apigenin, cirsimaritin, and isothy- musin), flavonoids (orientin and vicenin), and aromatic compounds (methyl chavicol and methyl eugenol) [6]. Eugenol forms the major active constituent of OS, even though other minor constituents like fixed oils and flavones have also been reported to have pharmacological activities [5]. Plants have always been an important source of drugs. A large number of the world’s populations, especially in developing countries, depend upon medicinal plants as an alternative and complimentary drugs therapy for various ailments. Some of the most common practices involve the use of crude plant extracts, which may contain a broad diversity of molecules with often unknown biological effects [1]. Since the medicinal plants are being used indiscriminately without notifying to their possible unhealthy or toxic effects, the World Health Organization has recommended that traditional plants used for the treatment of diseases need further scientific investiga- tion on their toxic side effects [2]. Plants produce bioactive compounds which act as defense mechanisms against any disease, and at the same time, may be toxic in nature [3]. However, the general acceptability of herbal medicines has been limited by a lack of defined chemical characterization, dose regimen, and adequate toxicity data to evaluate their safety [4]. Therefore, it has become essential to assess the safety of plants used for medicinal purposes for possible toxicity. 2 Journal of Toxicology 2 Journal of Toxicology (a) (b) (c) (d) Figure 1: Histology of liver (H&E, 100x) of control and OSE-treated animals. (a) Section of liver from control animals revealed normal architecture and hepatic cells with granulated cytoplasm; ((b), (c), and (d)) liver from OSE (200, 400, and 800 mg/kg)-treated animals exhibited normal architecture of hepatocytes and hepatic cells with granulated cytoplasm. 1. Introduction (a) (b) (b) (a) (d) (c) (d) (c) Figure 1: Histology of liver (H&E, 100x) of control and OSE-treated animals. (a) Section of liver from control animals revealed normal architecture and hepatic cells with granulated cytoplasm; ((b), (c), and (d)) liver from OSE (200, 400, and 800 mg/kg)-treated animals exhibited normal architecture of hepatocytes and hepatic cells with granulated cytoplasm. As to the best of our knowledge, there is no reference about the safe dosage of Ocimum sanctum Linn in traditional medicine so it was thought worthwhile to do the acute toxicity (mortality and CNS/ANS toxicities) in mice and subacute toxicity (biochemical, hematological, and histopathological) in rats. Hindu University) were collected during October-December and identified with the standard sample preserved in the Department of Dravyaguna, Institute of Medical Sciences, Banaras Hindu University, Varanasi. 50% ethanolic extract of OS (OSE) was prepared by adding 500 g of dried, crushed, and powdered leaves of OS in 1000 mL of 50% ethanol in a round bottom flask and was kept at room temperature for 3 days in shade. The extract was filtered and the above procedure was repeated twice. The extract filtrate so obtained was pooled and evaporated on water bath till it dried. The yield of OSE was about 5.00% (w/w). 2.2. Plant Material and Preparation of Extract. The leaves of Ocimum sanctum (OS) (Ayurvedic Gardens, Banaras 2. Materials and Methods 2.1. Experimental Animals. Inbred Charles-Foster albino rats (160–180 g) and Swiss albino mice (20–25 g) of either sex were obtained from the central animal house of the Institute of Medical Sciences, Banaras Hindu University, Varanasi. They were kept in the departmental animal house at 26 ± 20∘C and relative humidity of 44–56%, with light and dark cycles of 10 and 14 h, respectively, for one week before and during the experiments. Animals were provided with standard rodent pellet diet (Pashu Aahar Vihar, Ramnagar, Varanasi) and water ad libitum. “Principles of laboratory animal care” (NIH publication number 82-23, revised 1985) guidelines were followed. Approval from the Central Animal Ethical Committee of the University was taken prior to the experimental work (Notification number-Dean/2010-11/173 dated 23.07.2010). 2.3. Acute Toxicity Study. Adult Swiss albino mice of either sex, weighing between 20 and 25 g, fasted overnight and were used for acute toxicity study, as per the Organization for Economic Co-Operation and Development (OECD 423) guideline [8]. Four groups of mice (𝑛= 6) of both sexes was fasted overnight. The first control group mice received 0.5% carboxymethyl cellulose (CMC) suspension in distilled water while the other three groups received OSE suspended in 0.5% CMC at doses of 200, 600, and 2000 mg/kg. The above doses were selected on the basis of our previous reported work on OSE where 400 mg/kg was found to have good ulcer protective effects [7]. Animals were observed closely for first 4 hours, for any toxicity manifestation, like increased motor activity, salivation, convulsion, coma, and death. Sub- sequently observations were made at regular intervals for 2.3. Acute Toxicity Study. Adult Swiss albino mice of either sex, weighing between 20 and 25 g, fasted overnight and were used for acute toxicity study, as per the Organization for Economic Co-Operation and Development (OECD 423) guideline [8]. Four groups of mice (𝑛= 6) of both sexes was fasted overnight. The first control group mice received 0.5% carboxymethyl cellulose (CMC) suspension in distilled water while the other three groups received OSE suspended in 0.5% CMC at doses of 200, 600, and 2000 mg/kg. The above doses were selected on the basis of our previous reported work on OSE where 400 mg/kg was found to have good ulcer protective effects [7]. Animals were observed closely for first 4 hours, for any toxicity manifestation, like increased motor activity, salivation, convulsion, coma, and death. 2. Materials and Methods Sub- sequently observations were made at regular intervals for 2.2. Plant Material and Preparation of Extract. The leaves of Ocimum sanctum (OS) (Ayurvedic Gardens, Banaras 3 3 Journal of Toxicology (a) (b) (c) (d) Figure 2: Histology of kidney (H&E, 100x) of control and OSE-treated animals. (a) Section of kidney from control animal showed normal size of glomeruli with normal tubules; ((b), (c), and (d)) kidney from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal size of glomeruli with normal tubules. (b) (a) (a) (b) (d) (c) (d) (c) Figure 2: Histology of kidney (H&E, 100x) of control and OSE-treated animals. (a) Section of kidney from control animal showed normal size of glomeruli with normal tubules; ((b), (c), and (d)) kidney from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal size of glomeruli with normal tubules. 24 h. The animals were under further investigation up to a period of 14 days and the number of mice that died within the study period was noted [9]. 2.4.1. Haematological Parameters. Haemoglobin, total leuko- cyte count, and differential leukocyte count (polymorph, lymphocyte, and eosinophil) [11] were determined in control and OSE-treated groups. 2.4. Subacute Toxicity Study. The repeated doses (28 days) for oral toxicity studies were carried out in rats according to the OECD test guideline 407 [10]. Rats were divided randomly into 4 groups of 6 animals each (3 males and 3 females). After an overnight fast, animals in group I received 0.5% CMC suspension (control group), whereas rats in groups II, III, and IV (test groups) received OSE suspended in 0.5% CMC at the doses of 200, 400, and 800 mg/kg body weight, respectively. Doses of OSE and CMC were administered daily by oral gavage in the volume of 10 mL/kg body weight, once daily for 28 consecutive days. The animals were observed daily for any abnormal clinical signs and death during the study period. Body weights were measured and recorded at the beginning and then after every week of the experiment. At the end of the study, all animals fasted overnight (water ad libitum) and, on 29th day, the animals were weighed. Blood was collected from retroorbital technique with or without EDTA for haematological and biochemical analysis, respectively. The animals were sacrificed with an overdose of ether and other body organs were taken out for detailed weight and histopathological changes. 2.4.2. Biochemical Estimations. 2. Materials and Methods The serum was carefully aspi- rated with a Pasteur pipette into sample bottles for the various biochemical assays. Assay kits (Span diagnostic reagent kit and Precichem diagnostic kit) were employed for aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphate (ALP), creatinine, blood glucose, total protein, and total cholesterol and bilirubin analysis was determined in the serum following the procedure described in the kits. 2.4.2. Biochemical Estimations. The serum was carefully aspi- rated with a Pasteur pipette into sample bottles for the various biochemical assays. Assay kits (Span diagnostic reagent kit and Precichem diagnostic kit) were employed for aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphate (ALP), creatinine, blood glucose, total protein, and total cholesterol and bilirubin analysis was determined in the serum following the procedure described in the kits. 2.4.3. Organs Weight and Histology. The rats were quickly dis- sected and the liver, kidneys, stomach, spleen, lung, heart, and testis or ovary were excised and weighed. The specimens for histopathology were fixed in 10% neutral, buffered formalin for 18 h at 4∘C. 3-4 𝜇m in thickness of each specimen of liver, kidney, heart, spleen, and testis/ovary was cut and stained with hematoxylin and eosin stain following the standard laboratory procedures. The stained sections were examined under microscope for any cellular damage or change in morphology of that particular tissue. Journal of Toxicology (a) (b) (c) (d) Figure 3: Histology of heart (H&E, 100x) of control and OSE-treated animals. (a) Section of heart from control animal showed normal muscle fibers with acidophilic cytoplasm and centrally located nuclei; ((b), (c), and (d)) heart from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal muscle fibers with acidophilic cytoplasm and centrally located nuclei. (b) (a) (a) (b) (d) (c) (d) (c) Figure 3: Histology of heart (H&E, 100x) of control and OSE-treated animals. (a) Section of heart from control animal showed normal muscle fibers with acidophilic cytoplasm and centrally located nuclei; ((b), (c), and (d)) heart from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal muscle fibers with acidophilic cytoplasm and centrally located nuclei. control group after 28 days of study period in rats (Tables 1 and 2). 2.5. Statistical Analysis. Statistical comparison was per- formed using one way analysis of variance (ANOVA) fol- lowed by Dunnett’s test for multiple comparisons. All statisti- cal analysis was performed using SPSS statistical version 16.0 software package (SPSS Inc., USA). 3.2.3. Hematological Studies. 2. Materials and Methods Hematological parameters like mean haemoglobin content, WBC, RBC, and differential cell counts were not significantly different with OSE-treated rats from control group (Table 3). 3. Result 3.1. Acute Toxicity Study. The limit dose of 2000 mg/kg did not cause death or any toxic signs in treated male and female mice. All six mice were normal throughout the study and survived until the end of the 14-day experiment period. 3.2.4. Biochemical Analysis. Biochemical parameters for liver and kidney function test like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatinine, albumin, blood glucose, total protein, total choles- terol, and bilirubin did not show any difference with the above doses of OSE compared to control group (Table 3). 3.2. Subacute Toxicity Study OSE 400 160.6 ± 5.38 162.5 ± 4.22 171.2 ± 6.39 188.8 ± 3.75 206.3 ± 5.95 222.6 ± 5.21 OSE 800 163.1 ± 5.58 166.3 ± 4.97 173.8 ± 3.75 190.3 ± 3.71 204.4 ± 3.46 223.8 ± 2.45 Values are expressed as mean ± SEM of 6 rats in each group. (a) (b) (c) (d) Figure 4: Histology of spleen (H&E, 100x) of control and OSE-treated animals. (a) Section of spleen from control animal showed normal granular hemosiderin pigment predominantly within macrophages in the red pulp; ((b), (c), and (d)) spleen from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal hemosiderin pigment predominantly within macrophages in the red pulp with normal structure. (b) (a) (b) (a) (d) (c) (d) (c) Figure 4: Histology of spleen (H&E, 100x) of control and OSE-treated animals. (a) Section of spleen from control animal showed normal granular hemosiderin pigment predominantly within macrophages in the red pulp; ((b), (c), and (d)) spleen from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal hemosiderin pigment predominantly within macrophages in the red pulp with normal structure. 3.2. Subacute Toxicity Study 3.2.1. General Observations. Oral administration of OSE at doses of 200, 400, or 800 mg/kg body weight daily for 28 day, did not produce any mortality. All the treated and control mice were normal throughout the study. The animals did not show any changes in general behavior or other physiological activities. 3.2.5. Organs Weight and Histology. The organs like liver, kidney, heart, spleen, and testis or ovary isolated in various groups did not reveal any abnormalities in their gross exam- inations and difference in their mean weights both in treated and control groups (Table 4). The histological studies with liver, spleen, kidney, heart, and testis/ovary did not reveal any pathological changes after treatment even with higher dose of 800 mg dose of OSE when administered for 28 days (Figures 1, 2, 3, 4, 5, and 6). 3.2.5. Organs Weight and Histology. The organs like liver, kidney, heart, spleen, and testis or ovary isolated in various groups did not reveal any abnormalities in their gross exam- inations and difference in their mean weights both in treated and control groups (Table 4). The histological studies with liver, spleen, kidney, heart, and testis/ovary did not reveal any pathological changes after treatment even with higher dose of 800 mg dose of OSE when administered for 28 days (Figures 1, 2, 3, 4, 5, and 6). 3.2.2. Physical Parameters. Little or no change was observed in body weight, food consumption, and water intake in OSE (200, 400, and 800 mg/kg)-treated groups compared with 5 Journal of Toxicology Table 1: Effect on body weight after 28 days oral administration of OSE. Table 1: Effect on body weight after 28 days oral administration of OSE. ft Oral treatment (mg/kg, od) Body weight (g) 0 day 3 days 7 days 14 days 21 days 28 days Control 0.5% CMC 162.5 ± 6.34 166.3 ± 5.32 173.4 ± 5.46 185.0 ± 5.09 199.4 ± 5.78 215.0 ± 5.00 OSE 200 164.4 ± 6.01 165.0 ± 6.12 171.3 ± 4.20 186.9 ± 3.53 203.1 ± 7.49 220.0 ± 4.72 OSE 400 160.6 ± 5.38 162.5 ± 4.22 171.2 ± 6.39 188.8 ± 3.75 206.3 ± 5.95 222.6 ± 5.21 OSE 800 163.1 ± 5.58 166.3 ± 4.97 173.8 ± 3.75 190.3 ± 3.71 204.4 ± 3.46 223.8 ± 2.45 Values are expressed as mean ± SEM of 6 rats in each group. 4. Discussion and safe in mice. Subacute toxicity study in rats with 200, 400, and 800 mg/kg of OSE when administered for 28 days did not produce any mortality, change in food and water intake, body and organ weights, or histopathological changes in the organs like liver, kidney, spleen, heart, testis or ovary further strengthen the safety profile of OSE. The administration of herbal preparations without any stan- dard dosage, coupled with a scarcity of adequate scientific studies on their safety, has raised concerns regarding their toxicity [12]. Recently we reported teratological effects of Asparagus racemosus in rats which have been advocated in indigenous system of medicine during pregnancy and lactation [13], which thus indicated that herbal drugs are not safe as thought otherwise. To the best of our knowledge, there are no published studies on Ocimum sanctum toxicological profile following subacute exposure. In oral acute toxicity study, as high dose of OSE at 2000 mg/kg did not show any observable toxic effects in the mice in terms of any deaths or abnormal symptoms which points to its being nontoxic i Subacute studies in rats did not show any change in hematological, liver functions and spleen with 800 mg/kg OSE when administered for 28 days. The hematopoietic system/bone marrow is one of the most sensitive targets for toxic compounds and an important index of physiological and pathological status in man and animal [14]. Analysis of blood parameters is relevant to risk evaluation as the changes in the hematological system have a higher predictive value for human toxicity, when the data are translated from animal 6 Journal of Toxicology 6 Table 2: Effect on food intake and water intake after 28 days of the oral administration of OSE. 4. Discussion Oral treatment (mg/kg, od) Food intake (g/day) Water intake (mL/day) 0 day 3 days 7 days 14 days 21 days 28 days 0 day 3 days 7 days 14 days 21 days 28 days Control 0.5% CMC 11.4 ± 0.70 11.63 ± 0.55 12.2 ± 0.61 12.9 ± 0.66 13.9 ± 0.79 15.2 ± 0.90 12.3 ± 0.46 12.5 ± 0.62 13.1 ± 0.74 14.2 ± 0.63 14.6 ± 0.76 15.5 ± 0.99 OSE 200 11.5 ± 0.77 12.2 ± 0.69 12.7 ± 0.59 13.8 ± 0.76 14.5 ± 0.67 15.6 ± 0.82 13.1 ± 0.5 13.7 ± 0.58 14.1 ± 0.76 14.9 ± 0.74 16.0 ± 0.85 16.4 ± 0.91 OSE 400 11.6 ± 0.63 12.1 ± 0.49 13.0 ± 0.57 13.7 ± 0.65 14.5 ± 0.78 15.9 ± 0.86 12.9 ± 0.55 13.7 ± 0.65 14.1 ± 0.67 15.2 ± 0.77 16.1 ± 0.91 17.2 ± 0.98 OSE 800 10.9 ± 0.27 11.4 ± 0.44 13.1 ± 0.77 13.3 ± 0.99 14.5 ± 0.94 15.2 ± 1.10 13.3 ± 0.53 13.6 ± 0.60 14.3 ± 0.72 15.2 ± 0.76 16.1 ± 0.83 16.1 ± 0.95 Values are expressed as mean ± SEM of 6 rats in each group. Table 2: Effect on food intake and water intake after 28 days of the oral administration of OSE. 4. Discussion Oral treatment (mg/kg, od) Food intake (g/day) Water intake (mL/day) 0 day 3 days 7 days 14 days 21 days 28 days 0 day 3 days 7 days 14 days 21 days 28 days Control 0.5% CMC 11.4 ± 0.70 11.63 ± 0.55 12.2 ± 0.61 12.9 ± 0.66 13.9 ± 0.79 15.2 ± 0.90 12.3 ± 0.46 12.5 ± 0.62 13.1 ± 0.74 14.2 ± 0.63 14.6 ± 0.76 15.5 ± 0.99 OSE 200 11.5 ± 0.77 12.2 ± 0.69 12.7 ± 0.59 13.8 ± 0.76 14.5 ± 0.67 15.6 ± 0.82 13.1 ± 0.5 13.7 ± 0.58 14.1 ± 0.76 14.9 ± 0.74 16.0 ± 0.85 16.4 ± 0.91 OSE 400 11.6 ± 0.63 12.1 ± 0.49 13.0 ± 0.57 13.7 ± 0.65 14.5 ± 0.78 15.9 ± 0.86 12.9 ± 0.55 13.7 ± 0.65 14.1 ± 0.67 15.2 ± 0.77 16.1 ± 0.91 17.2 ± 0.98 OSE 800 10.9 ± 0.27 11.4 ± 0.44 13.1 ± 0.77 13.3 ± 0.99 14.5 ± 0.94 15.2 ± 1.10 13.3 ± 0.53 13.6 ± 0.60 14.3 ± 0.72 15.2 ± 0.76 16.1 ± 0.83 16.1 ± 0.95 Values are expressed as mean ± SEM of 6 rats in each group. Table 2: Effect on food intake and water intake after 28 days of the oral administration of OSE. 4. Discussion Oral treatment (mg/kg, od) Food intake (g/day) Water intake (mL/day) 0 day 3 days 7 days 14 days 21 days 28 days 0 day 3 days 7 days 14 days 21 days 28 days Control 0.5% CMC 11.4 ± 0.70 11.63 ± 0.55 12.2 ± 0.61 12.9 ± 0.66 13.9 ± 0.79 15.2 ± 0.90 12.3 ± 0.46 12.5 ± 0.62 13.1 ± 0.74 14.2 ± 0.63 14.6 ± 0.76 15.5 ± 0.99 OSE 200 11.5 ± 0.77 12.2 ± 0.69 12.7 ± 0.59 13.8 ± 0.76 14.5 ± 0.67 15.6 ± 0.82 13.1 ± 0.5 13.7 ± 0.58 14.1 ± 0.76 14.9 ± 0.74 16.0 ± 0.85 16.4 ± 0.91 OSE 400 11.6 ± 0.63 12.1 ± 0.49 13.0 ± 0.57 13.7 ± 0.65 14.5 ± 0.78 15.9 ± 0.86 12.9 ± 0.55 13.7 ± 0.65 14.1 ± 0.67 15.2 ± 0.77 16.1 ± 0.91 17.2 ± 0.98 OSE 800 10.9 ± 0.27 11.4 ± 0.44 13.1 ± 0.77 13.3 ± 0.99 14.5 ± 0.94 15.2 ± 1.10 13.3 ± 0.53 13.6 ± 0.60 14.3 ± 0.72 15.2 ± 0.76 16.1 ± 0.83 16.1 ± 0.95 Values are expressed as mean ± SEM of 6 rats in each group 7 Journal of Toxicology 7 Table 3: Effect on hematological and biochemical parameters after 28 days of the oral administration of OSE. 4. Discussion Parameters Control group OSE 200 mg/kg OSE 400 mg/kg OSE 800 mg/kg RBC (million/mm3) 9.2 ± 1.1 9.1 ± 1.1 8.9 ± 0.8 9.1 ± 1.0 Hb (g/dL) 12.2 ± 1.2 12.2 ± 1.1 12.3 ± 0.7 12.1 ± 0.9 WBC (million/mm3) 8.8 ± 0.9 8.7 ± 1.2 8.9 ± 1.4 9.0 ± 1.1 Neutrophils % 22.35 ± 6.3 23.38 ± 9.3 22.58 ± 5.4 23.51 ± 8.7 Eosinophils % 2.37 ± 1.0 2.34 ± 1.3 2.82 ± 1.1 2.47 ± 1.4 Basophils % 0.18 ± 0.06 0.18 ± 0.08 0.15 ± 0.09 0.19 ± 0.06 Lymphocytes % 76.33 ± 2.7 74.46 ± 3.4 75.56 ± 2.5 73.35 ± 3.1 Monocytes % 3.38 ± 0.61 3.87 ± 0.72 4.04 ± 0.65 3.26 ± 0.73 AST (U/L) 195.90 ± 1.30 194.98 ± 1.90 196.85 ± 1.87 197.87 ± 1.08 ALT (U/L) 81.46 ± 1.23 80.72 ± 1.03 81.17 ± 2.07 83.5 ± 2.23 ALP (U/L) 232.28 ± 1.28 231.14 ± 1.60 232.26 ± 1.52 231.08 ± 1.07 Creatinine (mg/dL) 0.91 ± 0.07 0.90 ± 0.04 0.92 ± 0.02 0.92 ± 0.05 Albumin (g/dL) 2.84 ± 0.05 2.74 ± 0.07 2.83 ± 0.09 2.84 ± 0.08 Total protein (g/dL) 7.3 ± 1.3 7.2 ± 1.4 6.9 ± 1.5 7.1 ± 1.5 Glucose (mg/dL) 94.86 ± 4.63 89.53 ± 5.34 90.68 ± 6.39 91.53 ± 4.87 Total cholesterol (mg/dL) 122.8 ± 3.6 121.2 ± 4.4 118.9 ± 2.8 121.1 ± 3.9 Bilirubin total (mg/dL) 1.26 ± 0.21 1.23 ± 0.22 1.22 ± 0.31 1.21 ± 0.41 Bilirubin direct (mg/dL) 0.71 ± 0.02 0.68 ± 0.01 0.72 ± 0.01 0.70 ± 0.01 Values are expressed as mean ± SEM of 6 rats in each group. Table 3: Effect on hematological and biochemical parameters after 28 days of the oral administration of OSE. Values are expressed as mean ± SEM of 6 rats in each group. (a) (b) (c) (d) Figure 5: Histology of testis (H&E, 100x) of control and OSE-treated animals. (a) Section of testis from control animal showed well-layered seminiferous tubules with germ cell; ((b), (c), and (d)) testis from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal seminiferous tubules with germ cell. (b) (a) (a) (a) (b) (d) (c) (d) (c) Figure 5: Histology of testis (H&E, 100x) of control and OSE-treated animals. 4. Discussion Oral treatment (mg/kg, od) Isolated organs weight/100 g body weight rat Liver Right kidney Heart Spleen Testis Ovary Control 0.5% CMC 2.7 ± 0.09 0.338 ± 0.01 0.360 ± 0.03 0.327 ± 0.01 0.649 ± 0.26 0.265 ± 0.17 OSE 200 2.6 ± 0.07 0.345 ± 0.02 0.354 ± 0.02 0.319 ± 0.01 0.668 ± 0.27 0.257 ± 0.20 OSE 400 2.7 ± 0.12 0.343 ± 0.01 0.352 ± 0.00 0.326 ± 0.02 0.656 ± 0.28 0.261 ± 0.16 OSE 800 2.7 ± 0.16 0.319 ± 0.01 0.364 ± 0.02 0.320 ± 0.01 0.661 ± 0.28 0.263 ± 0.18 Values are expressed as mean ± SEM of 6 rats in each group. (b) (a) (b) (a) (c) (d) (d) (c) Figure 6: Histological of ovary (H&E, 100x) of control and experimental group of animals. (a) Section of ovary from control animal showed normal small follicles and large follicles; ((b), (c) and (d)) ovary from OSE 200, 400, and 800 mg/kg treated exhibit normal small follicles and large follicles in the histology. studies [15]. Subacute exposure of rat to the lower doses of the OSE produced small and transient changes in some biochemical and hematological parameters. of both transaminases in the blood [16]. In our study, both AST and ALT did not show any treatment related increase even at the 800 mg/kg dose compared to the control group. In addition, AST found in the serum is of both mitochondrial and cytoplasmic origin and any rise can be taken as a first sign of cell damage that leads to the outflow of the enzymes into the serum [19]. Thus, no significant increases observed in ALT and AST activities strongly suggest that the subacute administration of OSE did not alter the hepatocytes and consequently the metabolism in the rats. Further, OSE did not show any histological changes in spleen and liver indicating no effect on reticuloendothelial system.if Liver is the major site for metabolism including drugs. Liver is a site of cholesterol disposal or degradation and its major site of synthesis. In the same perspective, it controls glucose synthesis and generates free glucose from hepatic glycogen stores [16]. Since no significant changes were observed in glucose and cholesterol levels this study, it suggests that OSE had no effect on the lipid and carbohydrate metabolism in rats. 4. Discussion (a) Section of testis from control animal showed well-layered seminiferous tubules with germ cell; ((b), (c), and (d)) testis from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal seminiferous tubules with germ cell. Figure 5: Histology of testis (H&E, 100x) of control and OSE-treated animals. (a) Section of testis from control animal showed well-layered seminiferous tubules with germ cell; ((b), (c), and (d)) testis from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal seminiferous tubules with germ cell. 8 Journal of Toxicology 8 Table 4: Effect on isolated organs weight after 28 days of the oral administration of OSE. Oral treatment (mg/kg, od) Isolated organs weight/100 g body weight rat Liver Right kidney Heart Spleen Testis Ovary Control 0.5% CMC 2.7 ± 0.09 0.338 ± 0.01 0.360 ± 0.03 0.327 ± 0.01 0.649 ± 0.26 0.265 ± 0.17 OSE 200 2.6 ± 0.07 0.345 ± 0.02 0.354 ± 0.02 0.319 ± 0.01 0.668 ± 0.27 0.257 ± 0.20 OSE 400 2.7 ± 0.12 0.343 ± 0.01 0.352 ± 0.00 0.326 ± 0.02 0.656 ± 0.28 0.261 ± 0.16 OSE 800 2.7 ± 0.16 0.319 ± 0.01 0.364 ± 0.02 0.320 ± 0.01 0.661 ± 0.28 0.263 ± 0.18 Values are expressed as mean ± SEM of 6 rats in each group. (a) (b) (c) (d) Figure 6: Histological of ovary (H&E, 100x) of control and experimental group of animals. (a) Section of ovary from control animal showed normal small follicles and large follicles; ((b), (c) and (d)) ovary from OSE 200, 400, and 800 mg/kg treated exhibit normal small follicles and large follicles in the histology. Table 4: Effect on isolated organs weight after 28 days of the oral administration of OSE. Table 4: Effect on isolated organs weight after 28 days of the oral administration of OSE. 4. Discussion Further, drugs showing any toxicity in liver affect the transaminases, (aspartate aminotransferase) AST, and (alanine amino transferase) ALT which are well- known enzymes used as good indicators of liver function [17] and biomarkers predicting possible toxicity [18]. Generally, any damage to the parenchymal liver cells results in elevations f y OSE neither showed any significant difference in the weight of the organs or color of organs nor affected the histopathological changes in organs like heart and testis/ovary indicating least cumulative toxic effects on 9 Journal of Toxicology [8] OECD, “Acute Oral Toxicity: Acute Toxic Class Method,” Test Guideline 423, 2001. reproductive and cardiac tissues. Further, no change was also observed in kidney function as evidenced by little or no change in serum creatinine level as well as histological changes in treated kidneys. [9] M. K. Gautam, A. Singh, C. V. Rao, and R. K. Goel, “Toxico- logical evaluation of Murraya paniculata (L.) leaves extract on rodents,” The American Journal of Pharmacology and Toxicology, vol. 7, no. 2, pp. 62–67, 2012. Conflict of Interests [17] J. El Hilaly, Z. H. Israili, and B. Lyoussi, “Acute and chronic toxicological studies of Ajuga iva in experimental animals,” Journal of Ethnopharmacology, vol. 91, no. 1, pp. 43–50, 2004.f The authors declare that they have no conflict of interests. [18] M. F. Rahman, “Effects of Vepacide (Azadirachta indica) on aspartate and alanine aminotransferase profiles in a subchronic study with rats,” Human and Experimental Toxicology, vol. 20, no. 5, pp. 243–249, 2001. Abbreviations CNS: Central nervous system ANS: Autonomous nervous system RBC: Red blood cell Hb: Hemoglobin WBC: White blood cell AST: Aspartate transaminase ALT: Alanine transaminase ALP: Alkaline phosphate. CNS: Central nervous system ANS: Autonomous nervous system RBC: Red blood cell Hb: Hemoglobin WBC: White blood cell AST: Aspartate transaminase ALT: Alanine transaminase ALP: Alkaline phosphate. CNS: Central nervous system ANS: Autonomous nervous system RBC: Red blood cell Hb: Hemoglobin WBC: White blood cell AST: Aspartate transaminase ALT: Alanine transaminase ALP: Alkaline phosphate. CNS: Central nervous system ANS: Autonomous nervous system RBC: Red blood cell Hb: Hemoglobin WBC: White blood cell AST: Aspartate transaminase ALT: Alanine transaminase ALP: Alkaline phosphate. [14] J. T. Mukinda and J. A. Syce, “Acute and chronic toxicity of the aqueous extract of Artemisia afra in rodents,” Journal of Ethnopharmacology, vol. 112, no. 1, pp. 138–144, 2007. [15] H. Olson, G. Betton, D. Robinson et al., “Concordance of the toxicity of pharmaceuticals in humans and in animals,” Regulatory Toxicology and Pharmacology, vol. 32, no. 1, pp. 56– 67, 2000. ALT: Alanine transaminase ALP: Alkaline phosphate. [16] N. Anderson and J. Borlak, “Molecular mechanisms and thera- peutic targets in steatosis and steatohepatitis,” Pharmacological Reviews, vol. 60, no. 3, pp. 311–357, 2008. 5. Conclusion [10] OECD (The Organization of Economic Co-operation Develop- ment), “Test No. 407: repeated Dose 28-day oral toxicity study in rodents,” in OECD Guidelines For the Testing of Chemicals, Section 4: Health Effects, OECD Publishing, 2008. This study provides valuable data on the acute and subacute oral toxicities profile of Ocimum sanctum Linn leaves (a com- mon household remedy for number of ailments) that could be very useful in its future clinical study. The 50% ethanol extract of Ocimum sanctum leaves seemed to be nontoxic as was seen after its acute and subacute oral administrations. Further, teratogenic, mutagenic and carcinogenic studies with this plant are needed to complete the safety profile of this plant. [11] M. M. Wintrobe, Clinical Haemotology, Lea and Febigeer, Philadelphia, Pa, USA, 1961. [12] B. Saad, H. Azaizeh, G. Abu-Hijleh, and O. Said, “Safety of tra- ditional Arab herbal medicine,” Evidence-Based Complementary and Alternative Medicine, vol. 3, no. 4, pp. 433–439, 2006. [13] R. K. Goel, T. Prabha, M. Mohan Kumar, M. Dorababu, P. Prakash, and G. Singh, “Teratogenicity of Asparagus racemosus Willd. root, a herbal medicine,” Indian Journal of Experimental Biology, vol. 44, no. 7, pp. 570–573, 2006. References [1] H. K. Yakob, A. M. Uyub, and S. F. Sulaiman, “Toxicological evaluation of 80% methanol extract of Ludwigia octovalvis (Jacq.) P.H. Raven leaves (Onagraceae) in BALB/cmice,” Journal of Ethnopharmacology, vol. 142, pp. 663–668, 2012. [19] J. T. Mukinda and P. F. K. Eagles, “Acute and sub-chronic oral toxicity profiles of the aqueous extract of Polygala fruticosa in female mice and rats,” Journal of Ethnopharmacology, vol. 128, no. 1, pp. 236–240, 2010. [2] WHO (World Health Organization), WHO Guidelines on Safety Monitoring of Herbal Medicines in Pharmacovigilance Systems, WHO (World Health Organization), Geneva, Switzerland, 2008. [3] A. B. D. Roch, R. M. Lopes, and G. Schwartsmann, “Natural products in anticancer therapy,” Current Opinion in Pharmacol- ogy, vol. 1, pp. 364–369, 2001. [4] Y. Denga, M. Caob, D. Shia et al., “Toxicological evaluation of neem (Azadirachta indica) oil: acute and subacute toxicity,” Environmental Toxicology and Pharmacology, vol. 35, pp. 240– 246, 2013. [5] P. Prakash and N. Gupta, “Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: A short review,” Indian Journal of Physiology and Pharmacology, vol. 49, no. 2, pp. 125–131, 2005. [6] P. Pattanayak, P. Behera, D. Das, and S. Panda, “Ocimum sanctum Linn: a reservoir plant for therapeutic applications: an overview,” Pharmacognosy Reviews, vol. 4, no. 7, pp. 95–105, 2010. [7] K. Sairam, C. H. V. 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State of the Art: trxG Factor Regulation of Post-embryonic Plant Development
Frontiers in plant science
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Keywords: trxG, PcG, development, chromatin, histone methylation, transcription, epigenetics, Arabidopsis Keywords: trxG, PcG, development, chromatin, histone methylation, transcription, epigenetics, Arabidopsis Reviewed by: Isabel Bäurle, University of Potsdam, Germany Keqiang Wu, National Taiwan University, Taiwan Reviewed by: Isabel Bäurle, University of Potsdam, Germany Keqiang Wu, National Taiwan University, Taiwan *Correspondence: Jennifer C. Fletcher jfletcher@berkeley.edu State of the Art: trxG Factor Regulation of Post-embryonic Plant Development Jennifer C. Fletcher1,2* 1 Plant Gene Expression Center, United States Department of Agriculture – Agricultural Research Service, Albany, CA, United States, 2 Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, United States Multicellular organisms rely on the precise and consistent regulation of gene expression to direct their development in tissue- and cell-type specific patterns. This regulatory activity involves arrays of DNA-binding transcription factors and epigenetic factors that modify chromatin structure. Among the chromatin modifiers, trithorax (trxG) and Polycomb (PcG) group proteins play important roles in orchestrating the stable activation and repression of gene expression, respectively. These proteins have generally antagonistic functions in maintaining cell and tissue homeostasis as well as in mediating widespread transcriptional reprogramming during developmental transitions. Plants utilize multiple trxG factors to regulate gene transcription as they modulate their development in response to both endogenous and environmental cues. Here, I will discuss the roles of trxG factors and their associated proteins in post-embryonic plant development. INTRODUCTION The development of multicellular organisms is driven by precise patterns of gene transcription that are tightly regulated in a spatial and temporal manner. Establishing and sustaining specific transcription states at gene loci are complex, multi-step processes. They require repertoires of sequence-specific DNA-binding transcription factors as well as epigenetic factors that alter chromatin structure and thereby affect accessibility by the transcriptional machinery. Epigenetic regulators classified as trithorax group (trxG) and Polycomb group (PcG) factors are critical for maintaining the stable transcription patterns at developmental regulatory loci by organizing chromatin in an active or inactive state, respectively (Schwartz and Pirrota, 2007). trxG and PcG factors generally act in large, multi-component complexes that function antagonistically to generate and maintain a balanced state of gene expression (Piunti and Shilatifard, 2016). *Correspondence: Jennifer C. Fletcher jfletcher@berkeley.edu Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Received: 25 September 2017 Accepted: 24 October 2017 Published: 14 November 2017 Citation: Fletcher JC (2017) State of the Art: trxG Factor Regulation of Post-embryonic Plant Development. Front. Plant Sci. 8:1925. doi: 10.3389/fpls.2017.01925 Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Received: 25 September 2017 Accepted: 24 October 2017 Published: 14 November 2017 TrxG genes were first identified in Drosophila as positive regulators of PcG developmental target genes (Ingham, 1988; Kennison, 1995), and their protein products operate in multiple complexes that affect gene expression on a global scale (Piunti and Shilatifard, 2016). Because transcription activation involves numerous steps, trxG factors are heterogeneous and fall into several functional categories: chromatin remodeling proteins, histone modifying methyltransferase and demethylase proteins, and DNA-binding and accessory proteins (Xiao et al., 2016) (Table 1). The chromatin remodeling proteins include members of the SWI/SNF, ISWI, and CHD families that utilize ATP to alter nucleosome assembly and distribution (Gentry and Hennig, 2014). The histone modifying enzymes deposit Edited by: Edited by: Stefan A. Rensing, Philipps University of Marburg, Germany MINI REVIEW MINI REVIEW published: 14 November 2017 doi: 10.3389/fpls.2017.01925 published: 14 November 2017 doi: 10.3389/fpls.2017.01925 Citation: November 2017 | Volume 8 | Article 1925 1 Frontiers in Plant Science | www.frontiersin.org trxG Factors in Plant Development Fletcher ROLE OF trxG FACTORS IN ROOT AND SHOOT APICAL MERISTEM MAINTENANCE H3K4me2/3 and/or H3K36me2/3 marks associated with transcription activation to counteract the activity of PcG complexes such as POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) that deposit H3K27me3 as the major repressive mark for transcription (Piunti and Shilatifard, 2016). H3K4me2/3 and/or H3K36me2/3 marks associated with transcription activation to counteract the activity of PcG complexes such as POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) that deposit H3K27me3 as the major repressive mark for transcription (Piunti and Shilatifard, 2016). Plants are sessile organisms that grow continuously and alter their development in response to changes in their environment. Organogenesis occurs throughout the life cycle from specialized structures at the growing shoot and root tips, called apical meristems (Steeves and Sussex, 1989). Both the root and shoot apical meristems (SAMs) contain small reservoirs of stem cells that constantly replenish themselves as well as provide progeny cells for continuous organ formation. The flexible regulation of gene expression via chromatin remodeling is essential for maintaining these pluripotent stem cell populations whose progeny can assume different fates. Animal stem cells possess special chromatin signatures (Bernstein et al., 2006) that permit plasticity in stem cell dynamics, and much is known about the epigenetic factors and mechanisms involved (Spivakov and Fisher, 2007). In contrast, the roles of epigenetic factors in p Plant trxG factors have been identified either by homology to known trxG factors in animals or by genetic characterization based on their ability to suppress PcG mutant phenotypes. Given their fundamental roles in the epigenetic regulation of gene expression states, mutations in plant trxG genes often cause pleiotrophic developmental phenotypes, including defects in seedling growth, anther and ovule formation, and gametophyte development (Grini et al., 2009; Guo et al., 2010; Carter et al., 2016; Chen et al., 2017). They also play key roles during developmental transitions when widespread gene reprogramming occurs. Here I will summarize our current understanding of trxG protein function in plant meristems, which drive post-embryonic development. TABLE 1 | Biological functions of the trxG factors and their accessory proteins in post-embryonic development. ROLE OF trxG FACTORS IN ROOT AND SHOOT APICAL MERISTEM MAINTENANCE trxG factor Biological function(s) Reference Chromatin remodelers BRM Maintains RAM and SAM activity; Farrona et al., 2004; Yang et al., 2015 Represses floral transition; Farrona et al., 2011; Li et al., 2015 Specifies floral organ identity Farrona et al., 2004; Wu et al., 2012 CHR11 Promotes floral morphogenesis Smaczniak et al., 2012 CHR17 Promotes floral morphogenesis Smaczniak et al., 2012 PKL Maintains RAM activity Aichinger et al., 2011 SYD Maintains SAM activity; Kwon et al., 2005 Specifies floral organ identity Wagner and Meyerowitz, 2002; Wu et al., 2012 Histone methyltransferases ATX1/SDG27 Maintains RAM activity; Napsucialy-Mendivil et al., 2014 Represses floral transition; Pien et al., 2008 Specifies floral organ identity Alvarez-Venegas et al., 2003 ATXR3/SDG2 Maintains RAM activity Yao et al., 2013 ATXR7/SDG25 Represses floral transition Berr et al., 2009; Tamada et al., 2009 SDG8 Represses floral transition; Shafiq et al., 2014; Yang et al., 2014 Specifies floral organ identity Grini et al., 2009 SDG26 Promotes floral transition Berr et al., 2015 SDG701 Promotes floral transition in rice Liu et al., 2017 SDG708 Promotes floral transition in rice Liu et al., 2016 AtCOMPASS core components ASH2R Represses floral transition Jiang et al., 2011 RBL Represses floral transition Jiang et al., 2011 WDR5 Represses floral transition Jiang et al., 2009 Histone demethylases ELF6 Represses floral transition Yang et al., 2016 REF6/JMJ12 Promotes floral transition Noh et al., 2004; Yang et al., 2016 DNA-binding and accessory proteins ALP1 Restricts FM activity Liang et al., 2015 NF-Y Promotes floral transition Hou et al., 2014 SEP3 Specifies floral organ identity Pelaz et al., 2000 ULT1/2 Restricts SAM activity; Carles et al., 2004, 2005 Restricts FM activity Carles and Fletcher, 2009 Biological function(s) Biological function(s) November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org Reference November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 2 trxG Factors in Plant Development Fletcher regulating plant stem cell activity are only beginning to be revealed. Maintenance of stem cell reservoirs in Arabidopsis shoot and floral meristems occurs via a spatial negative feedback loop mediated by the WUSCHEL (WUS)-CLAVATA (CLV) signal transduction pathway. The homeobox TF gene WUS is expressed in the core of the meristem and confers stem cell identity on the overlying cells (Laux et al., 1996). The stem cell-inducing activity of WUS is antagonized by the secreted polypeptide CLV3 (Fletcher et al., 1999; Rojo et al., 2002), which is produced by the stem cells and activates a signal transduction pathway in the meristem interior that limits the accumulation of WUS-expressing cells (Brand et al., 2000). This feedback loop mediates stem cell homeostasis, balancing the loss of stem cells to organ formation with their replenishment via cell division. Recent genetic studies have uncovered roles for multiple trxG factors in root apical meristem (RAM) maintenance. The RAM generates the entire underground root system and has a stereotypical organization. Four rarely dividing cells known as the quiescent center (QC) act as a niche (van den Berg et al., 1997) that maintains the surrounding cells as stem cells, aka initial cells, which undergo asymmetric cell divisions to generate the distinct root cell lineages. A gradient of the hormone auxin across the root tip exists due to the activity of members of the PIN-FORMED (PIN) family of auxin transport proteins (Blilou et al., 2005; Grieneisen et al., 2007). The auxin concentration maximum coincides with the QC and promotes the expression of the PLETHORA (PLT) AP2 domain transcription factor (TF) genes, which are essential for root stem cell niche maintenance (Aida et al., 2004). Although little is known about trxG activity in the SAM, the ratio of H3K4me3 active to H3K27me3 repressive marks is known to be important for reproductive SAM development in rice (Liu et al., 2015). The SWI2/SNF2 trxG factors BRM and SYD both act to sustain SAM activity in Arabidopsis (Farrona et al., 2004; Kwon et al., 2005), with SYD shown to bind to the WUS promoter and elevate its transcription in the SAM (Kwon et al., 2005) (Figure 1B). Reference In contrast, the SAND domain-containing proteins ULTRAPETALA1 (ULT1) and ULT2 restrict shoot and floral stem cell activity by limiting the size of the WUS expression domain (Carles et al., 2004, 2005). The SAND domain occurs in a small number of eukaryotic proteins including the human AIRE transcriptional regulator that is implicated in autoimmune diseases (Abramson and Goldfarb, 2016). ULT1 antagonizes the repressive activity of PRC1 and PRC2 PcG complex components (Carles and Fletcher, 2009; Pu et al., 2013), and physically associates with the H3K4 methyltransferase ATX1 (Carles and Fletcher, 2009) as well as the transcription factors KANADI1 (KAN1), KAN2 and ULTRAPETALA1 INTERACTING FACTOR (UIF1) (Pires et al., 2014; Moreau et al., 2016). Because both ATX1 and the AIRE protein form complexes with RNA Pol II and RNA-processing components (Abramson et al., 2010; Ding et al., 2012), the ULT proteins may coordinate chromatin mark modification at transcriptionally priming loci with basal transcription machinery recruitment by stage- and tissue-specific transcription factors (Carles and Fletcher, 2010; Engelhorn et al., 2014a). Two H3K4 histone methyltransferase trxG factors have been implicated in Arabidopsis RAM maintenance (Figure 1A). The SET domain protein SET DOMAIN GROUP 2 (SDG2) is the major H3K4 trimethyltransferase in Arabidopsis and is necessary for genome-wide H3K4me3 deposition (Guo et al., 2010). In the RAM SDG2 is required to maintain the auxin gradient and QC maximum, and to sustain cell identity and stem cell activity in the QC and surrounding initial cells (Yao et al., 2013). These functions correlate with a requirement for SDG2 to promote PLT1 expression and global H3K4me3 deposition in root cells (Yao et al., 2013). The ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1/SDG27) protein contributes ∼15% of genome-wide H3K4 trimethylation (Alvarez-Venegas and Avramova, 2005). ATX1 is needed for TATA binding protein (TBP) and RNA Polymerase II recruitment to its target promoters (Ding et al., 2011) and is also critical for H3K4me3 deposition associated with transcription elongation (Ding et al., 2012). Like SDG2, ATX1 is necessary for normal RAM organization, but also restricts the expression of QC markers such as WOX5 to the stem cell niche in an auxin-independent fashion (Napsucialy- Mendivil et al., 2014), indicating that the two H3K4 histone methyltransferases have distinct as well as shared roles in RAM maintenance. The SWI2/SNF2 chromatin remodeling complex ATPase genes PICKLE (PKL) and BRAHMA (BRM) also regulate RAM activity in Arabidopsis. Reference PKL acts antagonistically to the PRC2 PcG factor CURLY LEAF (CLF) to maintain RAM stem cell activity (Aichinger et al., 2011). PKL does not induce the activity of the root stem cell niche by affecting auxin accumulation. Instead, PKL elevates the expression levels of and limits CLF-mediated H3K27me3 deposition at PLT1, PLT2, and WOX5 (Aichinger et al., 2011). BRM likewise maintains the RAM stem cell niche by promoting expression of PLT1 and PLT2 (Yang et al., 2015). However, BRM unlike PKL affects auxin accumulation in the root tip by directly binding to and up-regulating the transcription of five PIN loci (Yang et al., 2015). Thus the evidence to date suggests that the auxin-dependent and auxin-independent regulatory pathways utilize distinct trxG factors to sustain RAM activity. ROLES OF trxG FACTORS IN FLORAL INDUCTION AT THE SHOOT APICAL MERISTEM During vegetative development FLC directly represses the transcription of the flowering time integrators FLOWERING LOCUS T (FT), FLOWERING LOCUS D (FD) and SOC1, which promote the transition to flowering (Michaels and Amasino, 2001). FT protein is produced in the rosette leaves and moves to the SAM, where it interacts with the FD protein. The FT-FD complex then induces the floral transition by activating the expression of TF genes such as SOC1, LEAFY (LFY) and APETALA1 (AP1), which confer floral meristem (FM) identity on the primordia that form on the flanks of the primary SAM (Irish, 2010). In Arabidopsis, chromatin modifications at key regulatory loci such as FLC, SOC1 and FT are crucial to the timing of the floral transition, and a number of trxG factors are involved in these processes (Figure 1C). Because the role of epigenetic factors in FLC regulation during vernalization has been extensively reviewed (Jarillo and Pineiro, 2011; Andres and Coupland, 2012; He, 2012; Hepworth and Dean, 2015), I will focus here on the control of flowering through other pathways. To prevent premature flowering during vegetative growth, the FLC locus is maintained in a transcriptionally active state marked by H3K36 tri-methylation (Zhao et al., 2005; Yang et al., 2014), which inhibits accumulation of H3K27me3 repressive marks (Shafiq et al., 2014). SDG8 is the major H3K36me3 methyltransferase in Arabidopsis (Yang et al., 2014) and is required for H3K36me3 deposition at the FLC locus (Shafiq et al., 2014; Yang et al., 2014). SDG8 associates with components of the transcription machinery, including RNA Pol II and PAF1, as well as with the H3K27 demethylase EARLY FLOWERING 6 (ELF6) (Yang et al., 2016). These physical associations (Figure 2) couple removal of repressive histone marks with deposition of active marks and transcription initiation/elongation to sustain high levels of FLC expression. ATXR7/SDG25 also represses the floral transition by binding to the FLC promoter and augmenting both H3K36me3 and H3K4me3 accumulation (Berr et al., 2009; Tamada et al., 2009), but whether SDG8 and ATXR7 function in concert to induce FLC transcription is unknown. The chromatin remodeling ATPase BRM prevents premature flowering by regulating FLC and FLC-related gene expression (Farrona et al., 2011; Li et al., 2015). Loss of function brm seedlings exhibit elevated H3K4me3 levels and reduced H3K27me3 levels in the FLC promoter, indicating that BRM imposes a repressive chromatin configuration at the FLC locus (Farrona et al., 2011). ROLES OF trxG FACTORS IN FLORAL INDUCTION AT THE SHOOT APICAL MERISTEM The floral induction process directs the SAM to transition from generating vegetative organs (leaves) to reproductive organs (flowers). The timing of this dynamic meristem cell fate switch is critical for plant reproductive success and occurs in response to endogenous pathways such as the age, GA and autonomous pathways (APs) as well as environmental cues including photoperiod, vernalization and temperature (Amasino, 2010; Srikanth and Schmid, 2011; Andres and Coupland, 2012). The MADS domain TF FLOWERING LOCUS C (FLC) is the main floral repressor in Arabidopsis seedlings (Michaels and Amasino, 1999) and is a key target of both endogenous November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 3 trxG Factors in Plant Development Fletcher Fletcher FIGURE 1 | Regulatory targets of trxG factors in post-embryonic development. (A) Root apical meristem maintenance. (B) Shoot apical meristem maintenance. (C) Vegetative to reproductive meristem transition. (D) Floral meristem patterning. Gene targets shown in green type. Arrows indicate positive and bars indicate negative regulatory interactions. The SAM image in (B) is reprinted from Fiume et al. (2010). No permission is required for its reproduction. FIGURE 1 | Regulatory targets of trxG factors in post-embryonic development. (A) Root apical meristem maintenance. (B) Shoot apical meristem maintenance. (C) Vegetative to reproductive meristem transition. (D) Floral meristem patterning. Gene targets shown in green type. Arrows indicate positive and bars indicate negative regulatory interactions. The SAM image in (B) is reprinted from Fiume et al. (2010). No permission is required for its reproduction. ATXR7/SDG25 does act independently of the H3K4me3 methyltransferase ATX1 in repressing the flowering transition (Tamada et al., 2009), although ATX1 also directly binds to the FLC locus to deposit H3K4me3 and elevate expression of the floral repressor gene (Pien et al., 2008). ATX1 physically associates with WDR5a (Figure 2), a core component of the Arabidopsis COMPASS-like complex (AtCOMPASS) that also binds the FLC locus and promotes its expression by elevating H3K4me3 levels (Jiang et al., 2009). Two other core AtCOMPASS components, ASH2R and RbBP5-LIKE (RBL), also repress the floral transition by enhancing FLC expression, with ASH2R availability apparently being the rate-limiting factor in H3K4 tri-methylation at FLC and other target loci (Jiang et al., 2011). Therefore both H3K36me3 and H3K4me3 contribute to the maintenance of active FLC transcription in Arabidopsis. and environmental signaling pathways (Sheldon et al., 2000; Michaels and Amasino, 2001). ROLES OF trxG FACTORS IN FLORAL INDUCTION AT THE SHOOT APICAL MERISTEM REF6 also induces transcription of the floral activator genes SOC1 and FT in an FLC-independent fashion (Noh et al., 2004; Lu et al., 2011). It is recruited by the nuclear factor Y (NF-Y) transcription factor complex to demethylate the SOC1 locus in response to the photoperiod and GA pathways (Hou et al., 2014), indicating that REF6 is a component of both endogenous and environmental signaling modules. Like REF6, the SDG26 histone methyltransferase also binds to and induces SOC1 transcription, augmenting the deposition of both H3K4me3 and H3K36me3 at the locus to promote the floral transition (Berr et al., 2015). the identity of each floral organ – sepals, petals, stamens, and carpels – from the outside to the inside of the flower (Coen and Meyerowitz, 1991). The activation of the floral homeotic genes at the onset of flower patterning requires counteracting the PcG-mediated repressive state that has persisted throughout vegetative development (Pu and Sung, 2015), an activity that is associated with increases in H3K4me3 levels at PcG target genes (Engelhorn et al., 2017) and involves multiple trxG factors. g p The plant specific TF LFY and the MADS domain TF SEPALLATA3 (SEP3) play crucial roles in activating the expression of MADS box-containing floral homeotic genes such as APETALA3 (AP3) and AGAMOUS (AG) that specify petal, stamen, and carpel identity in the developing flower (Weigel and Meyerowitz, 1993; Pelaz et al., 2000). SYD and BRM physically associate with the LFY and SEP3 proteins (Figure 2), which recruit SYD to the AP3 and AG loci (Wu et al., 2012). At the onset of flower patterning, SYD and BRM redundantly regulate floral organ identity specification (Wagner and Meyerowitz, 2002; Farrona et al., 2004) (Figure 1D) by activating AP3 and AG expression, antagonizing CLF activity at the two loci by reducing H3K27me3 deposition and enhancing H3K4me3 deposition (Wu et al., 2012). ATX1 and SDG8 also specify floral organ identity by maintaining floral homeotic gene expression levels (Alvarez- Venegas et al., 2003; Grini et al., 2009), although the mechanistic details are as yet unknown. Finally, REF6 and PKL as well as two ISWI-type chromatin remodelers, CHR11 and CHR17, occur in floral MADS domain protein complexes and affect floral organ morphogenesis (Smaczniak et al., 2012). ROLES OF trxG FACTORS IN FLORAL INDUCTION AT THE SHOOT APICAL MERISTEM In addition, BRM directly activates the expression of the FLC-related MADS box TF gene SHORT VEGETATIVE PHASE (SVP) (Li et al., 2015). SVP forms a complex with FLC to repress flowering under non-inductive conditions (Fujiwara et al., 2008; Li et al., 2008). SVP expression is regulated by the AP, GA and temperature pathways, and directly represses SOC1 and FT transcription (Li et al., 2008). BRM represses flowering largely by inducing SVP transcription in vegetative tissues, binding to the SVP locus where it limits H3K27me3 accumulation by restricting CLF occupancy and activity (Li et al., 2015). Thus the early flowering phenotype of brm mutants can be accounted for by a reduction in SVP mRNA levels leading to lower abundance of the FLC-SVP repressor complex, resulting in elevated FT transcript levels that induce precocious flowering. November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org Frontiers in Plant Science | www.frontiersin.org 4 trxG Factors in Plant Development Fletcher FIGURE 2 | Association networks of trxG factors and interacting proteins. Solid bars designate physical associations between trxG factors (blue), transcription factors (green), AtCOMPASS components (yellow), and transcription machinery components and accessory proteins (purple). FIGURE 2 | Association networks of trxG factors and interacting proteins. Solid bars designate physical associations between tr factors (green), AtCOMPASS components (yellow), and transcription machinery components and accessory proteins (purple). works of trxG factors and interacting proteins. Solid bars designate physical associations between trxG factors (blue), transcription S components (yellow), and transcription machinery components and accessory proteins (purple). FIGURE 2 | Association networks of trxG factors and interacting proteins. Solid bars designate physical associations between trxG factors (blue), transcription factors (green), AtCOMPASS components (yellow), and transcription machinery components and accessory proteins (purple). Repression of FLC transcription is crucial for the transition from the vegetative to the reproductive state. The JmjC domain- containing H3K27 demethylase REF6/JMJ12 promotes flowering independently of photoperiod by repressing FLC transcription (Noh et al., 2004). Because REF6 acts antagonistically to CLF by removing repressive H3K27me2/3 marks (Lu et al., 2011), binding its target genes in a sequence-specific fashion via its C2H2 zinc-finger domains (Cui et al., 2016) and facilitating recruitment of BRM (Li et al., 2016), the repression of FLC by REF6 is likely to be indirect (Yang et al., 2016). ROLES OF trxG FACTORS IN FLORAL INDUCTION AT THE SHOOT APICAL MERISTEM SEP3 and several other floral homeotic TFs bind their target genes prior to detectable increases in DNA accessibility (Pajaro et al., 2014), suggesting that they work closely with epigenetic factors to facilitate transcription initiation during early flower development by modulating chromatin accessibility at target loci. Finally, two methyltransferases that accelerate the floral transition independent of photoperiod have recently been characterized in rice. SDG708 encodes a methyltransferase that deposits up to three methyl groups on H3K36, and promotes flowering by catalyzing H3K36 methylation at the key flowering time regulatory genes H3Da and RFT1, which are closely related homologs of Arabidopsis FT, and Ehd1 (Liu et al., 2016). SDG701 encodes an H3K4 di- and tri-methyltransferase that likewise promotes flowering by depositing H3K4me3 to elevate the expression of H3Da and RFT1 (Liu et al., 2017). ROLES OF trxG FACTORS IN PATTERNING THE FLORAL MERISTEM The ULT1 trxG protein induces AG transcription in the center of the FM at stage 3 of flower formation (Alvarez-Venegas et al., 2003; Carles and Fletcher, 2009), binding directly to the locus to limit CLF-mediated H3K27me3 deposition and enhance H3K4me3 deposition (Carles and Fletcher, 2009). ULT1 thus sets the timing of the transition of the AG locus from a repressed Flowering signals induce reproductive development in plants by reprogramming the SAM into an inflorescence meristem (IM) that produces floral meristems (FMs) instead of leaves. A small suite of floral homeotic transcription factor genes then specifies November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 5 trxG Factors in Plant Development Fletcher to an active state, helping trigger a molecular pathway that ultimately terminates FM activity (Carles and Fletcher, 2009; Engelhorn et al., 2014b; Cao et al., 2015; Sun and Ito, 2015). Mutations in the domesticated transposase gene ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) enhance ult1 FM phenotypes, and ALP1 promotes floral organ identity gene expression in the absence of LFY (Liang et al., 2015). ALP1 antagonizes CLF function, acting genetically as a trxG factor, and is required for the activity of PcG target genes such as AP3 and AG. Notably, the ALP1 protein complex lacks known trxG factors but consists of core components of PRC2 and accessory factors such as EMF1 and LHP1. ALP1 is therefore proposed to antagonize PcG activity by blocking the interaction between PRC2 and accessory factors that stimulate its activity (Liang et al., 2015). Although significant progress has been made in determining the roles of trxG factors in plant development, many gaps in our understanding remain. It still remains to be determined how many trxG complexes exist in plants, not to mention their full composition and whether that composition is static or changes depending on the developmental stage or tissue. The DNA binding proteins that recruit trxG factors to developmental regulatory loci are only beginning to be identified, while elucidating the chromatin signatures of plant stem cell populations can provide a valuable starting point for determining how tissue- and stage-specific epigenetic states are ultimately achieved during development. Finally, much work remains to decipher how developmental switches between trxG and PcG activities are implemented at individual loci as well as broadly across the genome to coordinate widespread transcriptional reprogramming. REFERENCES Amasino, R. (2010). Seasonal and developmental timing of flowering. Plant J. 61, 1001–1013. doi: 10.1111/j.1365-313X.2010.04148.x Abramson, J., Giraud, M., Benoist, C., and Mathis, D. (2010). 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Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity. Science 289, 617–619. doi: 10.1126/science.289.5479.617 Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., et al. (2005). The PIN auxin efflux carrier facilitator network controls growth and patterning in Arabidopsis roots Nature 433 39 44 doi: 10 1038/nature03184 Alvarez-Venegas, R., and Avramova, Z. (2005). Methylation patterns of histone H3 Lys 4, Lys 9 and Lys 27 in transcriptionally active and inactive Arabidopsis genes and in atx1 mutants. Nucleic Acids Res. 33, 5199–5207. AUTHOR CONTRIBUTIONS The author confirms being the sole contributor of this work and approved it for publication. ROLES OF trxG FACTORS IN PATTERNING THE FLORAL MERISTEM Further investigation in these areas will provide a more complete picture of how plants are able to maintain and as necessary adjust their gene expression programs during development in response to a wealth of endogenous and environmental cues. ACKNOWLEDGMENTS The author apologizes for references omitted due to space limitations. This work was supported by research grants from the USDA (CRIS 2030-21000-041-00D) and the NSF (IOS-105- 2050). CONCLUSION A variety of trxG factors exist in plants that carry out diverse biochemical activities to promote active gene expression states: chromatin-remodeling ATPases, histone methyltransferases, AtCOMPASS core components, histone demethylases, as well as DNA-binding and accessory proteins (Table 1). It is becoming clear from recent research that members of all of these categories of proteins play important roles in regulating landmark post-embryonic developmental processes such as meristem maintenance and floral induction. Moreover some trxG factors, such as BRM and ATX1, mediate multiple developmental processes during plant growth whereas others appear to have more restricted roles. The coupling of a core set of reiteratively used trxG components together with stage-, tissue- or process- specific trxG components may provide a flexible mechanism for tailoring the basic process of transcription activation to discrete gene networks in response to changing endogenous and environmental signals during the life cycle. Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., et al. (2005). The PIN auxin efflux carrier facilitator network controls growth and patterning in Arabidopsis roots. Nature 433, 39–44. doi: 10.1038/nature03184 Brand, U., Fletcher, J. 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U.S.A. 97, 3753–3758. doi: 10.1073/pnas.97.7. 3753 Copyright © 2017 Fletcher. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Smaczniak, C., Immink, R. G. H., Muino, J. M., Blanvillain, R., Busscher, M., Busscher-Lange, J., et al. (2012). Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 8
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ISSN 0103-6440 ISSN 0103-6440 Brazilian Dental Journal (2017) 28(6): 726-730 http://dx.doi.org/10.1590/0103-6440201701372 1Department of Restorative Dentistry, Dental Materials Division, Piracicaba Dental School, UNICAMP - Universidade Estadual de Campinas, Piracicaba, SP, Brazil 2Department of Prosthodontics, UNIVAG - Centro Universitário de Várzea Grande, Cuiaba, MT, Brazil 3Dental School, UNISA - Universidade de Santo Amaro, São Paulo, SP, Brazil 4Department of Pediatric Dentistry, Piracicaba Dental School, UNICAMP - Universidade Estadual de Campinas, Piracicaba, SP, Brazil 5Restorative Dental Science Department, Operative Dentistry Division, College of Dentistry, University of Florida, Gainesville, FL, USA Correspondence: Mário Alexandre Coelho Sinhoreti, Avenida Limeira, 901, 13414-903 Piracicaba, SP, Brasil. Tel: +55-19-2106-5374. e-mail: sinhoreti@unicamp.br Influence of Different Concentration and Ratio of a Photoinitiator System on the Properties of Experimental Resin Composites Caio Vinícius Signorelli Grohmann1, Eveline Freitas Soares1, Eduardo José Carvalho Souza-Junior2, William Cunha Brandt3, Regina Maria Puppin- Rontani4, Saulo Geraldeli5, Mário Alexandre Coelho Sinhoreti1 The aim in this study was to evaluate the influence of different ratio of camphorquinone/ tertiary amine concentration on the flexural strength (FS), elastic modulus (EM), degree of conversion (DC), yellowing (YL), water sorption (WS) and water solubility (WSL) of experimental composites. Thus, acrylate blends were prepared with different camphorquinone (CQ) and amine (DABE) concentrations and ratios by weight: (CQ/DABE%): 0.4/0.4% (C1), 0.4/0.8% (C2), 0.6/0.6% (C3), 0.6/1.2% (C4), 0.8/0.8% (C5), 0.8/1.6% (C6), 1.0/1.0% (C7), 1.0/2.0% (C8), 1.5/1.5% (C9), 1.5/3.0% (C10). For the FS and EM, rectangular specimens (7x2x1 mm, n=10) were photo-activated by single-peak LED for 20 s and tested at Instron (0.5 mm/ min). Then, the same specimens were evaluated by FTIR for DC measurement. For YL, disks (5x2 mm, n=10) were prepared, light-cured for 20 s and evaluated in spectrophotometer using the b aspect of the CIEL*a*b* system. For WS and WSL, the volume of the samples was calculated (mm³). For WS and WSL, composites disks (5x0.5 mm, n=5) were prepared. After desiccation, the specimens were stored in distilled water for 7 days and again desiccated, in order to measure the WS and WSL. Data were submitted to one-way ANOVA and Tukey’s test (5%). The groups C8, C9 and C10 showed higher DC, EM and YL means, compared to other composites. Therefore, the FS and WS values were similar among all groups. Also, C1, C2 and C3 presented higher WSL in 7 days, compared to other composites. In general, higher concentrations of camphorquinone promoted higher physical-mechanical properties; however, inducing higher yellowing effect for the experimental composites. Key Words: camphorquinone, dental resin, curing performance. Key Words: camphorquinone, dental resin, curing performance. Introduction material (8). According to Musanje et al. (4), higher quantities of CQ promote a rapid generation of large amounts of free radicals, but this may result in a reduced ability to form an adequate polymer network, since polymers with relatively lower molecular weight are formed. On the other hand, in accordance with Schneider et al. (5), the high concentration of the photoinitiator system may guarantee a high monomer conversion and consequently higher physical and mechanical properties of the composites. Therefore, the intense yellow hue of CQ places practical limits on the concentration of photoinitiators that can be used, because of the esthetic properties, but this may not limit the degree of polymerization and depth of cure that can be attained (9). To achieve satisfactory polymerization of the resin composites, a proper combination of photoinitiator/co- initiator system, which are usually camphorquinone (CQ) and a tertiary amine, respectively (1,2) should be used. Light absorption raises CQ to an excited state that interacts with a tertiary amine, forming a triplet exciplex that disintegrates into free radicals. These radicals initiate the polymerization of methacrylates, which success conducts on obtaining a reliable bond and good polymerization (3). The concentration of CQ and amine, as well as the CQ/amine ratio, play important roles in the polymerization efficiency and ultimately in the physical and mechanical properties of resin composite materials (4-6). Conversely, if the concentration of photoinitiators is kept too low, an inadequately polymerized resin composite may result, and the physical-mechanical properties and durability of the restoration can be compromised (10). Also, an incomplete photocuring process leaves the composite with more leachable residual monomers and initiators that can increase its biological incompatibility and jeopardize the color stability (11,12). Therefore, it is important to find an optimal photoinitiator and co-initiator concentration and ratio that guarantee a satisfactory polymerization CQ is inherently yellow and can cause a color mismatch in aged aesthetic resin-based composite restorations. This mismatch becomes even more pronounced if bleaching is performed in those restored teeth (7). Flexural Strength and Elastic Modulus Immediately after the DC analysis, the same bar-shaped specimens (n=10) were used for the three-point bending flexural test. The flexural test was performed according to ISO 4049:2009, except for the specimen’s dimensions (7 mm length x 2 mm width x 1 mm thickness). The specimens were photo-activated by a LED curing unit (Radii-Cal, 900 mW/ cm2) for 20 s and dry stored in light-proof containers for 24 h at 37 °C. The three-point bending test was performed in a universal testing machine (Instron, Canton, USA. span between supports=5 mm) at a cross-head speed of 0.5 mm/ min. The maximum load for the specimens at fracture was recorded and the flexural strength (FS) calculated using the following equation: FS=3FL/(2bh2), where F is the maximum load (N) exerted on the specimens; L the distance (mm) between the supports; b is the width (mm) of the specimens measured immediately prior testing; h is the height (mm) of the specimens measured immediately prior testing. Yellowing Effect The experimental resin composites were inserted in The experimental resin composites were inserted in 727 Table 1. Concentration and ratio of the photo-initiator system of the experimental composites Composite CQ wt% DABE wt% Composite 1 (C1) 0.4 0.4 Composite 2 (C2) 0.4 0.8 Composite 3 (C3) 0.6 0.6 Composite 4 (C4) 0.6 1.2 Composite 5 (C5) 0.8 0.8 Composite 6 (C6) 0.8 1.6 Composite 7 (C7) 1.0 1.0 Composite 8 (C8) 1.0 2.0 Composite 9 (C9) 1.5 1.5 Composite 10 (C10) 1.5 3.0 Table 1. Concentration and ratio of the photo-initiator system of the experimental composites Concentration and ratio of photoinitiator system Concentration and ratio of photoinitiator system - Sigma-Aldrich Inc); and triethyleneglycol dimethacrylate Concentration and ratio of photo The elastic modulus was measured as the slope of the strain x strain curve in the linear portion, using the following equation: E=L1D310-3/4bh3d, where L1 is load (N), d: distance (mm) between the supports, b: width (mm), h: height (mm) and D: displacement (mm). Introduction Moreover, due to the tendency for internal discoloration of the resin composite, the amount of the tertiary amine may be a concern, since these amines have double bonds, which are capable of absorbing UV light and react with oxygen or other aromatic groups, causing the yellowing of the Braz Dent J 28(6) 2017 aliphatic carbon-carbon double-bond absorbance peak intensity (located at 1638 cm−1) and that of the aromatic (C…C) (located at 1608 cm−1; reference peak) were collected. The DC (%) was calculated using the following equation: DC (%)=100×[1−(R polymerized/R non-polymerized)], where R represents the ratio between the absorbance peak at 1638 cm−1 and 1608 cm−1. behavior and consequently better chemical-mechanical properties without increasing the yellowing of the composite restorations. The purpose in this study was to determine the optimal concentration and ratio of camphorquinone (CQ) and tertiary amine (DABE) in relation to the degree of conversion (DC), color stability (CS), elastic modulus (EM), flexural strength (FS), water sorption (SP) and solubility (SL) of microhybrid experimental composites. The tested hypotheses were: 1) The higher the content of CQ, the higher are the physical-mechanical properties, but also the yellowing effect of the experimental composites; 2) The higher the DABE ratio, the higher are the properties, but also the yellowing effect. Materials and Methods Resin Preparation Ten experimental resin formulations were tested in this study. The resin matrix for all formulations consisted of a combination of bisphenol glycidyl methacrylate – 29.0 wt% (BisGMA – Sigma-Aldrich Inc, St Louis, MO, USA); urethane dimethacrylate – 32.5 wt% (UDMA - Sigma-Aldrich Inc); bisphenol ethoxylate dimethacrylate – 32.5 wt% (BisEMA - Sigma-Aldrich Inc); and triethyleneglycol dimethacrylate – 6.0 wt% (TEGDMA - Sigma-Aldrich Inc). Composites were loaded at 75% by volume with silanized filler (20 wt% of 0.04 μm colloidal silica and 80 wt% of 0.7 µm Ba-Al-silicate glass with 0.7 µm – FGM, Joinville, SC, Brazil). The inhibitor, BHT (butylated hydroxytoluene; Sigma-Aldrich Inc), was added to the organic matrix in a concentration of 0.1 wt% to avoid spontaneous polymerization of the monomers. From this base-composition, ten experimental composites were produced, varying the concentration and ratio of the photo-initiator system (CQ - camphorquinone and DABE - N,N-dimethyl-p-aminobenzoic acid ethylester; Sigma- Aldrich Inc). The experimental groups are shown in Table 1. Ten experimental resin formulations were tested in this study. The resin matrix for all formulations consisted of a combination of bisphenol glycidyl methacrylate – 29.0 wt% (BisGMA – Sigma-Aldrich Inc, St Louis, MO, USA); urethane dimethacrylate – 32.5 wt% (UDMA - Sigma-Aldrich Inc); bisphenol ethoxylate dimethacrylate – 32.5 wt% (BisEMA Degree of Conversion For the degree of conversion (DC) analysis, bar-shaped specimens (7 mm length x 2 mm width x 1 mm thickness) (n=10) were made in molds of silicon and photo-activated by a LED curing unit (Radii Cal, SDI, Victoria, Australia - 900 mW/cm2), for 20 s. The energy density was standardized at 16 J/cm2. After polymerization, the specimens were removed from the molds and dry stored in light-proof containers at 37 °C for 24 h. DC was measured on the top surface of each specimen using Fourier Transformed Infrared spectroscopy with attenuated total reflectance (FTIR/ATR - Spectrum 100; PerkinElmer, Shelton, CA, USA). The absorption spectra of non-polymerized and polymerized composites were obtained from the region between 4000 and 650 cm−1 with 32 scans at 4 cm−1. The Braz Dent J 28(6) 2017 circular Teflon molds (6 mm diameter x 0.5 mm thickness) with placement of a Mylar strip on the top and bottom surfaces (n=10). Then, the composites were photoactivated for 20 using a LED curing unit (SDI, 900 mW/cm2; Radii Cal) and were dry stored in light-proof containers for 24 h at 37 °C. For the yellowing effect analysis, a spectrophotometer CM-700d (Minolta, Corp., Ramsey, NJ, USA) was utilized to measure the b-axis parameter of the CIELab system. This parameter measures the yellow color of a material, with higher b-values relating to a greater yellowing effect. no weight change) this value was recorded as m2 and the samples were returned into the desiccators. The entire mass reconditioning cycle was repeated and the constant mass (two days of no weight change) was recorded as m3. The values for water sorption (WS) and solubility (WSB), in micrograms per cubic millimeters, were calculated using the following equations: WS=(m2–m3)/V WS=(m2–m3)/V Water Sorption and Solubility Data were analyzed by one-way ANOVA and Tukey’s post-hoc test. Statistical significance was established at α=0.05 for all tests. This study was performed in compliance with ISO 4049:2009 standard specifications (except for the specimen dimensions and curing protocol) as follows. To verify the sorption (WS) and solubility (WBS), circular samples (0.5 mm thickness x 6 mm diameter) (n=5) were photo-activated by a LED curing unit (SDI, 900 mW/cm2; Radii Cal) for 20 s. The specimens were stored in desiccators containing silica gel at 37 °C and weighed daily on an analytical balance (Tel Marke; Bel Quimis, Sao Paulo, SP, Brazil) accurate to 0.0001 g, until a constant mass (m1) was obtained (i.e. three days of no weight change). Thickness (four measurements at four equidistant points on the circumference) and diameter of each specimen were measured using a digital electronic caliper (Mitutoyo Corporation, Tokyo, Japan). Mean values were used to calculate the volume (V) of each specimen (in mm³). Thereafter, the samples were stored in plastic containers with 6 mL of distilled water at 37 °C for 7 days and weighed daily after carefully wiping with absorbent paper. When constant weight was obtained (two days of Results FS, EM, DC and yellowing effect means are shown in Table 2. The composites C6, C7, C8, C9 and C10 showed higher DC when compared to the other composites (p=0.00001). For the flexural strength (FS), all composites showed similar values (p=0.41797). In relation to the elastic modulus (EM), the composites containing CQ/DABE in the 1.5/1.5 wt% (C9) and 1.5/3.0 wt% (C10) showed higher values (p=0.00001). As expected, the yellowing effect (YL) was higher for the composites having higher ratio of phothoinitiators (C9 and C10, p=0.00001). As normal distribution was not possible, the data for water sorption (WS) and water solubility (WSB) were log transformed (log x+0). After, the normalized data were submitted to one-way ANOVA and Tukey’s test (α=0.05). The means are shown in the Table 3. All groups showed similar WS means, after 7 Means followed by same capital letter in the same column are not statistically different at 5%, by Tukey’s test. Concentration and ratio of photoinitiator system For the DC analysis, Alonso et al. (15), using similar composite formulations as those in this study, found a significant reduction of monomer conversion when a experimental composite containing 0.5 wt% of CQ was compared to the one containing 1.5 wt%, i.e., the higher the initiator concentration, the higher the DC. This is consistent with the present study in which higher CQ concentrations resulted in higher DC. On the other hand, Musanje et al. (4) found that composites with 1.44 wt% of CQ, the polymer matrix would interfere badly and decline the physical properties of the resin material. In general, the DABE ratio did not influence the polymerization performance of the experimental composites, except for the composites C5 and C6 in DC. Based on this, the second hypothesis was rejected, since for the majority of the composites the DABE concentration did not influence the selected properties. This result indicates that the amount of CQ has a greater influence on the curing efficiency for this resin system than the amine content and that hydrogen abstraction with the higher amine content does not help or interfere in forming the polymer network (16). It might be suggested that the production of free radicals is not limited by the concentration of the co-initiator, but duration of curing light exposure. In this study was used a high energy density (16 J/cm2) for photoactivate the specimens, which may have contributed to compensate the effect of amine content. This is in accordance with previous investigations (24,25) where DC and rate of polymerization were independent of co- initiator concentration. Moreover, it has been suggested that hydrogen abstraction necessary for polymerization may take place without a co-initiator, possibly by direct interaction between the initiator and resin monomer, which contains active hydrogen ions (25). The higher DC for composites with higher concentration of CQ/DABE may be explained by the fact that they allow the generation of more free radicals during light application, promoting increased monomer conversion (16-18). In this study, the threshold level of photoinitiator concentration was 0.8% of CQ and 1.6% of the co-initiator (C6), since this composite reached the maximum DC with good physical properties and a low yellowing effect. However, for the EM, the higher the CQ concentration, the higher the EM values for the tested composites. This finding it is also supported by the higher DC found in these composites. C.V.S. Grohmann et al. C.V.S. Grohmann et al. 728 Table 2. Means (standard-deviation) of elastic modulus (EM), flexural strength (FS), yellowing (YL) and degree of conversion (DC) for the tested experimental composites Composite EM (GPa) FS (MPa) YL (b*) DC (%) C1 2.4 (0.5)CD 104.8 (27.5)A 13.5 (1.1)EF 56.9 (1.2)C C2 2.1 (0.4)D 107.0 (19.8)A 12.6 (2.8)F 54.9 (3.4)C C3 2.6 (0.3)CD 117.1 (23.1)A 16.3 (1.2)CDE 57.7 (3.1)C C4 2.8 (0.5)BC 110.2 (32.0)A 15.7 (1.6)ED 56.9 (3.0)C C5 2.7 (0.5)CD 121.2 (22.1)A 17.9 (0.8)BCD 61.0 (2.2)B C6 2.6 (0.2)CD 127.4 (10.7)A 16.2 (0.3)CDE 65.2 (2.2)A C7 2.6 (0.1)CD 122.9 (13.6)A 19.8 (0.6)B 67.7 (1.2)A C8 2.7 (0.2)BC 122.1 (19.9)A 19.0 (1.5)BC 66.8 (1.5)A C9 3.3 (0.4)AB 119.3 (19.3)A 24.4 (1.9)A 66.1 (1.1)A C10 3.9 (0.2)A 125.5 (21.0)A 24.2 (0.7)A 66.7 (2.5)A Means followed by same capital letter in the same column are not statistically different at 5%, by Tukey’s test Table 2. Means (standard-deviation) of elastic modulus (EM), flexural strength (FS), yellowing (YL) and degree of conversion (DC) for the tested experimental composites Table 3. Means (standard-deviation) of water sorption (WS) and water solubility (WSL) for the tested experimental composites Composite WS (µg/mm3) WSL (µg/mm3) C1 25.1 (6.9) A 13.6 (1.2) A C2 16.3 (4.0) A 13.4 (1.5) A C3 19.2 (3.0) A 8.8 (2.1) B C4 19.8 (6.0) A 4.2 (2.8) B C5 17.5 (5.5) A 3.5 (2.0) B C6 12.4 (5.0) A 3.6 (1.6) B C7 20.6 (5.3) A 9.2 (2.0) B C8 21.5 (7.0) A 6.8 (3.4) B C9 17.3 (4.2) A 4.0 (2.0) B C10 16.9 (4.3) A 1.17 (0.9) C Means followed by same capital letter in the same column are not statistically different at 5%, by Tukey’s test. Table 3. Means (standard-deviation) of water sorption (WS) and water solubility (WSL) for the tested experimental composites Braz Dent J 28(6) 2017 concentrations to maximize cure. However, the yellowing effect causing by the presence of CQ may cause difficulty in matching dental restorations, as well as result in a tendency for internal discoloration due to the amine (1, 22). Therefore, the CQ/amine concentration should be as low as possible to produce a satisfactory DC but without causing esthetic problems; this is why the formulation of C6 may be the optimal concentration of CQ/DABE in the present study, since it presents high DC with low yellowing effect. days of immersion in distilled water (p=0.15213). C.V.S. Grohmann et al. However, for the WSB, the composites containing the lower amount of the photoinitiator system presented higher WSB values after 7 days of immersion in distilled water (p=0.00001). Discussion The type and concentration of the photoinitiator system are fundamental parameters that determine the polymerization characteristics of a resin composite (1). Commercial resin composites are supplied with a wide range of concentration of these molecules and their effects on the polymerization behavior and physical-mechanical properties have not been completely elucidated. Some studies have evaluated CQ concentrations ranging from 0.05-3 wt% (4,5,13,14). In this study, the concentration of CQ ranged from 0.4 to 1.5 wt% because they are the concentrations most commonly observed in the formulation of the current composite resins. The outcomes was shown that this range affected the tested mechanical properties and the yellowing effect. Thus, the first hypothesis tested was accepted. While water sorption was similar among the composites, the composites with higher photoinitiator concentration resulted in less solubility. This result is consistent with the DC outcomes. The WS may be more dependent upon the chemical composition of the monomers, which was equivalent in the ten composites, and thus they did not show a difference in water uptake. The solubility may be more related to formulation (monomers type/concentration) and polymer network formation (monomer conversion). Composites presenting higher DC have fewer residual monomers to be leached (23). Also, these composites would be less degraded through hydrolytic degradation by the water than would composites with poorer conversion (5, 24). Resumo of model resin composites. J Esthet Restor Dent 2015;27:S41-S4 O objetivo foi avaliar a influência da concentração e proporção de canforoquinona (CQ)/amina terciária (DABE) na resistência à flexão (RF), módulo flexural (MF), grau de conversão (GC), amarelamento (AM), sorpção (SA) e solubilidade em água (SL) de compósitos experimentais. Blendas acrilatas foram preparadas com diferentes concentrações e proporções de CQ/DABE em peso, como segue: (CQ/DABE%): 0,4/0,4% (C1); 0,4/0,8% (C2); 0,6/0,6% (C3); 0,6/1,2% (C4); 0,8/0,8% (C5); 0,8/1,6% (C6); 1,0/1,0% (C7); 1,0/2,0% (C8); 1,5/1,5% (C9); 1,5/3,0% (C10). Para RF e MF, espécimes retangulares (7x2x1 mm, n=10) foram fotoativados com LED de pico único (Radii Cal) por 20 s e testados em máquina Instron (0,5 mm/min). Após, o GC dos mesmos espécimes (fragmentos) foi mensurado por FTIR. Para AM, discos de compósito (5x2 mm, n=10) foram preparados, fotoativados por 20 s e imediatamente avaliados em espectrofotômetro, considerando o aspecto b do sistema CIEL*a*b*. Para SA e SL, discos de compósito (5x0,5 mm, n=5) foram preparados e seus volumes calculados (mm³). Após desidratação, as amostras foram pesadas e armazenadas em água destilada por 7 dias, pesadas e novamente desidratadas e pesadas, para se calcular o SA e SL. Os dados foram submetidos a one-way ANOVA e teste de Tukey (5%). Os grupos C8, C9 e C10 mostraram maior GC, MF e AM, comparado aos outros grupos. No entanto, RF e SA foram similares entre todos os grupos. Ainda, C1, C2 e C3 apresentaram maior SL em 7 dias comparado aos outros grupos. Em geral, maiores concentrações de CQ promoveram melhores propriedades físico-mecânicas; no entanto, levaram ao maior amarelamento dos compósitos. 10. Oliveira DC, Rocha MG, Gatti A, Correr AB, Ferracane JL, Sinhoreti MA. Effect of different photoinitiators and reducing agents on cure efficiency and color stability of resin-based composites using different LED wavelengths. J Dent 2015;43:1565-1572. 11. Nomura Y, Teshima W, Kawahara T, Tanaka N, Ishibashi H, Okazaki M, et al.. Genotoxicity of dental resin polymerization initiators in vitro. J Mater Sci Mater Med 2006;17:29-32. 12. Oliveira DC, Rocha MG, Correa IC, Correr AB, Ferracane JL, Sinhoreti MA.The effect of combining photoinitiator systems on the color and curing profile of resin-based composites. Dent Mater 2016;32:1209- 1217. 13. Shin D-H, Rawls HR. Degree of conversion and color stability of the light curing resin with new photoinitiators systems. Dent Mater 2009;25:1030-1038. 14. Schroeder WF, Vallo CI. References 18. Schneider LFJ, Pfeifer CSC, Consani S, Prahl SA, Ferracane JL. Influence of photoinitiator type on the rate of polymerization, degree of conversion, hardness and yellowing of dental resin composites. Dent Mater 2008;24:1169-1177. 1. Park YJ, Chae KH, Rawls HR. Development of a new photoinitiation system for dental light-cure composite resins. Dent Mater 1999;15:120-127. C.V.S. Grohmann et al. 2. Pereira SG, Telo JP, Nunes TG. Towards a controlled photopolymerization of dental dimethacrylate monomers: EPR studies on effects of dilution, filler loading, storage and aging. J Mater Sci Mater Med 2008;19:3135- 3144. 19. Gonçalves F, Pfeifer CCS, Stanbury JW, Newman SM, Braga RR. Influence of matrix composition on polymerization stress development of experimental composites. Dent Mater 2010;26:697-703. 20. Brandt WC, Tomaselli LO, Correr-Sobrinho L, Sinhoreti MAC. Can phenyl-propanedione influence Knoop hardness, rate of polymerization and bond strength of resin composite restorations? J Dent 2011;39:438-447. 3. Segreto DR, Naufel FS, Brandt WC, Guiraldo RD, Correr-Sobrinho L, Sinhoreti MA. Influence of photoinitiator and light-curing source on bond strength of experimental resin cements to dentin. Braz Dent J 2016;27:83-89. 21. Geurtsen W, Spahl W, Leyhausen G. Residual monomer/additive release and variability in cytotoxicity of light-curing glass-ionomer cements and compomers. J Dent Res 1998;77:2012-2019. 4. Musanje L, Ferracane JL, Sakagushi RL. Determination of the optimal photoinitiator concentration in dental composites based on essential material properties. Dent Mater 2009;25:994-1000. 22. Moin Jan C, Nomura Y, Urabe H, Okazaki M, Shintani H. The relationship between leachability of polymerization initiator and degree of conversion of visible light-cured resin. J Biomed Mater Res 2001;58:42- 46. 5. Schneider LFJ, Cavalcante LM, Consani S, Ferracane JL. Effect of co-initiator ratio on the polymer properties of experimental resin composites formulated woth camphorquinone and phenyl- propanedione. Dent Mater 2009;25:369-375. 23. Schneider LF, Moraes RR, Cavalcante LM, Sinhoreti MA, Correr- Sobrinho L, Consani S. Cross-link density evaluation through softening tests: effect of ethanol concentration. Dent Mater 2008;24:199-203. 6. Furuse AY, Mondelli J, Watts DC. Network structures of Bis-GMA/ TEGDMA resins differ in DC, shrinkage-strain, hardness and optical properties as a function of reducing agent. Dent Mater 2011;27:497- 506. 24. Asmussen E, Peutzfeldt A. Influence of composition on rate of polymerization contraction of light-curing resin composites. Acta Odontol Scand 2002;60:146–150. 7. Alvim HH, Alecio AC, Vasconcellos WA, Furlan M, Oliveira JE, Saad JRC. Analysis of camphorquinone in composite resins as a function of shade. Dent Mater 2007;23:1245-1249. 25. Yoshida K, Greener EH. Concentration and ratio of photoinitiator system The higher DC should have improved the cohesive forces of the polymer network of these composites, which became more rigid to deformation and can ensure good clinical performance of these composites (19). Based on the results of the present study, the higher concentration of camphorquinone presented higher properties; however, it also induced a greater yellowing effect. Also, the co-initiator concentration (1:1 or 1:2) did not influence the polymerization behavior or the physical- mechanical properties of the experimental microhybrid resin composites. The photoinitiator content of a composite must be sufficient to allow an adequate polymerization (18,20). An inadequate polymerization affects biocompatibility due to the release of monomers that may damage cells in oral tissues (21). This would argue for higher CQ/amine 729 Braz Dent J 28(6) 2017 9. Salgado VE, Borba MM, Cavalcante LM, Moraes RR, Schneider LF. Effect of photoinitiator combinations on hardness, depth of cure, and color of model resin composites. J Esthet Restor Dent 2015;27:S41-S48. Resumo Effect of different photoinitiator systems on conversion profiles of a model unfilled light-cured resin. Dent Mater 2007;23:1313-1321. 15. Alonso RC, Souza-Júnior EJ, Dressano D, Araújo GA, Rodriguez JM, Hipólito VD, et al.. Effect of photoinitiator concentration on marginal and internal adaptation of experimental composite blends photocured by modulated methods. Eur J Dent 2013;7:S1-S8. 16. Ogunyinka A, Palin WM, Shortall AC, Marquis PM. Photoinitiation chemistry affects light transmission and degree of conversion of curing experimental dental resin composites. Dent Mater 2007;23:807-813. 17. Chen Y-C, Ferracane JL, Prahl SA. Quantum yield of conversion of the photoinitator camphorquinone. Dent Mater 2007;23:655-664. References Effect of photoinitiator on degree of conversion of unfilled light cured resin. J Dent 1994;22:296–299. 8. Oliveira DC, Silva CB, Muniz BV, Volpato MC, Costa AR, Sinhoreti MA. Effect of 4-(N,N-dimethylamino)phenethyl alcohol on degree of conversion and cytotoxicity of photo-polymerized CQ-based resin composites. Braz Dent J 2014;25:538-542. 8. Oliveira DC, Silva CB, Muniz BV, Volpato MC, Costa AR, Sinhoreti MA. Effect of 4-(N,N-dimethylamino)phenethyl alcohol on degree of conversion and cytotoxicity of photo-polymerized CQ-based resin composites. Braz Dent J 2014;25:538-542. Received August 2, 2017 Accepted September 11, 2017 Received August 2, 2017 Accepted September 11, 2017 730
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Effects of Psychological Capital, Coping Style and Emotional Intelligence on Self-Rated Health Status of College Students in China During COVID-19 Pandemic
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Associations of psychological capital, coping style and emotional intelligence with self-rated health status of college students in China during COVID-19 pandemic Xiangzhi Jing  Xinyang Vocational and Technical college Hongling Meng  Zhengzhou Normal University Yuping Li  Zhengzhou Normal University Lingeng Lu  Yale University School of Public Health yongcheng yao  (  806306944@qq.com ) Zhengzhou Normal University https://orci Abstract Background COVID-19 lockdown can lead to mental health problem, and the problem is heterogeneous across individuals. In this study, we aimed to explore the association between the self-reported health status, emotional coping style, emotional intelligence and positive psychological state of college students during the COVID-19 pandemic in China. Methods The questionnaires of Self-rated Health Measurement Scale (SRHMS), Emotional Intelligence Scale (EIS), Psychological Capital Questionnaire (PCQ), Simplified Coping Style Questionnaire (SCSQ) were used for online survey. The study included 367 undergraduates. Results Undergraduate sex, sibling and birth of place were associated with either psychological capital scores, or/and emotional intelligence. Online time per day had significantly negative associations with self-rated health and psychological capital scores. Multivariate analysis showed that the interaction between emotional intelligence, psychological capital and coping styles was statistically significant (β = -0.112,p =0.045) in health status with psychological capital ranking the top (β = 0.428). Mediation analysis showed that psychological capital and coping style modified the effect of emotional intelligence on health status. Conclusions: Psychological capital and coping style modified the effect of emotional intelligence on health status in college students during the COVID-19 pandemic. In this pandemic emergency, a comprehensive intervention, such as positive coping styles with optimistic emotional perception and understanding as well as social support, is an important measure against the pandemic-induced mental health problem in college students in China. Research Article Keywords: college students, coping style, emotional intelligence, health status, psychological capital Posted Date: September 9th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-141892/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Psychology Research and Behavior Management on September 7th, 2022. See the published version at https://doi.org/10.2147/PRBM.S383743. Page 1/20 Introduction An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), occurred in early January 2020 in Wuhan, China, resulting in the complete lockdown of Wuhan right before the eve of Chinese (or Lunar) New Year. To help slow the spread of highly contagious COVID-19, most of the cities in China later on took a series of prevention and control measures such as the restriction of traveling, party gathering and business operation, and social distancing, isolation and contact tracing. To meet the measurement for containing COVID-19, the Ministry of Education of China made a decision, closing all schools for the spring semester until reopen. Consequently, all students were not allowed to return to campuses for in-person class, instead stay at home and opt for virtual online courses. Such physical isolation made individuals fewer opportunities to engage with peers. In addition, the communications with others via internet increased the probability of excessive online surfing, exposing individuals to a large number of complex information streams. Therefore, the long-term isolation and lockdown or closures and stressful information on COVID-19 could result in anxiety, fear, grief and other emotional problems, consequently affecting physical, social and mental health issues 1. Mental health issues, such as difficulty in sleeping or eating, abuse alcohol An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), occurred in early January 2020 in Wuhan, China, resulting in the complete lockdown of Wuhan right before the eve of Chinese (or Lunar) New Year. To help slow the spread of highly contagious COVID-19, most of the cities in China later on took a series of prevention and control measures such as the restriction of traveling, party gathering and business operation, and social distancing, isolation and contact tracing. To meet the measurement for containing COVID-19, the Ministry of Education of China made a decision, closing all schools for the spring semester until reopen. Consequently, all students were not allowed to return to campuses for in-person class, instead stay at home and opt for virtual online courses. Such physical isolation made individuals fewer opportunities to engage with peers. In addition, the communications with others via internet increased the probability of excessive online surfing, exposing individuals to a large number of complex information streams. Introduction Therefore, the long-term isolation and lockdown or closures and stressful information on COVID-19 could result in anxiety, fear, grief and other emotional problems, consequently affecting physical, social and t l h lth i 1 M t l h lth i h diffi lt i l i ti b l h l Consequently, all students were not allowed to return to campuses for in-person class, instead stay at home and opt for virtual online courses. Such physical isolation made individuals fewer opportunities to engage with peers. In addition, the communications with others via internet increased the probability of excessive online surfing, exposing individuals to a large number of complex information streams. Therefore, the long-term isolation and lockdown or closures and stressful information on COVID-19 could result in anxiety, fear, grief and other emotional problems, consequently affecting physical, social and mental health issues 1. Mental health issues, such as difficulty in sleeping or eating, abuse alcohol Page 2/20 Page 2/20 consumption or substance, worsening chronic conditions, increased due to worry and stress over the pandemic 2-5. To alleviate this potential health problem, mental health experts provided a series of shareable resources on how to cope with COVID-19 and how to manage stress during this difficult time, educating individuals how to stabilize emotions with a positive attitude to fight against COVID-19-related stress. Although college students are young with relatively less susceptible to severe COVID-19 in comparison to the elderly (greater than 65 years old), campus closure, physical isolation (or home- confinement) and online instruction still brought negative impacts on their mental health 6. A recent study consisting a small sample size conducted in Poland reported that the COVID-19 outbreak led to high stress and low general self-rated health in University students 7. As a special population, college students’ health always can affect their parents’ and the community’s health and emotional distress. Thus, it is essential to evaluate college students’ health and assess factors influencing mental health in the context of the COVID-19 pandemic, so that strategies could be made to improve their health and achievements. Emotional intelligence refers to the ability of an individual to properly perceive and understand emotions, regulate emotions through certain strategies, and use emotional knowledge to deal with difficulties 8. Introduction In stressful situations, individuals with high emotional intelligence generally can interact with others in a receptive and appropriate way, thereby optimally adapting themselves to surrounding environments with motivation, perseverance, empathy and mental agility 9. In contrast, those with low emotional intelligence are vulnerable to stress and burnout 10. During the pandemic, nurses with high emotional intelligence have been shown to have better job performance in a study conducted in Saudi Arabia 11. Emotional intelligence could reduce the anxiety in international students studying in China during the pandemic12. Wang and colleagues reported that professional students who had a high level of emotional intelligence could keep mental health better during the pandemic13. Psychological capital refers to a set of resources an individual can use to help improve their performance on the job and their success. It consists of four aspects: self-efficacy (confidence), optimism, hope and resilience. It has been shown that psychological capital could ameliorate the negative consequences of stress on mental health, and enhance positive psychological outcome in undergraduate students 14. High psychological capital empowers individuals with resilience strength to cope up with adverse situations. The results from another study also suggest that increasing psychological capital has a protective effect on the risk of burnout and traumatic stress in social workers, augmenting the sustainability of their working conditions15. Similar results were reported in a previous study on Chinese nurses, showing that psychological capital reduced job burnout with the help from organizational commitment16. Psychological resilience, an ability to “bounce back” from a negative emotional experience and to confront stressful situations, is a major protective factor of burnout in nurses 17,18. Moreover, a recent study reported that psychological support was positively associated with better mental health19, which might alleviate psychological impact in Bangladesh university students during COVID-19 pandemic20. Psychological capital refers to a set of resources an individual can use to help improve their performance on the job and their success. It consists of four aspects: self-efficacy (confidence), optimism, hope and resilience. It has been shown that psychological capital could ameliorate the negative consequences of stress on mental health, and enhance positive psychological outcome in undergraduate students 14. High psychological capital empowers individuals with resilience strength to cope up with adverse situations. Introduction The results from another study also suggest that increasing psychological capital has a protective effect on the risk of burnout and traumatic stress in social workers augmenting the sustainability of their Page 3/20 Page 3/20 Mediating analyses showed that psychological capital had direct and indirect effect on mental health via social capital during COVID-19 pandemic21. When individuals confront stress or difficulties, different persons may take different coping strategies of either task-, emotion- and avoidance-oriented coping. Previous studies have shown that a lower burnout is accompanied with task-oriented coping, whereas an increased burnout results from emotion- oriented coping, and the long-term avoidance-oriented coping can lead to severe mental problem 22-25. Over the period COVID-19 pandemic, individuals with poor coping styles had adverse mental health of anxiety and depression 26,27. However, the anxiety and depression could be substantially alleviated if an individual had a plenty of resources (psychological capital) to use, which could make the one to take an appropriate coping strategy 28. Moreover, individuals with a strong emotional intelligence have an ability to restrain negative emotions, low self-esteem and anxiety, and they can courageously face the challenge of stress-related issues 29. Previous studies also showed that sex, marital status, and birth place were also associated with job-related stress and burnout in nurses 30,31. Female students were more anxious and stressed than males during COVID-19 pandemic in Bangladesh20. Taken together, we hypothesize that emotional intelligence, psychological capital and coping style are associated with self-rated health of college students during the COVID-19 pandemic in China. Thus, the purposes of this study were to investigate the associations among emotional intelligence, psychological capital, coping style  and health status in college students during the COVID-19 by conducting a cross- sectional survey in Henan, China, so that we can provide theoretic basis to make strategies to keep students healthy in the context of COVID-19. Participants And Methods In this study, the Cronbach’s α coefficient of the scale was 0.890. Psychological capital: The psychological capital questionnaire (PCQ) developed by Luthans et al. was used to comprehensively evaluate psychological capital. The Chinese version of PCQ was translated by Li and colleagues 34. The questionnaire contains 24 items in four measure dimensions: self-efficacy, hope, resilience and optimism. Each item uses Likert 6-level scoring method, 1 means "strongly disagree" and 6 for "strongly agree". The higher the score is, the higher the level of positive psychology for individuals, and the more resources they can use to improve their performance on the job and their success. The Cronbach’s α coefficient of the scale in this study was 0.958. Coping style:  The Simplified Coping Style Questionnaire (SCSQ) was used to measure the coping style of college students35. There are 20 items in two dimensions of positive and negative coping. The score each item is graded 0 to 3. A high score of the positive coping indicates that the individuals are more inclined to adopt positive coping styles with efforts to solve a stressful situation/problem, and a high score of the negative coping refers that the individuals are more inclined to adopt negative coping styles with distancing or avoiding a stressful situation. The sum score of coping style is equal to the positive coping score minus the negative coping score. The Cronbach’s α coefficient of the positive coping and negative coping in this study was 0.863 and 0.741, respectively. Statistical analysis:  All data analyses were performed using SPSS 18.0 software. The measurement data showed an approximate normal distribution using the normality test with both Skew and kurtosis methods. Online surfing time was grouped based on the cutoff of 3 and 6 h as previously described elsewhere36. Pearson correlation analysis was performed to examine the correlation between self-rated health, emotional intelligence, psychological capital, and coping style. ANOVA test was used to compare the difference in numerical variables with Bonferroni correction for multiple comparisons. Multivariate regression analysis was used to explore the effect of emotional intelligence, psychological capital, and coping style on and their interaction in self-rated health 37. The PROCESS plugin for SPSS was used to assess the strength of the indirect effects in mediation analyses 38. In PROCESS, total, direct and indirect effects were calculated and tested for significance 39-41. Participants And Methods Study subjects: In May 2020, we conducted a survey on the impact of COVID-19 pandemic on health status in college students in China using online questionnaires via the platform of “Questionnaire Star”. There are 25 universities in Zhengzhou, and one college was chosen by randomly sampling based on the first letter of the sorted school names, each of the schools was assigned a number for selection. The study team first contacted the class teacher for explanation of the study project. Then through WeChat class group for each class, the questionnaire was delivered to each student in the group with the instruction. A total number of 367 undergraduate students voluntarily participated and provided valid questionnaires. Among the participants, 88 (24.0%) were men, and 279 (76%) women. The age ranged from 18-24 years old, with an average of 20 years old. The basic demographics and questions include such as sex, age, birthplace, and time length spent on internet surf per day. Informed consent was obtained from all participants included in the study. Health status: Using the self-rated health measurement scale in Chinese version 32 (self-rated health measurement scale, SRHMS), the health status of each participant was evaluated. The measurement scale includes 48 items in three dimensions: physical, mental, and social health. Using 1 to 5 scoring Page 4/20 Page 4/20 method for each item, the scores of each dimension are the sum of all items in the dimension. The higher the score is, the better the health status. The Cronbach’s α coefficient of the scale in this study was 0.939. method for each item, the scores of each dimension are the sum of all items in the dimension. The higher the score is, the better the health status. The Cronbach’s α coefficient of the scale in this study was 0.939. Emotional Intelligence: The Emotional Intelligence Scale (EIS), which was developed by Wong and Law 33, has 16 items in 4 measure dimensions of emotional awareness and management of one’s own and others: self-emotional evaluation, emotional evaluation of others, emotional use and management. Using the 5-point rating method for each item, 1 represents "strongly disagree", and 5 for "strongly agree". The higher the score of the subject is, the higher the level of emotional intelligence is, and the higher ability to regulate one’s own emotions is when necessary and help others to do the same. Participants And Methods The mean value for the ab (a × b) product across the bootstrapped samples provided a point estimate of the indirect effect and 95% confidence intervals (CI). Each analysis was based on 5000 bootstrapped samples, as suggested by Preacher 40. A p value less than 0.05 was considered statistically significant. Page 5/20 Page 5/20 Page 5/20 Results Common-method variance (CMV) test Common-method variance (CMV) test Common-method variance (CMV) test The Harman’s single factor test was used to measure the common-method variance. The result showed no severe common-method variance existing in this study. Twenty-two factors had eigenvalues greater than 1, and 27.67% of the variance was explained by the first factor, which is less than the criterion of 40%42. Characteristics of self-rated health, emotional intelligence, psychological capital and coping style in college students during COVID-19 pandemic Table 1 illustrates the associations of self-rated health, emotional intelligence, psychological capital and coping style scores with demographic variables in college students of a university in China during the COVID-19 pandemic period. Male students had a significantly higher score of psychological capital than female (p < 0.05). The students who have no siblings had a significantly higher score of both emotional intelligence and psychological capital than those having siblings (p < 0.05). There were significant differences in emotional intelligence scores between the students from different areas (p < 0.05). Pairwise comparisons showed that the students from urban areas had a significantly higher emotional intelligence score than those from rural areas (p < 0.05). Significant differences were also observed in self-rated health status, and psychological capital scores between students who spent different hours per day in internet surfing (p < 0.05). With online surfing hours per day increasing, the scores of self-rated health and psychological capital significantly decreased. Students who spent less than 3 h online surfing per day had significantly better health status or higher psychological capital scores than those who spent more than 6 h (p < 0.05). Table 1 illustrates the associations of self-rated health, emotional intelligence, psychological capital and coping style scores with demographic variables in college students of a university in China during the COVID-19 pandemic period. Male students had a significantly higher score of psychological capital than female (p < 0.05). The students who have no siblings had a significantly higher score of both emotional intelligence and psychological capital than those having siblings (p < 0.05). There were significant differences in emotional intelligence scores between the students from different areas (p < 0.05). Pairwise comparisons showed that the students from urban areas had a significantly higher emotional intelligence score than those from rural areas (p < 0.05). Significant differences were also observed in self-rated health status, and psychological capital scores between students who spent different hours per day in internet surfing (p < 0.05). Common-method variance (CMV) test With online surfing hours per day increasing, the scores of self-rated health and psychological capital significantly decreased. Students who spent less than 3 h online surfing per day had significantly better health status or higher psychological capital scores than those who spent more than 6 h (p < 0.05). Page 6/20 Table 1. Associations of self-rated health, emotional intelligence, psychological capital and coping style with demographic variables in college students(mean ± SD) Variable N Self-rated health Emotional intelligence Psychological capital Coping style Sex           Male 88 335.31±50.54 46.05±6.09 107.24±16.86 0.77±0.64 Female 279 335.76±42.65 44.91±4.77 102.72±15.99 0.83±0.61 t   ﹣0.076 1.816 2.283﹡ ﹣0.824 Having Siblings           No 61 342.56±44.09 46.70±5.18 109.21±16.13 0.90±0.69 Yes 306 334.27±44.64 44.88±5.08 102.72±16.14 0.80±0.60 t   1.326 2.558﹡ 2.869﹡﹡ 1.226 Birthplace           Urban 104 338.42±45.90 46.39±4.69 106.38±16.08 0.81±0.63 Suburban 41 342.34±43.23 45.02±5.94 104.44±16.59 1.00±0.65 Rural 222 333.12±44.60 44.64±5.09 102.47±16.25 0.78±0.61 F   1.021  4.216﹡ 2.089  2.19  Online surfing time           <3h 88 342.00±43.59 45.72±5.28 107.90±16.44 0.89±0.65 3~6 h 178 338.27±42.50 45.35±5.15 104.53±14.83 0.82±0.59 >6 h 101 325.50±47.72 44.43±4.92 98.95±17.53 0.74±0.65 F   3.872﹡ 1.67  7.708﹡﹡ 1.237  ﹡P<0.05,﹡﹡P< 0.01; ﹡P<0.05,﹡﹡P< 0.01; Correlations between health status, emotional intelligence, psychological capital and coping style scores in college students Correlations between health status, emotional intelligence, psychological capital and coping style scores in college students The results of Pearson correlation analyses are shown in Table 2. There were significantly positive correlations between self-rated health, emotional intelligence, psychological capital and coping style scores in college students during the COVID-19 pandemic (p < 0.01). A moderate correlation strength was observed between either emotional intelligence, or psychological capital or coping style, or self-rated health, as well as between emotional intelligence and psychological capital (correlation coefficients ranged from 0.50 to 0.65). A relatively weak positive correlation existed between coping style score and either emotional intelligence or psychological capital (correlation coefficients 0.35 and 0.42). Page 7/20 Table 2. Pearson correlations between health status, emotional intelligence, psychological capital and coping style in college students (N = 367) Variable Mean SD Self-rated health Emotional intelligence Psychological capital Coping style   Self-rated health 335.65 44.60  1         Emotional intelligence 45.18 5.13 0.50﹡﹡ 1       Psychological capital 103.80  16.29  0.63﹡﹡ 0.65﹡﹡ 1     Coping style 0.82 0.62 0.50﹡﹡ 0.35﹡﹡ 0.42﹡﹡ 1   ﹡﹡p< 0.01. Table 2. Pearson correlations between health status, emotional intelligence, psychological capital and coping style in college students (N = 367) Table 2. Common-method variance (CMV) test Pearson correlations between health status, emotional in psychological capital and coping style in college students (N Association of self-rated health with emotional intelligence, psychological capital and coping style To investigate the effect of emotional intelligence, psychological capital and coping style on and their interactions in self-rated heath in college students during the period of COVID-19 pandemic, we performed multivariate regression analyses by constructing three different models. The results are illustrated in Table 3. All the tolerances were greater than 0.1 and VIF <10, so there is no collinearity of the variable. In the model 1, we included the main effects of three variables with sex and having siblings as covariates only. We found that the main effects explained 47.5% variation of self-rated health. The emotional intelligence, psychological capital and coping style had significantly positive effects, whereas sex and siblings were not statistically significant. The coefficient of psychological capital was the largest, suggesting that psychological capital was the largest contributor to self-rated health in the model. In the model 2, then we added pairwise interaction terms of emotional intelligence, psychological capital and coping style besides the main effects. No pairwise interaction was found statistically significant. We then added a three-way interaction term beyond the model 2 in the model 3, and we found the three-way interaction term was statistically significant (p = 0.045). The three-way interaction independently explained 0.6% variation of self-rated health. Page 8/20 Page 8/20 Table 3. Common-method variance (CMV) test diating effect of psychological capital and coping style on emotional intelligence and self-rated health Note: ∗P<0.05,∗∗P<0.01,∗∗∗P<0.001; a, the effect of the independent variable on mediating variable; b, the effect of mediating variable on self-rated health; c, the total effect of the independent variable on self-rated health; c', the direct effect of the independent variable on self-rated health after the introduction of mediating variable; a × b, the mediating effect of mediating variable between emotional intelligence and self-rated health To visualize the effect of emotional intelligence, psychological capital and coping style and its interaction in self-rated health in college students during the period of COVID-19, we further constructed a figure showing the relationship between three variables and self-rated health based on the method for a three- way interaction as described by Dawson and Richter37 (Figure 1). With the increased emotional intelligence, the self-rated health score increased regardless of psychological capital and coping style. At the same level of emotional intelligence, the self-rated health status was better in college students with high vs. low psychological capital. At the same levels of emotional intelligence and psychological capital, the self-rated health score was better in college students who actively took positive vs. negative coping styles. Overall, individuals with high psychological capital and coping style show the best health status regardless of emotional intelligence. High emotional intelligence improves health status in comparison to low one at the same level of psychological capital and coping style. Common-method variance (CMV) test Effect of emotional intelligence, psychological capital and coping style on self- rated health in college students  Variable b p value Tolerance VIF Model 1 Sex 0.056 0.146 0.961 1.040 Having Siblings 0.024 0.527 0.964 1.037 Emotional Intelligence 0.170  0.001 0.546 1.830 Psychological capital 0.419 <0.001 0.524 1.909 Coping style 0.250  <0.001 0.767 1.304 Adjusted R2 0.475       Model 2 Sex 0.053 0.176 0.936 1.069 Having Siblings 0.024 0.532 0.959 1.043 Emotional Intelligence 0.171 0.001 0.543 1.841 Psychological capital 0.415 <0.001 0.501 1.995 Coping style 0.253 <0.001 0.744 1.345 Emotional intelligence × psychological capital 0.053  0.335 0.473 2.114 Emotional intelligence × coping style -0.071 0.283 0.334 2.993 Psychological capital × coping style -0.015 0.799 0.404 2.476 Adjusted R2 0.474  ΔR2 0.003  0.517 Model 3 Sex 0.051 0.194 0.935 1.070 Having Siblings 0.028 0.476 0.957 1.045 Emotional Intelligence 0.218 <0.001 0.450 2.224 Psychological capital 0.428 <0.001 0.495 2.022 Coping style 0.289 <0.001 0.636 1.573 Emotional intelligence × psychological capital 0.068 0.217 0.464 2.154 Emotional intelligence × coping style -0.061 0.354 0.332 3.009 Psychological capital × coping style -0.018 0.757 0.404 2.478 Emotional intelligence × Psychological capital × coping style -0.112 0.045 0.457 2.186 Adjusted R2 0.478 ΔR2 0.006 0.045 To further investigate the mediating effect of psychological capital and coping style on emotional intelligence and self-rated health, we performed mediation analysis using the Bootstrap method. The results are shown in Table 4. We found that the indirect effects from both psychological capital and coping style were statistically significant. The contribution of psychological capital accounted for 58.8% and coping style for 26.1%. Page 9/20 Page 9/20 Table 4. Mediating effect of psychological capital and coping style on emotional intelligence and self-rated health    Mediating  Variable Emotional intelligence (a) Self-rated health (b) Total effect (c) Direct Effect (c’) Indirect effect (ab) (95%CI)   Percentage (ab/c) % Psychological capital 2.05∗∗∗ 1.36∗∗∗ 4.74∗∗∗ 1.95∗∗∗ 2.79 (2.10,3.51) 58.8 Coping style 0.05∗∗∗ 24.41∗∗∗ 4.74∗∗∗ 3.50∗∗∗ 1.24 (0.86,1.66) 26.1 Note: ∗P<0.05,∗∗P<0.01,∗∗∗P<0.001; a, the effect of the independent variable on mediating variable; b, the effect of mediating variable on self-rated health; c, the total effect of the independent variable on self-rated health; c', the direct effect of the independent variable on self-rated health after the introduction of mediating variable; a × b, the mediating effect of mediating variable between emotional intelligence and self-rated health. Discussion In this study, we demonstrated the associations between emotional intelligence, psychological capital, coping style and self-rated health in college students in China during the period of COVID-19 pandemic. We found all three variables were significantly positively associated with self-rated health, and there was a statistically significant three-way interaction. We also found that almost 60% of total effect of emotional intelligence on health status could be explained by psychological capital, whereas coping style accounted for 26.1%. The results suggest that college students who had high emotional intelligence, psychological capital and coping style scores had better health conditions than their peers with low ones. Particularly, psychological capital provided more resources to help students to overcome the stress challenge. We also found that psychological capital and coping style modified the effect of emotional intelligence on health status. This result extends the previous findings reported by another study recently conducted in China, in which social capital mediated the effect of psychological capital on mental health in Chinese residents during COVID-19 pandemic21. To our knowledge, this is the first study to investigate Page 10/20 the effect of emotional intelligence, psychological capital and coping style, and their interaction on college student health during the pandemic. Emotional intelligence is an important determinant in an individual’s life success, and affects a person's mental health and social status. The stronger the individual's ability to regulate emotion is, the more helpful it is for the person to relieve stress and stabilize his/her emotional state 43. A recent study demonstrated that the emotional intelligence workshops and seminars could improve academic engagement and performance, and reduce academic burnout in pharmacy students during the lockdown and quarantine 44. Individuals with a positive psychological capital score showed a more determined attitude in the face of difficulties 45, and believed that they had abilities and more resources to use to overcome difficulties and achieve success when facing the challenges. Lebares and colleagues reported that a high psychological resilience was significantly positively associated with a low risk of emotional exhaustion-related burnout and low job performance 46. Similarly, Hafsa and colleagues also observed a positive correlation between emotional intelligence and loneliness in adolescents 47. High emotional intelligence could significantly alleviate the anxiety, maintaining mental health for student during the pandemic12,13. Discussion Moreover, it has been shown that psychological support might mitigate the effect of COVID-19-caused stress in university students in Bangladesh during COVID-19 pandemic20, and that individuals with more psychological support had better mental health status19. In consistence with this principal, our study showed that at the same level of emotional intelligence, college students with a high psychological capital score had better self-rated health state than those with a low one. Coping style refers to the cognitive and behavioral styles that individuals take to deal with the frustrations and difficulties when they face in daily life. The choice of coping styles, task-oriented, emotion-oriented and avoidance-oriented, determines the consequent effect of stress events on individuals’ health 48. Individuals taking task-oriented coping will actively find a solution to solve the stress problems by such as learning new skills or obtaining more information to manage and consequently eliminate it. Emotion- oriented coping involves an emotional response to the perception of stress, alleviating the stressor- associated unpleasant stressful feelings. Avoidance-oriented coping instead involves activities and cognition to avoid the stressful situations, always accompanying with distraction and social diversion. Repeated or long-term avoidance or distancing from a stressful situation can result in a detrimental consequence to mental health. Individuals with a positive coping style usually actively try to find a problem solution (active coping), face the challenge, and seek social support with optimistic attitude. A previous study demonstrated that the positive coping styles could promote individual’s mental health with a positive attitude, and reduce psychological harm 49. For example, a negative correlation was found between positive coping style and psychological stress in infertile women 50. A similar finding was found in patients with either breast cancer or osteoarthritis, the coping style affected the mental health 51,52. Another study showed that undergraduate nursing students with higher personal resilience (positive coping style) had a lower stress during COVID-19 pandemic in India53. Similarly, students who had an Page 11/20 Page 11/20 emotion-focused coping style had a higher level of anxiety during the COVID-19 pandemic in Poland54. A two-phase longitudinal study showed that during the COVID-19 pandemic, Chinese children with active- avoidance coping style had a low level of anxiety and depression55. Negative coping style associated with psychological distress in Chinese college students56. Discussion A recent study conducted in a large university of the Mid-Atlantic region in the United States showed that extraversion and avoidance coping style moderated the effect of COVID-19 stressful life events on mental health57. Moreover, a positive correlation was observed between positive coping style and less psychological distress in Indian population during lockdown58. Furthermore, when an individual has more psychological capitals during the grow-up, it is easier for him/her to take a positive coping style to find solutions with more positive optimism when facing challenges 59, promoting the individual's health. In this study, we found that the psychological capital was a major effect on self-rated health with a statistically significant three-way interaction analysis. Seeking social support and taking a positive coping style are important in maintaining a good health status when they face a stressful situation, and it is a more active strategy even at the same level of emotional intelligence. In this study, we also found that sex, having sibling or not, birthplace and online surfing time were associated with emotional intelligence, psychological capital and coping style. Previous studies have shown that women had a higher emotional intelligence than men in the nursing profession 60,61. However, we did not find a significant difference in emotional intelligence in college students. This discrepancy is most likely duo to the difference in social experience. Undergraduate Students, particularly in China, have much less experience in society in comparison to nurses or other professionals. Male college students have a relatively more social relation, and more resources to use. Thus, when they face challenges and difficulties, generally, they are more active with courage and optimism to solve the problems in comparison to their female peers. The college students from only-one child family (no siblings) usually had a higher emotional intelligence and psychological capital score, since they probably started earlier to play together with peers from other families, learning mutual and social relationships through the interaction. Similarly, the students from urban area usually had more skills to identify and manage their own emotions and might affect others as necessary, since their family incomes were relatively higher with more opportunities to expose themselves to different cultures in urban than in rural area. In addition, although the internet emergency facilitates the access to obtain knowledge and information, the internet also provides a virtual village for users, allowing online interactions behind screen but with isolating themselves from others. Conclusion In summary, this study demonstrated the associations of emotional intelligence, psychological capital and coping style with health status in college students in China during the period of COVID-19 pandemic We found that interaction existed between psychological capital, coping style and emotional intelligence in college students’ health with the psychological capital and coping style modifying the effect of emotional intelligence on health status in college students regardless of sex and whether they had siblings or not under the pandemic stress. The findings provide knowledge for our educational administrators how to improve and maintain college students’ health, keeping them mentally healthy during the period of COVID-19 pandemic. A comprehensive intervention measure targeting emotional intelligence, coping style and psychological capital (e.g., social support) is warranted. Discussion It has been reported that dissociation positively associated with depression in married women 62. Internet addiction made college students insufficient personal communication with peers, thereby leading to mental health problem56. Interestingly, the significance of sex and siblings in the Univariate analyses disappeared in the multivariate analyses, suggesting that the associations of sex and siblings with mental health are dependent. There was a significantly positive correlation among the variables of college students’ emotional intelligence, psychological capital, and coping styles as expected. The findings suggest that college students with a high emotional intelligence have more resources and adopt a positive coping style, since Page 12/20 Page 12/20 they have a strong ability to perceive, evaluate, understand, express their own emotions and influence others’ 63. When they had more psychological capitals to use, they took more positive coping styles with optimism to face difficulties 64. Thus, a better health status was maintained. Some limitations exist in this study. This survey is a cross-sectional study, and it is difficult to make causal inference from the association between the factors we investigated and self-rated health. Secondly, the sample size is relatively small, and the survey was conducted in one college only, bias may exist and the participants may not well represent the whole population of college students. However, the findings warrant further studies with a relatively large sample size. Longitudinal or intervention studies can be carried out Some limitations exist in this study. This survey is a cross-sectional study, and it is difficult to make causal inference from the association between the factors we investigated and self-rated health. Abbreviations SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2; COVID, contagious coronavirus disease;  SRHMS, Self-rated Health Measurement Scale; EIS, Emotional Intelligence Scale; PCQ, Psychological Capital Questionnaire; SCSQ, Simplified Coping Style Questionnaire. Ethics approval and consent to participate The procedures followed were in accordance with the ethical standards of the Committee on Zhengzhou Normal University (ZZNU-2020-006). The questionnaire had an introductory paragraph explaining the purpose of the survey and the name of the research center undertaking the research, and that responses were anonymous. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Consent for publication Consent for publication Page 13/20 Availability of data and materials The dataset supporting the conclusions of this article can be shared with the corresponding author by email. Funding This study was supported by Henan Province Educational Science “13th Five-year Plan” Fund (2020YB0256). All the authors listed have approved the manuscript that is enclosed. All the authors listed have approved the manuscript that is enclosed. Authors’ contributions YY and XJ designed the research, carried out data analysis, wrote the paper, and YY is the corresponding author. HM provided guidance in study design, organized the investigation. LL, YL and XJ provided help with the data analysis, interpretation, and write-up. YL provided help with the data collection and interpretation. All authors read and approved the final manuscript. Acknowledgements: We thank all participants who voluntarily completed the questionnaires in this study. We also wish all students stay well during the pandemic and achieve their goals in their careers. Acknowledgements: We thank all participants who voluntarily completed the questionnaires in this study. W l i h ll t d t t ll d i th d i d hi th i l i th i Competing interests No conflicts of interest are declared. Page 13/20 Page 13/20 References 1. Zhang R, Jiang T, Li N, et al. The negative psychology for the public in Zhejiang province during the epidemic of human H7N9 avian influenza. Zhonghua yu fang yi xue za zhi Chinese journal of preventive medicine. 2015;49(12):1073. 1. Zhang R, Jiang T, Li N, et al. The negative psychology for the public in Zhejiang province during the epidemic of human H7N9 avian influenza. Zhonghua yu fang yi xue za zhi Chinese journal of preventive medicine. 2015;49(12):1073. Page 14/20 . Zhang R, Jiang T, Li N, et al. The negative psychology for the public in Zhejiang province during he epidemic of human H7N9 avian influenza. 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The mediating effect of emotional intelligence on adult attachment and professional adaptability of nursing college students. Journal of Nursing. 2019;26(4):75-78. Figures Figure 1 Page 19/20 Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line represents the individuals with high psychological capital and coping style; grey line represents those with high psychological capital but low coping style; tangerine line represents low psychological capital but high coping style, and orange line represents low psychological capital and coping style. Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line represents the individuals with high psychological capital and coping style; grey line represents those with high psychological capital but low coping style; tangerine line represents low psychological capital but high coping style, and orange line represents low psychological capital and coping style. Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line represents the individuals with high psychological capital and coping style; grey line represents those with high psychological capital but low coping style; tangerine line represents low psychological capital but high coping style, and orange line represents low psychological capital and coping style. Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line represents the individuals with high psychological capital and coping style; grey line represents those with high psychological capital but low coping style; tangerine line represents low psychological capital but high coping style, and orange line represents low psychological capital and coping style. Page 20/20
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A Unifying Model for Capture–Recapture and Distance Sampling Surveys of Wildlife Populations
Journal of the American Statistical Association
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1. INTRODUCTION tion is not certain, effective area sampled is obtained by in- tegrating under an estimated detection probability surface. DS methods estimate the detection probability surface by using ob- served distances to detections to estimate detection probability as a function of distance from detector. CR methods have un- til recently had no statistically rigorous method for estimating density, but this changed with the advent of spatially explicit capture–recapture (SECR) methods (Efford 2004; Borchers and Efford 2008; Royle and Young 2008; Royle et al. 2013a). SECR data do not include distances to animal locations; instead SECR methods use the distances between detectors at which animals are (and are not) detected to estimate a distance-based detection probability surface. Estimating animal population density is crucial for success- ful and efficient management and conservation of wildlife re- sources. As a complete census is rarely feasible, this usually requires survey sampling, most often using one of the two dom- inant survey methods: capture–recpature (CR) or distance sam- pling (DS; see Schwarz and Seber 1999; Borchers, Buckland, and Zucchini 2002; Williams, Nichols, and Conroy 2002; Royle and Dorazio 2008, for overviews of methods). In CR, a series of detectors (e.g., traps or cameras) are deployed on multiple sam- pling occasions. The resulting “capture history” of occasions on which each uniquely identified animal was detected is used to estimate the probability of detection, and hence account for un- detected animals. DS requires only a single survey occasion and uses the distances of detected animals from detectors to estimate the detection probability and hence account for animals missed. As it involves a distance-based detection function, SECR is closer to DS than is traditional CR, and in fact SECR methods have borrowed detection function forms from DS. At the same time, there have been developments in DS that bring it closer to CR methods. For example, standard DS methods have been extended to use two independent observers, generating capture history as well as DS data—a method known as mark recap- ture distance sampling (MRDS; Manly, McDonald, and Garner 1996; Borchers, Zucchini, and Fewster 1998). Both methods sample a subset of the area occupied by the population of interest and both require some measure of ef- fective area sampled to estimate animal density. A Unifying Model for Capture–Recapture and Distance Sampling Surveys of Wildlife Populations D. L. BORCHERS, B. C. STEVENSON, D. KIDNEY, L. THOMAS, and T. A. MARQUES A fundamental problem in wildlife ecology and management is estimation of population size or density. The two dominant methods in this area are capture–recapture (CR) and distance sampling (DS), each with its own largely separate literature. We develop a class of models that synthesizes them. It accommodates a spectrum of models ranging from nonspatial CR models (with no information on animal locations) through to DS and mark-recapture distance sampling (MRDS) models, in which animal locations are observed without error. Between these lie spatially explicit capture–recapture (SECR) models that include only capture locations, and a variety of models with less location data than are typical of DS surveys but more than are normally used on SECR surveys. In addition to unifying CR and DS models, the class provides a means of improving inference from SECR models by adding supplementary location data, and a means of incorporating measurement error into DS and MRDS models. We illustrate their utility by comparing inference on acoustic surveys of gibbons and frogs using only capture locations, using estimated angles (gibbons) and combinations of received signal strength and time-of-arrival data (frogs), and on a visual MRDS survey of whales, comparing estimates with exact and estimated distances. Supplementary materials for this article are available online. KEY WORDS: Abundance estimation; Acoustic survey; Closed population; Measurement error; Visual survey. Supplementary materials for this article are available online. Please go to www.tandfonline.com/r/JASA upplementary materials for this article are available online. Please go to www.tandfonline.com/r/JASA © David Borchers, Ben Stevenson, Darren Kidney, Len Thomas, Tiago Marques Published with license by Taylor & Francis Journal of the American Statistical Association March 2015, Vol. 110, No. 509, Theory and Methods DOI: 10.1080/01621459.2014.893884 © David Borchers, Ben Stevenson, Darren Kidney, Len Thomas, Tiago Marques This is an Open Access article distributed under the terms of the Cre- ative Commons Attribution-Non-Commercial License (http://creativecommons. org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distri- bution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. D. L. Borchers (E-mail: dlb@st-andrews.ac.uk), B. C. Stevenson (E-mail: bcs5@st-andrews.ac.uk), D. Kidney, (E-mail: dk253@st-and.ac.uk), L. Thomas (E-mail: dk253@st-and.ac.uk), and T. A. Marques (E-mail: tiago@mcs.st-and.ac.uk), Centre for Research into Ecological and Environ- mental Modelling, University of St. Andrews and The Observatory, Buchanan Gardens, Fife, KY16 9LZ, Scotland. We are grateful to the National Geographic Society/Waitt Grants Program for a grant (W184-11) for funding the frog sur- vey, to John Measey and Res Altwegg for conducting it, to Ben Rawson and Conservation International for the gibbon data, and the Marine Research In- stitute of Iceland for the minke whale data. Marques was part-funded by the Fundacao Nacional para a Ciencia e Tecnologia, Portugal (FCT) under the project PEst OE/MAT/UI0006/2011. This work was part-funded by EPSRC grant EP/I000917/1. We are grateful to two anonymous referees, which sub- stantially improved the article. C l i f f th fi i th ti l b f d li g ( ) D. L. Borchers (E-mail: dlb@st-andrews.ac.uk), B. C. Stevenson (E-mail: bcs5@st-andrews.ac.uk), D. Kidney, (E-mail: dk253@st-and.ac.uk), L. Thomas (E-mail: dk253@st-and.ac.uk), and T. A. Marques (E-mail: tiago@mcs.st-and.ac.uk), Centre for Research into Ecological and Environ- mental Modelling, University of St. Andrews and The Observatory, Buchanan Gardens, Fife, KY16 9LZ, Scotland. We are grateful to the National Geographic Society/Waitt Grants Program for a grant (W184-11) for funding the frog sur- vey, to John Measey and Res Altwegg for conducting it, to Ben Rawson and Conservation International for the gibbon data, and the Marine Research In- stitute of Iceland for the minke whale data. Marques was part-funded by the Fundacao Nacional para a Ciencia e Tecnologia, Portugal (FCT) under the project PEst OE/MAT/UI0006/2011. This work was part-funded by EPSRC grant EP/I000917/1. We are grateful to two anonymous referees, which sub- stantially improved the article. This is an Open Access article distributed under the terms of the Cre- ative Commons Attribution-Non-Commercial License (http://creativecommons. org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distri- bution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/r/jasa. 3.2 Probability Model and Likelihood Gibbons are difficult to detect visually in forest but can be detected quite easily acoustically when they make territo- rial calls. An acoustic survey with human detectors, of north- ern yellow-cheeked gibbon (Nomascus annamensis) was con- ducted in northeastern Cambodia by Conservation International in 2010. The design involved three people standing in a line spaced approximately 500 m apart, recording estimated angles to all gibbon groups they heard. Observers who detected a group comprise the group’s capture history, while the estimated angles to detected groups provide additional data on group location. Use of the additional data is shown to improve inference. Consider a survey of a region with surface area A in which K detectors are deployed on S occasions. Following Borchers and Efford (2008), we assume that animals are independently dis- tributed in this region according to a nonhomogeneous Poisson process (NHPP) with parameter vector φ and intensity D(x; φ) at x. We denote the probability that an animal at x is detected by at least one detector on the survey as p·(x; θ), with unknown parameter vector θ. It follows that the locations of detected an- imals, X = (x1, . . . , xn), are realizations of a filtered NHPP with intensity D(x; φ)p·(x; θ) at x. We construct a probability model for the outcomes of a survey via a product of conditional probabilities, which are developed below. The first component of the model is the probability of detecting n animals: P(n; φ, θ). The second is the probability density function (pdf) of animal locations, X, conditional on detection, which we write as fX(X; φ, θ). 2.2 Frog Survey An acoustic survey of Lightfoot’s moss frog (Arthroleptella lightfooti) in a water seepage on Table Mountain, South Africa, was conducted using six microphones in a roughly rectangular arrangement. The survey is similar to the gibbon survey in that spatial capture histories consist of the locations of detectors (mi- crophones) at which each vocalization (frog click) was detected. The time difference of arrival (TDOA) of the same click at dif- ferent detectors and the received SS at each detector provide additional data on animal location. Each of the additional data types improves inference in this case. The third component is the probability of observing the capture histories , conditional on detections and detected animal locations X, which we write as P( | X; θ). Here  = (ω1, . . . , ωn), where ωi is the capture history of the ith animal. The joint pdf of n, X, and  is then fn,X,(X, n, ; φ, θ) = P(n; φ, θ)fX(X; φ, θ)P( | X; θ). (1) (1) 2.3 Minke Whale Survey We now expand upon each of the terms on the RHS of this equation, after which we add a term for (possibly noisy) obser- vations of animal locations. As part of the 2001 North Atlantic Sightings Survey (NASS 2001; see Pike et al. 2009, for details), two independent ob- servers surveyed the same region of sea simultaneously from an aircraft, recording estimated distances to detected whale cues (blows). The detectors (the observers) were at the same loca- tion, and capture histories indicate which observer(s) detected each cue. Having the detectors at the same location has implica- tions for SECR analysis that we expand upon below. Additional data on whale location are contained in the estimated distances to detected cues, even though they are subject to measurement error. Use of these data is shown to substantially reduce density estimation bias. Note that our model assumes that each animal has a single x for the survey. This does not mean that animals do not move, just that x is the center of activity over the whole survey if they do move. We discuss this further in Section 5. 3.2.1 Capture History Given Location. P( | X; θ). We define an indicator variable ωiks that is 1 if animal i is detected by detector k on occasion s and is 0 otherwise, so that the capture history of animal i on occasion s is ωis = (ωi1s, . . . , ωiKs) and its full capture history is ωi = (ωi1, . . . , ωiS). It is convenient to define two indicator variables derived from ωiks, as follows: let ωi·s = 1 if animal i was detected on occasion s and ωi·s = 0 otherwise, and ωi·· = 1 if animal i is detected at all and ωi·· = 0 otherwise. Letting B(ω, p) indicate a Bernoulli probability mass function for ω, with parameter p, we can write P( | X; θ) as 2. MOTIVATING PROBLEMS tial capture history, and then extend this to include location observation data. 2.1 Gibbon Survey 1. INTRODUCTION When detec- In this article, we unify DS and CR methods and in doing so create a class of model that includes a range of models that can be viewed as hybrids of them. Examples include MRDS sur- veys with distance measurement error and SECR surveys that contain additional information about animal location, such as received acoustic signal strength (SS), precise time of acoustic detection, or estimated bearing to detected animals. We demon- strate the new class through a series of applications to both real and simulated datasets. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/r/jasa. 195 Journal of the American Statistical Association, March 2015 196 2. MOTIVATING PROBLEMS 3.1 Animal Location detaining them) and detectors that hold animals until the end of the occasion. Appendix A contains the details for each of these cases. It is also shown in this Appendix that in the case of proximity detectors with a single occasion and any survey with a single detector and multiple occasions, P( | X; θ) is identical to the conditional likelihood of Huggins (1989). 3.2.4 Location Observation Given Capture History. f (Y | X, ; γ ). Suppose now that in addition to observing ωiks for animal i on occasion s, we also observe a vector yiks = (yiks1, . . . , yiksM) containing M different kinds of data, each of which is a noisy observation of animal location. An example with M = 2 is an acoustic survey in which detectors are micro- phones and SS (yiks1) and time of arrival (yiks2) of the sound at a microphone are recorded. Writing the set of all observations yiks as Y, we write the conditional pdf of Y given X as fY|X(Y | X, ; γ ), where γ is a vector of parameters to be estimated. In the models we consider, the yiks’s are conditionally indepen- dent, given X. In general, yiksm may affect detection probability, and in this case pks(x; θ) must be replaced by pks(x, yiks; θ, γ ) in all of the above, and P(n; φ, θ), fX(X; φ, θ), P( | X; θ) become P(n; φ, θ, γ ), fX(X; φ, θ, γ ), P( | X; φ, θ, γ ). (See Efford, Dawson, and Borchers 2009b, and below.) So if the xi’s were observed, we could estimate abundance using the conditional likelihood approach of Huggins (1989), with x as the observed covariate vector. This implies that (unlike conventional CR) estimation is possible from multiple detectors on one occasion with proximity detectors, as recaptures within occasion are possible. (Efford, Borchers, and Byrom 2009a, first noted this fact.) Because animal location (x) is not observed on conventional CR studies (only locations of capture are observed), we cannot take the approach of Huggins (1989). But the location covari- ate x is observed on MRDS surveys, which involve a single occasion (S = 1) and typically two observers (K = 2), acting as independent detectors, recording locations of detections. In this case, we could use the approach of Huggins (1989). 3.1 Animal Location We use a generic notion of animal location, specified via Cartesian coordinates x = (x1, x2). In DS surveys, x is the ac- tual location of an animal at the time of the survey. If an animal moves during the survey its location x represents the average of its positions over the survey. In the context of trapping studies, these locations have variously been called, “home range cen- ters,” “centroids,” and “activity centers” (Borchers and Efford 2008; Royle and Young 2008; Royle et al. 2009a). Ideally, we would like to observe x, but this may not be possible. Below we derive a likelihood function that accommodates situations in which location is observed, in which it is partially observed or observed with error, and in which only locations of the detec- tors are observed. We develop the likelihood for SECR surveys without any information on animal locations other than the spa- P( | X; θ) = n  i=1 S s=1 B(ωi·s, p·s(xi; θ))Pr(ωis|ωi·s = 1; θ)ωi·s p·(xi; θ) (2) (2) where p·s(xi; θ) = 1 −K k=1{1 −pks(xi; θ)} is the probability that animal i at xi is detected on occasion s, pks(xi; θ) is the probability that animal i is detected by detector k on occasion s, and p·(xi; θ) = 1 −S s=1{1 −p·s(xi; θ)} is the inclusion prob- ability for animal i, that is, the probability that it is detected at all. P(ωis|ωi·s = 1; θ) is the probability that on occasion s detected animal i has capture history ωis. This probability is different for proximity detectors (which detect animals without where p·s(xi; θ) = 1 −K k=1{1 −pks(xi; θ)} is the probability that animal i at xi is detected on occasion s, pks(xi; θ) is the probability that animal i is detected by detector k on occasion s, and p·(xi; θ) = 1 −S s=1{1 −p·s(xi; θ)} is the inclusion prob- ability for animal i, that is, the probability that it is detected at all. P(ωis|ωi·s = 1; θ) is the probability that on occasion s detected animal i has capture history ωis. This probability is different for proximity detectors (which detect animals without 197 Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations is a fixed number N, then n is a binomial random variable with parameters N and p· =  R2 π(x; φ)p·(x; θ) dx. 3.3 The Likelihood Function The joint density of n, X,  and Y is f (n, X, , Y; φ, θ, γ ) = P(n; φ, θ, γ ) × fXY(X, , Y | n; φ, θ, γ ), (3) (3) × fXY(X, , Y | n; φ, θ, γ ), (3) 3.2.2 Animal Locations, Given Detection. fX(X; φ, θ). As noted above, MRDS methods assume an independent uniform distribution of animals within detectable range ( Borchers, Zucchini, and Fewster 1998). This distribution is consistent with animals being distributed according to a homogeneous Poisson process (HPP) in the plane. We make the more gen- eral assumption that animals occur according to an NHPP, with intensity D(x; φ) at x. As an animal at x is detected with prob- ability p·(xi; θ), it follows that detected animals occur accord- ing to a filtered NHPP with intensity D(xi; φ)p·(xi; θ). The pdf of x given detection is obtained using Bayes’ theorem as fx(xi; φ, θ) = D(xi; φ)p·(xi; θ)/λ(φ, θ), where λ(φ, θ) =  R2 D(x; φ)p·(x; θ) dx. Assuming independent detections, we have fX(X; φ, θ) = n i=1 fx(xi; φ, θ). The same fX(X; φ, θ) is obtained if we treat the number of animals in the area as fixed at N and assume that these animals are located independently in space with probability density π(x; φ) = D(x; φ)/  R2 D(x; φ) dx at x. where fXY(X, , Y | n; φ, θ, γ ) is the product of fX (X; φ, θ, γ ), P( | X; φ, θ, γ ) and fY|X(Y | X, ; γ ). In gen- eral, X is not observed and this density cannot therefore be evaluated. We obtain our likelihood by marginalizing over X in Equation (3): L(φ, θ, γ | n, , Y) = P(n; φ, θ, γ ) ×  R2 fXY(X, , Y; φ, θ, γ ) dX (4) L(φ, θ, γ | n, , Y) = P(n; φ, θ, γ )  (4) and we estimate φ, θ, γ by maximizing this equation with re- spect to φ, θ, γ . We obtain interval estimates using the inverse of the negative Hessian matrix, which is obtained from numeri- cal maximization of the likelihood. Model selection can be done using AIC or any other likelihood-based method. 3.3.1 Estimating Animal Location. 3.1 Animal Location This is, however, seldom done because on MRDS and other DS sur- veys with randomized sampler locations, animal locations in the vicinity of detectors can be treated as random variables with a known pdf determined by design (namely a uniform distribu- tion in the plane) and Borchers (1996) showed that using this pdf of locations in estimation usually improves MRDS estima- tor properties. Hence, the estimator of Huggins (1989), which conditions on locations, is not optimal for MRDS estimation and is generally not used for MRDS data. Instead MRDS inference is based on likelihood functions that treat X as random. These involve the conditional distribution of animal locations given detection, fX(X; φ, θ), which we now consider in more detail. Following Efford, Dawson, and Borchers (2009b), we model the expected value of the random variable ym (dropping the iks subscript for brevity here), given x, as E(ym | x) = μm(x) = g−1 m (β0m + β1mhmk(x)). Here gm is a link function, βm = (β0m, β1m) is a component of γ and the “proxy function” hmk(x) returns the component of location for which ym is a proxy, at detector k. For example, if ym is either the observed distance from detector to animal or the received SS, then hmk(X) is the true distance from detector k to the animal. 4.2 Frog Survey: SECR With Arrival Times and Signal Strength In this case, we have multivariate location data Y, comprising the TDOA and SS of detected frog clicks. We have one occasion (S = 1) and the arrangement of the six microphones (K = 6) is shown in Figure 4. Here S = 1 and we model the probability of detecting animal i with location xi in trap k on this occasion as pk1(xi; θ) = exp{−dk(xi)2/(2θ2)}, where θ ≡θ, dk(xi) is the distance from observer k (located at coordinates zk = (zk1, zk2)) to animal i at xi = (xi1, xi2): dk(xi) =  (zk2 −xi2)2 + (zk1 −xi1)2. We assume an HPP for animal locations with D(x; φ) = φ. We compare estimators using SECR methods with no location observations, using TDOA data, using SS data, and using both. We use the same forms for pk(xi; θ), dk(xi), and D(x; φ) as were used in the gibbon survey. Models for TDOA data and SS are specified below, followed by analysis and simulation results for each case. Supplementary data comprise recorded angles to animals, so M = 1 and, dropping subscripts s and m for brevity, we let yik denote the recorded angle between animal i and detector k, with respect to some reference direction. The proxy func- tion hk(xi) is the inverse tangent of (zk2 −xi2)/(zk1 −xi1)). We assume an identity link in the expectation function so that E(ym | x) = β0 + β1hk(x), and we assume angles are estimated without bias at all distances so that β0 = 0 and β1 = 1. A von Mises distribution with concentration parameter γ is used to model the angle observation error (γ ≡γ ). With independent angle observation errors, 4.2.1 TDOA Observation. As we have only one kind of supplementary location data (M = 1), we omit the m subscript and we let yik denote the time of arrival of the ith clicks at detector k. The proxy function hk(xi) is the distance function dk(xi) (in meters) used above. We assume normal errors in time of arrival, and constant variance σ 2 t of this error at all micro- phones, which is consistent with randomness in time of arrival being dominated by measurement error. 4. ANALYSES OF MOTIVATING PROBLEMS The continuum of increasingly spatially resolved spatial sam- pling models covered in this article is illustrated in Figure 1. SECR models without location observations Y are obtained by omitting fY|X(Y | X, ; γ ) from the model. Detection probability of an animal at distance zero from detectors (de- noted pks(x(k); θ), with x(k) being the location of the kth de- tector) may be constrained to be 1 or not, depending on the application. DS and MRDS models are obtained by defining fY|X(Y | X, ; γ ) to be unity at Y = X and zero elsewhere. MRDS models generally have K = 2 and S = 1 and allow pks(x(k); θ) < 1, while conventional DS models have K = 1, S = 1 and define pks(x(k); θ) = 1. To investigate the cause of the differences we plotted esti- mated locations of calling groups using Equation (5), and we conducted a simulation study (with 500 simulations) in which true parameter values were equal to those estimated using  and Y. Illustrative examples of location contours are shown in Figure 2 and the simulated sampling distributions of the two estimators is shown in Figure 3. The utility of angle data is apparent in Figure 2 in the form of much tighter contours when  and Y are used than for  alone. It is also apparent in Figure 3, which shows the “simple” estimator using only  to be biased (by about 15%), very much more dispersed and with a mode far below truth (“truth” being the density used in simulating). (Note that with three detectors there are only seven possible capture histories and hence the simple SECR model will estimate all animals to be at one of only seven locations, while with the angle data, an infinite number of locations is possible.) All the case studies below involve proximity detectors and a single occasion (so we omit subscript s), but the methods apply equally to multi-catch traps and multiple occasions. We do not include any covariates or individual random effects (other than x) in our applications for brevity and because our emphasis is on illustration of the effects of adding supplementary data. See Discussion for more on covariates. All analyses and plots were done with the R library admbsecr, written by authors of this article (see online sup- plementary material). 3.3 The Likelihood Function Given estimates ˆφ, ˆθ, and ˆγ , animal locations can be estimated from , Y by appli- cation of Bayes’ Theorem as follows (omitting ˆφ, ˆθ, and ˆγ for brevity and indicating estimates by “hats” on functions): 3.2.3 Number of Detections. P(n; φ, θ). If animals are in- dependently distributed in the plane according to an NHPP with parameter vector φ and intensity D(x; φ) at x, and they are in- dependently detected with probability p·(x; θ), it follows that n, the number of detected animals, is a Poisson random variable with rate parameter λ(φ, θ). If the number of animals in the area ˆfX|Y(X|, Y) = ˆfY|X(Y|X, ) ˆP(|X) ˆfX(X)  R2 ˆfY|X(Y|X, ) ˆP(|X) ˆfX(X) dX . (5) (5) Besides being of possible inherent interest, the pdf of animal locations, ˆfX|Y(X|, Y), is useful for illustrating the effect Journal of the American Statistical Association, March 2015 198 of the location observation data Y on the precision of location estimation, and we use it primarily for this purpose below. gibbon groups is estimated to be 0.83 groups per square kilome- ter, with a coefficient of variation (CV) of 44%, while using  and Y it is estimated to be 0.32 with CV of 23%. The differences arise as a consequence of the estimated detection function scale parameter θ being much smaller when only  is used ( ˆθ = 754 m; CV = 23%) than when Y is also used ( ˆθ = 1248 m; CV = 11%). 4.1 Gibbon Survey: SECR With Estimated Angles 4.1.1 The Model. Recall that the detectors are observers standing in a line spaced approximately 500 m apart (see Figure 2), recording estimated angles to gibbon groups they heard. We use SECR methods to estimate the density of calling groups from the locations of the observers who detected the group, both with and without the angle data. 4. ANALYSES OF MOTIVATING PROBLEMS Part of the problem is poor design: with detectors spaced only 500 m apart and scale parameter θ = 1248 the simple estimator has no information on how detection probability varies at dis- tances greater than 1000 m—because detections are never more than 1000 m apart. The angle data overcome this limitation: use of Y improves estimation. Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the article. Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the article. Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the article. incidental parameters, and to eliminate them we can base infer- ence on the likelihood of time differences of arrival (TDOAs) from the mean arrival time, conditional on the mean arrival time: σ 2 s , but unlike the time of arrival model, we estimate β0 and β1 in addition to σ 2 s , so that γ = (β0, β1, σ 2 s ). In addition, be- cause signals weaker than some specified strength c are fil- tered out at the acoustic processing stage, detection probability depends on received SS. We can write the probability of mi- crophone k detecting signal i made at a distance d(xi) from it as pk1(x, yk; θ, γ ) = 1 −Fk(c; xi, γ ), where Fk(c; xi, γ ) is the cumulative distribution function (CDF) of a normal ran- dom variable with mean exp{β0 + β1hk(xi)} and variance σ 2 s , evaluated at c. Then, fY|X(Y | X, ; γ ) ∝ n+  i=1 2πσ 2 t (1−mi)/2 exp mi k=1 (δk(xi) −¯δi)2 −2σ 2 t , (7) where n+ is the number of clicks detected on more than one microphone, mi is the number of microphones on which the ith of these was detected, γ ≡σ 2 t , δk(xi) = yik −E(yik | xi), and ¯δi = 1 mi mi k=1 δk(xi). The same likelihood can be obtained using a marginal approach, treating the β0s as random effects (see online supplementary material). For this reason, and for brevity, we do not explicitly show the conditioning on ¯δis on the LHS of the equation. fY|X(Y | X, ; γ ) = n  i=1 mi  k=1 Nk(yik; xi, γ ) 1 −Fk(c; xi, γ ), (8) (8) where mi is as before, the number of microphones on which click i was detected and Nk(yik; xi, γ ) is a normal pdf with mean exp{β0 + β1hk(xi)} and variance σ 2 s , evaluated at yik. 4.2.2 Signal Strength (SS) Observation. 4.2 Frog Survey: SECR With Arrival Times and Signal Strength We use an identity link so that E(yik | xi) = β0i + β1hk(xi), where β0i is the time the ith sound was generated and β1 is the inverse of the speed of sound in air (in meters per second). f (Y | X, ; γ ) = n  i=1 K  k=1 exp {γ cos [yik −hk(xi)]} 2πI0 (γ ) ωik , (6) (6) The time clicks are made is uninformative about location, as a click made at distance d(x) at time β0 has the same expected arrival time as one made at distance d(x) + c/β1 at time β0 −c, for any c. The β0i’s are what (Millar 2011, pp. 188–189) called where I0( ) is the modified Bessel function of order 0. where I0( ) is the modified Bessel function of order 0. 4.1.2 Results. A total of 123 detections of 77 calls were made. Using only capture histories (), the density of calling Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations 199 Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the article. Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Population 199 Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations 200 Journal of the American Statistical Association, March 2015 0 100 200 300 -0.01 0.01 0.03 0.05 Estimate as % deviation from Truth pdf of Estimate simple angle Figure 3. Smoothed simulated sampling distributions of estimated gibbon call density when only spatial capture history is used in estimation (“simple”) and when capture history and observed angles are used (“angle”). The down arrow marks true (simulated) density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions. 0 100 200 300 -0.01 0.01 0.03 0.05 pdf of Estimate simple angle Estimate as % deviation from Truth Figure 3. Smoothed simulated sampling distributions of estimated gibbon call density when only spatial capture history is used in estimation (“simple”) and when capture history and observed angles are used (“angle”). The down arrow marks true (simulated) density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions. Figure 3. Smoothed simulated sampling distributions of estimated gibbon call density when only spatial capture history is used in estimation (“simple”) and when capture history and observed angles are used (“angle”). The down arrow marks true (simulated) density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions. independent we have independent we have SECR+TDOA often differed substantially. Figure 4 shows an example for a specific click. The average difference in received SS for individual clicks was less than 2% of its mean value and it may be that the distances between microphones were too small for the contrast in received SS to be very informative about location. The same is not true of TDOA. fY|X(Y | X, ; γ ) = n+  i=1 2πσ 2 t (1−mi)/2 2T √mi exp mi k=1 (δk(xi) −¯δi)2 −2σ 2 t × n  i=1 mi  k=1 Nk(yik; xi, γ ) 1 −Fk(c; xi, γ ). (9) fY|X(Y | X, ; γ ) = n+  i=1 2πσ 2 t (1−mi)/2 2T √mi exp mi k=1 (δk(xi) −¯δi)2 −2σ 2 t × n  i=1 mi  k=1 Nk(yik; xi, γ ) 1 −Fk(c; xi, γ ). (9) We investigate estimator properties by simulation (500 simulations), using the parameter estimates from the SECR+SS+TDOA model as truth and mean sample size equal to that observed on the survey. The ideas of this section are taken from Efford, Dawson, and Borchers (2009b). M = 1 and we let yik denote the received SS at detector k. The proxy function hk(xi) is as above and we model the ex- pectation as E(yik|xi) = β0 + β1hk(xi), where β0 is the mean SS of clicks and β1 is a parameter quantifying SS loss with propagation distance. (We also tried a log link, E(yik|xi) = exp{β0 + β1hk(xi)}, but this was found to be inferior in terms of AIC: AIC = 18.) As with the time of arrival model, we assume that yik is normally distributed with constant variance, 4.2.3 TDOA and Signal Strength (SS) Observation. In this case, M = 2 and we let yik = (yik1, yik2) where yik1 is the time of arrival and yik2 is the received SS of click i at detector k. Both h1k(xi) and h2k(xi) are the distance function dk(xi) and we assume the same models as above so that E(yik|xi) = (β01 + β11h1k(xi), exp{β02 + β12h2k(xi)}), γ = (β11, σ 2 t , β02, β12, σ 2 s ), and assuming yik1, yik2 to be −2000 0 1000 3000 5000 −2000 0 1000 x y −2000 −1000 0 1000 2000 −2000 −1000 0 1000 x y Figure 2. Example location estimates, given capture, of two different gibbons. Detectors are crosses; circled detectors are those that detected the gibbon call. Arrows show estimated angles to detections. Dotted lines are the contours of the estimated probability of the group being contained within the contour, given only the spatial capture history data . Dashed lines are estimated contours, given only observed angles to detections. Solid lines are estimated contours, given capture history and angles. −2000 −1000 0 1000 2000 −2000 −1000 0 1000 x y −2000 0 1000 3000 5000 −2000 0 1000 x y x x Figure 2. Example location estimates, given capture, of two different gibbons. Detectors are crosses; circled detectors are those that detected the gibbon call. Arrows show estimated angles to detections. Dotted lines are the contours of the estimated probability of the group being contained within the contour, given only the spatial capture history data . Dashed lines are estimated contours, given only observed angles to detections. Solid lines are estimated contours, given capture history and angles. Simulated sampling distributions are shown in Figure 5. As expected, the addition of SS or TDOA reduces bias and improves precision, and there is a further small improvement in precision when both SS and TDOA data are used: the CVs for the SECR, SECR+SS, SECR+TDOA, and SECR+SS+TDOA models are 7.9%, 6.8%, 6.8%, and 6.1%, respectively. (9) 4.2.4 Comparison of Estimates With and Without TDOA, SS. A total of 590 detections of 345 frog clicks were made. Using SECR only, the click density is estimated to be 152.1 clicks per hectare per minute, with standard error 10.6 (CV = 7.0%). When SS is used these are reduced to 148.9 and 8.9 (CV = 6.0%), when TDOA is used they are reduced to 134.5 and 9.5 (CV=7.1%), and when both SS and TDOA are used, they reduce to 125.7 and 8.0 (CV = 6.4%). While both SS and TDOA reduce the point estimate of density and its standard error, the effect of SS on the point estimate is weaker. Investigation at the individual click level revealed that point estimates of click locations from the TDOA+SS model tended to agree well with those from the simple SECR model (but were more precise), while those from 4.3 Whale Survey: MRDS With Estimated Distances Smoothed simulated sampling distributions of estimated frog click density using only spatial capture history (“simple”), using capture history and time of arrival (“TDOA”), using capture history and signal strength (“SS”), and using capture history, time of arrival and signal strength (“joint”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions, expressed as percentage deviation from truth (some are almost coincident). Figure 5. Smoothed simulated sampling distributions of estimated frog click density using only spatial capture history (“simple”), using capture history and time of arrival (“TDOA”), using capture history and signal strength (“SS”), and using capture history, time of arrival and signal strength (“joint”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions, expressed as percentage deviation from truth (some are almost coincident). made one pass over animals. Standard SECR methods cannot be applied in this case because a distance-dependent detection function cannot be estimated from detectors at a single loca- tion. But with the addition of estimated distances to detections (yik for observer k’s estimate of distance to cue i), estimation is possible. (Buckland et al. 2001) and hence use a HPP for animal loca- tions with D(x; φ) = φ. This leads to the usual cue-counting pdf for radial distances of detected animals (see online sup- plementary material). We found it necessary to introduce detector-specific detection function parameters as one detec- tor was far more efficient than the other. We use pk1(xi; θk) = logit−1(θk2) exp{−dk(xi)2/(2θ2 k1)}, where θk = (θk1, θk2) (k = 1, 2) and θ = (θ11, θ12, θ21, θ22). The proxy function h1k(xi) is the distance function dk(xi). Following Borchers et al. (2009), we assume unbiased distance estimation with gamma errors, that is, E(yik|xi) = dk(xi) and MRDS survey models treat distances as being observed with- out error (see Borchers et al. 2009; Laake et al. 2011, for cue counting and point transect examples); our model readily al- lows distance measurement error to be incorporated in MRDS inference, estimating measurement error from the pairs of recorded distances of the two observers to recaptures, simul- taneously with density and detection function parameters. In this survey, measurement error is substantial, as can be seen from Figure 6. 4.3 Whale Survey: MRDS With Estimated Distances We estimate cue density allowing probability of detection at distance zero to be less than unity, both with and without the assumption of no measurement error. Were we to enforce certain detection at distance zero, we would have con- ventional distance sampling (CDS) models with and without measurement error. (See Borchers et al. 2010, for references to CDS models with measurement error.) 4.3 Whale Survey: MRDS With Estimated Distances 4.3.1 The Model. We estimate the number of minke whale cues per hectare over the sampling period from 71 detections ob- tained on the aerial cue-counting component of the NASS 2001 survey. K = 2 as there were two detectors and S = 1 as they -10 -5 0 5 10 15 0 5 10 15 -10 -5 0 5 10 15 0 5 10 15 Figure 4. Estimated location contours given capture history and SS (left) and capture history and TDOA (right), of a click. Detectors are crosses; circled detectors are those that detected the frog click. Dotted lines are the contours of the probability density of frog location given only spatial capture history data . Dashed lines in the right plot are contours given only TDOA. Solid lines are contours of location given capture history and SS (left) or capture history and TDOA (right). -10 -5 0 5 10 15 0 5 10 15 -10 -5 0 5 10 15 0 5 10 15 15 Figure 4. Estimated location contours given capture history and SS (left) and capture history and TDOA (right), of a click. Detectors are crosses; circled detectors are those that detected the frog click. Dotted lines are the contours of the probability density of frog location given only spatial capture history data . Dashed lines in the right plot are contours given only TDOA. Solid lines are contours of location given capture history and SS (left) or capture history and TDOA (right). Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations 201 -30 -20 -10 0 10 20 0.00 0.01 0.02 0.03 Estimate as % deviation from Truth pdf of Estimate simple TDOA SS joint Figure 5. Smoothed simulated sampling distributions of estimated frog click density using only spatial capture history (“simple”), using capture history and time of arrival (“TDOA”), using capture history and signal strength (“SS”), and using capture history, time of arrival and signal strength (“joint”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions, expressed as percentage deviation from truth (some are almost coincident). Estimate as % deviation from Truth Figure 5. fY|X(Y | X, ; γ ) fY|X(Y | X, ; γ ) = n  i=1 mi  k=1 dk(xi) α α (α) −1 yα−1 ik exp  −αyik dk(xi)  , (10) (10) where yik is the radial distance measurement from observer j to cue i. where yik is the radial distance measurement from observer j to cue i. For the case without measurement error, we define fY|X(Y | X, ; γ ) to be 1 if Y = X, and zero otherwise. 4.3.2 Results. When distance measurement error is accom- modated using an SECR model with estimated distance data, density is estimated to be 1.72 whale cues per hectare over the Following standard practice for DS surveys, we assume an independent uniform distribution of animals in the plane -1000 -500 0 500 1000 0 200 600 1000 x y 0 200 400 600 800 1000 1200 1400 0e+00 4e-05 8e-05 Radial distance (r) pdf(r) Figure 6. Estimated location contours (dotted) given capture history and recorded location (solid) of a whale detected by one of the two detectors. Contours are such that 100α% of the density falls between the two contours marked α. The left plot shows locations in perpendicular and forward distance space, the right curve shows it in radial distance space. Detectors are crosses. -1000 -500 0 500 1000 0 200 600 1000 x y 0 200 400 600 800 1000 1200 1400 0e+00 4e-05 8e-05 Radial distance (r) p ( ) pdf(r) Figure 6. Estimated location contours (dotted) given capture history and recorded location (solid) of a whale detected by one of the two detectors. Contours are such that 100α% of the density falls between the two contours marked α. The left plot shows locations in perpendicular and forward distance space, the right curve shows it in radial distance space. Detectors are crosses. 202 Journal of the American Statistical Association, March 2015 -50 0 50 100 0.00 0.01 0.02 0.03 Estimate as % deviation from Truth pdf of Estimate mrds dist Figure 7. Smoothed simulated sampling distributions of estimated whale cue density when capture history and exact distances are observed (“mrds”) and when capture history and estimated distances are used (“dist”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions. Estimate as % deviation from Truth Figure 7. 5.1 Model Extensions One topic that we have skirted, for lack of space, is how covariate data is incorporated into the models. Covariates can be incorporated in the density model D(x; φ) most naturally via a log link function, in the scale parameter of detection functions using a log link, and in the intercept parameter of detection functions using a logit link. Borchers and Efford (2008) and Royle et al. (2013b) contained SECR examples with a variety of explanatory variables and the former includes individual random effects. Marques and Buckland (2003) dealt with explanatory variables for DS models, and Borchers, Zucchini, and Fewster (1998) dealt with them for MRDS models. We conducted a simulation study (500 simulations) to in- vestigate the effect of neglecting measurement error on density estimates, using the parameters estimated from the model that incorporates measurement error, with mean sample size of 70, and with error CVs of 12%, 32%, and 50%. Results for the 32% case are shown in Figure 7. On the 1987 NASS survey measurement error CV was estimated to be 32% compared to 12% on the 2001 survey—see Borchers et al. (2009). All esti- mators were found to be positively biased but those from the MRDS model were (in order of increasing measurement error CVs) larger by 14%, 34%, and 68%, respectively. Biases us- ing the SECR model with measurement error were 7.7%, 7.0%, and 8.2%. As the model estimates six parameters from only 70 observations, we believe this to be small-sample bias. We have also not covered any detail of how NHPP or other models that involve nonuniform animal distribution might be implemented. Although animal distribution is typically not ho- mogeneous in space, it is usual to assume uniform spatial distri- bution in DS analyses (as a consequence of random placement of detectors), but DS estimators usually use this assumption only to estimate detection probability (estimating density con- ditional on detection probability using design-based methods). They have been found to be relatively robust to violation of the assumption at this level (see Buckland et al. 2001). Other methods may not be. Johnson, Laake, and Hoef (2010) imple- mented DS with an NHPP and Royle et al. (2013b) implemented a Bayesian version of SECR with an NHPP, with log-linear de- pendence on environmental covariates in both cases. We believe fY|X(Y | X, ; γ ) Smoothed simulated sampling distributions of estimated whale cue density when capture history and exact distances are observed (“mrds”) and when capture history and estimated distances are used (“dist”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are the means of the sampling distributions. duration of the survey (CV = 18%), and detection probability at distance zero for the two detectors (pk1(0; ˆθk), k = 1, 2) to be 1.0 (CV = 0.01%) and 0.30 (CV = 25%). When using an MRDS estimator in which distances are assumed to be error- free (as is the norm for such analyses), density is estimated to be 1.61 (CV = 17%) and pk1(0; ˆθk), k = 1, 2 to be 1.0 (CV = 0.01%) and 0.30 (CV = 23%). Figure 6 shows the contours of estimated location of a whale detected only by detector 2, when observed distance is assumed error free and when it is estimated with measurement error. when MRDS surveys are considered as SECR surveys, most have the worst possible design (detectors at the same location) and inference about density from them would be impossible without the additional location data. In the case of DS surveys, the new class of model provides a ready means for incorporating measurement error into in- ference, with or without the conventional DS assumption of certain detection at distance zero. The general model also pro- vides a framework for incorporating into SECR surveys the point independence (Laake 1999; Innes et al. 2002; Borchers et al. 2006) and limiting independence (Buckland, Laake, and Borchers 2009) methods developed in the DS literature, as a means of reducing bias due to unmodeled heterogeneity. Formulating the MRDS survey as an SECR estimation prob- lem with distance measurement error provides a ready means of accommodating both measurement error and estimation of pk1(0; θk)—something that has to date not been done in anal- yses of DS data, with the exception of a model developed by Hiby and Lovell (1998) which used distance interval data rather than continuous distance measurements. 5. DISCUSSION We have shown that DS and CR are special cases of a more general class of spatial sampling model that uses detection lo- cations to assist in estimating detection probability, and hence density. We have also shown that in the case of CR surveys, sup- plementing data on locations of captures with data on animal location (albeit noisy or incomplete) can substantially improve inference, particularly when designs are not optimal. Indeed, 203 Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations imply or require that animals do not move during the survey. Nor does it require that movement between occasions on a multi occasion survey (S > 1) be modeled; providing that either (a) single- or multi-catch traps are used, or (b) occasions are long enough that the distribution of points that an animal visits over the duration of an occasion is the same as that over the duration of the whole survey. In the former case, there is no information on animal movement within occasions and the location is by def- inition the center of activity across occasions. In the latter case, the center of activity across occasions is identical to that within occasions. If proximity detectors are used and (b) above does not hold, then the detection functions within occasion will differ from those across occasions (typically having shorter ranges for shorter occasions). Models that do not allow for this are mis- specified and may produce biased estimates. This problem can usually be avoided by having a design with occasions that are sufficiently long. there is a need for more flexible models that are not neces- sarily monotonic in their dependence on explanatory variables, and expect that these will be developed in the near future. This could be achieved using penalized regression splines, in a sim- ilar way to that in which Gimenez et al. (2006) used them to model nonmonotonic dependence of survival probability in an open-population capture–recapture model. Bayesian and frequentist versions of SECR have been devel- oped in parallel by different authors. Bayesian inference tends to be particularly useful in the presence of latent variables or ran- dom effects—and animal locations are latent variables in SECR models. However, marginalization over locations involves a sim- ple two-dimensional integral when locations are independent, making maximum likelihood inference straightforward. APPENDIX A: VARIETIES OF P( | X; θ) Multi-catch traps detain animals until the end of the sampling occasion in which they are trapped (and do not fill up). Proximity detectors are detectors that do not detain animals and therefore allow captures of the same animal on different traps within occasions. In some proximity detector applications, it is possible to detect the same animal more than once at the same detector. In this case, either binary capture histories of the sort used in the body of this article can be used or the capture frequency of each animal at each trap on each occasion can be recorded. 5.2 Robustness and Diagnostics A final important issue that remains to be resolved for this class of model, and indeed for many CR models of any sort, is how to deal with uncertain recapture identification, as this can be fraught with uncertainty when animals are not physically tagged. This general problem was addressed by Link et al. (2010) for example, while Bonner (2013) and work referenced therein ad- dressed the issue when there are multiple sources of individual identification. None of these methods explicitly use location in- formation and we expect that methods that use location data to quantify the probability that detections are recaptures will be useful, as they were in the MRDS analysis of Hiby and Lovell (1998). The robustness of estimators within the class of models de- veloped in this article to failures of assumptions is likely to be case-specific. DS point estimators of density tend to be robust to failure of the assumption of independent uniform animal dis- tribution (see Buckland et al. 2001, p. 36), although interval estimators are not. Efford, Borchers, and Byrom (2009a) found SECR point and interval estimators with multi catch traps to be robust to failure of assumptions of independence and uniformity (see Table 4, p. 266), and also found density estimates to be little affected by the form assumed for the detection function. Goodness-of-fit diagnostics are well developed for DS detec- tion function estimators, using observed locations (see Buckland et al. 2004, pp. 385–389). Similar diagnostics when locations are not observed remain to be developed (for both DS and SECR es- timators). Borchers and Efford (2008) proposed a Monte Carlo goodness-of-fit test based on scaled deviance for the overall fit of SECR models but this does not distinguish between lack of fit of the animal density model and lack of fit of the detection model. This is an area that would benefit from further research. 5. DISCUSSION In this case, both approaches work well and it is largely a matter of per- sonal preference which is used. Maximum likelihood estimation has to date proved to be much faster than the MCMC methods used for Bayesian inference, even when a random effect for un- modeled heterogeneity in detection probability is incorporated in the model. It seems likely that the Bayesian approach will come into its own when there is a more complicated latent vari- able structure—when there is dependence between latent vari- ables, for example. In such cases, the marginalization required for maximum likelihood inference may become infeasible. We expect that models that do not involve independent distribution of animal locations (as NHPPs do) will soon be developed, as animals are often not independently distributed. A simple but common case is when animals occur in groups; in this case, animals within the group may not be detected independently of one another. This can often be dealt with by treating the group as the detection unit while simultaneously estimating mean group size if individual animal density is of interest, but in other cases models for spatial dependence may be required. When activity centers move between occasions, an additional model layer for activity center movement will be required in general. The simplest such model is probably one in which the activity centers on each occasion are independent random effects with mean equal to an animal’s activity center across all occasions. But we believe that this will not be an adequate model in many applications, because activity centers on consec- utive occasions are likely to be correlated. If activity centers are observed on some (but not all) occasions, the methods of Lan- grock and King (2013) and of references therein may be useful for modeling activity centers that were not observed, conditional on those that were. (If animal activity centers are the same for all occasions and some but not all are observed, the likelihood is like Equation (4), but with integration over only those centers that were not observed.) 5.3 Animal Movement The methods of this article assume a single location (activity center) for each animal over the whole survey, but this does not In the case of multi catch traps, all but one of ωi1s, . . . , ωiKs are zero and Pr(ωis|ωi·s = 1; θ) is a multinomial distribution with index Journal of the American Statistical Association, March 2015 Journal of the American Statistical Association, March 2015 204 1 and probabilities pks(xi; θ)/  k pks(xi; θ) (k = 1, . . . , K). Model- ing pks(xi; θ) using a competing hazard formulation (see Borchers and Efford 2008), pks(xi; θ) = rks(xi; θ)p·s(xi; θ), where rks(xi; θ) is de- fined as hks(xi; θ)/ h·s(xi; θ), the relative hazard of detection at trap k on occasion s for an animal at xi, hks(xi) is the detection hazard at trap k and h·s(xi) =  k hks(xi) is the total hazard on the occasion. Hence,  k pks(xi; θ) = p·s(xi; θ) and the multinomial probabilities are r1s(xi; θ), . . . , rKs(xi; θ). 1 and probabilities pks(xi; θ)/  k pks(xi; θ) (k = 1, . . . , K). Model- ing pks(xi; θ) using a competing hazard formulation (see Borchers and Efford 2008), pks(xi; θ) = rks(xi; θ)p·s(xi; θ), where rks(xi; θ) is de- fined as hks(xi; θ)/ h·s(xi; θ), the relative hazard of detection at trap k on occasion s for an animal at xi, hks(xi) is the detection hazard at trap k and h·s(xi) =  k hks(xi) is the total hazard on the occasion. Hence,  k pks(xi; θ) = p·s(xi; θ) and the multinomial probabilities are r1s(xi; θ), . . . , rKs(xi; θ). Buckland, S. T., Anderson, D. R., Burnham, K. P., Laake, J. L., Borchers, D. L., and Thomas, L. J. (2001), Introduction to Distance Sampling, Oxford: Oxford University Press. [201,202,203] Buckland, S. T., Anderson, D. R., Burnham, K. P., Laake, J. L., Borchers, D. L., and Thomas, L. J. (2004), Advanced Distance Sampling, Oxford: Oxford University Press. [203] Buckland, S. T., Laake, J. L., and Borchers, D. L. (2009), “Double-Observer Line Transect Methods: Levels of Independence,” Biometrics, 66, 169–177. [202] Efford, M. G. (2004), “Density Estimation in Live-Trapping Studies,” Oikos, 106, 598–610. [195] In the case of proximity detectors with binary ωiks, Pr(ωis|ωi·s = 1; θ) is written as K k=1 B(ωiks, pks(xi; θ))/p·s(xi; θ). 5.3 Animal Movement In the case of proximity detectors with frequency data in which ωiks is the fre- quency of detection on detector k on occasion s, Royle et al. (2009b) proposed a Poisson model for ωi·s, such that Pr(ωis|ωi·s = 1; θ) is K k=1 Po(ωiks, λ0pks(xi; θ))/p·s(xi; θ), where Po(x, λ) is a Poisson dis- tribution with parameter λ. Efford, M. G., Borchers, D. L., and Byrom, A. E. (2009a), “Density Estima- tion by Spatially Explicit Capture-Recapture: Likelihood-Based Methods,” in Modeling Demographic Processes in Marked Populations, eds. D. L. Thompson, E. G. Cooch, M. J. Conroy, New York: Springer, pp. 255–269. [197,203] Thompson, E. G. Cooch, M. J. Conroy, New York: Springer, pp. 255–269. [197,203] Efford, M. G., Dawson, D. K., and Borchers, D. L. (2009b), “Population Density Estimated From Locations of Individuals on a Passive Detector Array,” Ecology, 90, 2676–2682. [197,199] With binary capture histories, Equation (2) reduces to Equation (A.1) below for proximity detectors when K = 1 and it reduces to Equation (A.2) with either kind of detector when S = 1. Gimenez, O., Crainiceanu, C., Barbraud, C., Jenouvrier, S., and Morgan, B. J. T. (2006), “Semiparametric Regression in Capture–Recapture Modelling,” Biometrics, 62, 691–698. [203] Hiby, L., and Lovell, P. (1998), “Using Aircraft in Tandem Formation to Estimate Abundance of Harbour Porpoise,” Biometrics, 54, 1280–1289. [202,203] P(K=1)( | X; φ, θ) = n  i=1 S s=1 B(ωi1s, p1s(xi; θ)) p·(xi; θ) (A.1) P(S=1)( | X; φ, θ) = n  i=1 K k=1 B(ωik1, pk1(xi; θ)) p·(xi; θ) . (A.2) P(K=1)( | X; φ, θ) = n  i=1 S s=1 B(ωi1s, p1s(xi; θ)) p·(xi; θ) (A.1) P(S=1)( | X; φ, θ) = n  i=1 K k=1 B(ωik1, pk1(xi; θ)) p·(xi; θ) . (A.2) P(K=1)( | X; φ, θ) = n  i=1 S s=1 B(ωi1s, p1s(xi; θ)) p·(xi; θ) (A.1) P(S=1)( | X; φ, θ) = n  i=1 K k=1 B(ωik1, pk1(xi; θ)) p·(xi; θ) . (A.2) Huggins, R. M. (1989), “On the Statistical Analysis of Capture Experiments,” Biometrika, 76, 133–140. [197,204] Innes, S., Heide-Jorgensen, M. P., Laake, J. L., Laidre, K. L., Cleator, H. J., Richard, P., and Stewart, R. E. A. (2002), “Surveys of Belugas and Narwhals in the Canadian High Arctic in 1996,” NAMMCO Scientific Publications, 4, 169–190. [202] Johnson, D. S., Laake, J. L., and Hoef, J. M. Ver. SUPPLEMENTARY MATERIALS Langrock, R., and King, R. (2013), “Maximum Likelihood Estimation of Mark- Recapture-Recovery Models in the Presence of Continuous Covariates,” Annals of Applied Statistics, 7, 1709–1732. [203] Conventional point transect likelihood as a special case; Derivation of random effect TDOA distribution; Details of the R library admbsecr. Link, W. A., Yoshizaki, J., Bailey, L. L., and Pollock, K. H. (2010), “Uncovering a Latent Multi-Nomial: Analysis of Mark-Recapture Data With Misidenti- fication,” Biometrics, 66, 178–185. [203] R library admbsecr. Manly, B. F. J., McDonald, L. L., and Garner, G. W. (1996), “Maximum Like- lihood Estimation for the Double-Count Method With Independent Ob- servers,” Journal of Agricultural, Biological and Environmental Statistics, 1, 170–189. [195] Marques, F. F. C., and Buckland, S. T. (2003), “Incorporating Covariates Into Standard Line Transect Analyses,” Biometrics, 59, 924–935. [202] [Received February 2013. Revised February 2014.] 5.3 Animal Movement (2010), “A Model-Based Approach for Making Ecological Inference From Distance Sampling Data,” Biometrics, 66, 310–318. [202] These equations have the same form as the conditional likelihood of Huggins (1989). Equation (A.1) corresponds to the conventional CR case—in which there is usually more than one trap but all traps together are treated as a single composite trap, effectively with one location. Laake, J. L. (1999), “Distance Sampling With Independent Observers: Reduc- ing Bias From Heterogeneity by Weakening the Conditional Independence Assumption,” in Marine Mammal Survey and Assessment Methods, eds. G. W. Amstrup, S. C. Garner, J. L. Laake, B. F. J. Manly, L. L. McDonald, and D. G. Robertson, Rotterdam: Balkema, pp. 137–148. [202] Laake, J. L., Collier, B. A., Morrison, M. L., and Wilkins, R. N. (2011), “Point- Based Mark-Recapture Distance Sampling,” Journal of Agricultural, Bio- logical and Environmental Statistics, 16, 389–408. [201] REFERENCES Millar, R. B. (2011), Maximum Likelihood Estimation and Inference, Chichester, UK: Wiley. [198] Bonner, S. (2013), “Response to: A New Method for Estimating Animal Abun- dance With Two Sources of Data in Capture-Recapture Studies,” Methods in Ecology and Evolution, 4, 585–588. [203] Pike, D. G., Paxton, C. G. M., Gunnlaugsson, Th., and Vikingsson, G. A. (2009), “Trends in the Distribution and Abundance of Cetaceans From Aerial Sur- veys in Icelandic Coastal Waters, 1986–2001,” NAMMCO Scientific Publi- cations, 7, 117–142. [196] Borchers, D. L. (1996), “Line Transect Abundance Estimation With Uncertain Detection on the Trackline,” PhD Thesis, University of Cape Town, Cape Town. [197] Royle, J. A., Chandler, R. B., Sollman, R., and Gardner, B. (2013a), Spatial Capture-Recapture, Boston: Academic Press. [195] Borchers, D. L., Buckland, S. T., and Zucchini, W. (2002), Estimating Animal Abundance: Closed Populations, London: Springer. [195] Royle, J. A., Chandler, R. B., Sun, C. C., and Fuller, A. K. (2013b), “Integrating Resource Selection Information With Spatial Capture-Recpature,” Methods in Ecology and Evolution, 4, 520–530. [202] Borchers, D. L., and Efford, M. G. (2008), “Spatially Explicit Maximum Like- lihood Methods for Capture-Recapture Studies,” Biometrics, 64, 377–385. [195,196,202,203] Royle, J. A., and Dorazio, R. M. (2008), Hierarchical Modeling and Inference in Ecology, London: Academic Press. [195] Borchers, D. L., Laake, J. L., Southwell, C., and Paxton, C. G. M. (2006), “Accommodating Unmodelled Heterogeneity in Double-Observer Distance Sampling Surveys,” Biometrics, 62, 372–378. [202] Royle, J. A., Karanth, K. U., Gopalaswamy, A. M., and Kumar, N. S. (2009a), “Bayesian Inference in Camera-Trapping Studies for a Class of Spatial Capture-Recapture Models,” Ecology, 90, 3233–3244. [196] Borchers, D. L., Marques, T. A., Gunlaugsson, Th., and Jupp, P. (2010), “Esti- mating Distance Sampling Detection Functions When Distances are Mea- sured With Errors,” Journal of Agricultural, Biological and Environmental Statistics, 15, 346–361. [201] Royle, J. A., Nichols, J. D., Karanth, K. U., and Gopalaswamy, A. M. (2009b), “A Hierarchical Model for Estimating Density in Camera-Trap Studies,” Journal of Applied Ecology, 46, 118–127. [204] Royle, J. A., and Young, K. V. (2008), “A Hierarchical Model for Spatial Capture–Recapture Data,” Ecology, 89, 2281–2289. [195,196] Borchers, D. L., Pike, D., Gunlaugsson, Th., and Vikingson, G. (2009), “Minke Whale Abundance Estimation From the NASS 1987 and 2001 cue Counting Surveys Taking Account of Distance Estimation Errors,” North Atlantic Marine Mammal Commission Publications, 7, 201–220. [201,202] p p gy Schwarz, C. J., and Seber, G. A. F. REFERENCES (1999), “Estimating Animal Abundance: Review III,” Statistical Science, 14, 427–456. [195] Review III,” Statistical Science, 14, 427–456. [195] Williams, B. K., Nichols, J. D., and Conroy, M. J. (2002), Analysis and Man- agement of Animal Populations, London: Academic Press. [195] Borchers, D. L., Zucchini, W., and Fewster, R. (1998), “Mark-Recapture Models for Line Transect Surveys,” Biometrics, 54, 1207–1220. [195,197,202]
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Short-term availability of adult-born neurons for memory encoding
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To cite this version: Jérémy Forest, Mélissa Moreno, Matthias Cavelius, Laura Chalençon, Anne Ziessel, et al.. Short- term availability of adult-born neurons for memory encoding. Nature Communications, 2019, 10 (1), ￿10.1038/s41467-019-13521-7￿. ￿hal-03370736￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03370736 https://hal.science/hal-03370736v1 Submitted on 8 Oct 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 ARTICLE 1 INSERM, U1028; CNRS, UMR5292; Lyon Neuroscience Research Center, Neuroplasticity and Neuropathology of Olfactory Perception Team, Lyon F-69000, France. 2 Claude Bernard University-Lyon1 and University of Lyon, Lyon F-69000, France. 3These authors contributed equally: Jérémy Forest, Mélissa Moreno. 4These authors jointly supervised this work: Anne Didier, Nathalie Mandairon. *email: nathalie.mandairon@cnrs.fr ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 A F(1,106) = 9.89; p = 0.002). Enriched animals were able to dis- criminate the two enantiomers of limonene from T1 to T3 (Paired t-test, p < 0.05 between Hab4 and Test) but not after the longer time periods T4 and T5 (Paired t-test, p > 0.05 between Hab4 and Test; Supplementary Table 1, Fig. 1b). To assess the specificity of learn- ing, discrimination of another pair of similar odorants, decanal/ dodecanone (dec/dodec), to which the animals had not been enri- ched was also tested. No discrimination of dec and dodec was seen after enrichment with +lim and −lim, (ANOVA group effect: F(5,90) = 1.18, p = 0.32; trial effect: F(1,90) = 0.05; p = 0.83, p > 0.05 between Hab4 and Test) (Supplementary Table 1; Fig. 1c). NE control animals did not discriminate any of the two odorant pairs (Paired t-test, T1: p > 0.05 between Hab4 and Test; Supplementary Table 1; Fig. 1b, c). A ll animals are exposed to a changing olfactory environ- ment requiring constant adjustments of odor perception and memory, in order for the olfactory system to effi- ciently guide the animal’s behavior. Adult-born neurons provided by neuroblasts formed in the subventricular zone1–4, constantly integrate the olfactory bulb (OB) and underlie olfactory learn- ing5–14. However it has been proposed that, in the hippocampus, adult neurogenesis promotes forgetting. Indeed, an increase in hippocampal neurogenesis tends to enhance memory clearance thereby reducing proactive interference while decreased neuro- genesis prevents forgetting and reduces behavioral flexibility15–17. Thus, the neuronal turnover provided by adult neurogenesis could sub-serve memory formation and regulate the transience of the memory trace. This raises the issue of how this ‘remembering/ forgetting’ balance is regulated as a function of environmental demand, allowing the animal to keep relevant information in memory for optimal behavioral adaptation. However, the para- meters determining memory persistence or forgetting such as information relevance or the time between successive inputs and to what extent they depend on adult-born neurons are unknown. More specifically, adult-born neurons show a critical period during which they can be recruited by learning6 but whether they can support successive memory traces within this critical period and whether they suffer from memory interference are unknown. ARTICLE g To assess adult-born neuron survival, a cohort of adult-born neurons present in the OB at the beginning of learning was labeled by injecting the DNA synthesis marker 5′-bromo-2′- deoxyuridine (BrdU) 8 days before learning8,9. BrdU-positive cells were counted in the granule cell layer for each time period post-learning (T1 to T5). We did not assess neurogenesis in the glomerular cell layer of the OB since we had previously found no modulation of the rate of neurogenesis in this layer after perceptual learning8. Analysis was performed based on learning performance, by comparing animals who discriminated after learning (T1, T2, and T3) to those who did not (T4 and T5) and to their corresponding NE groups (ANOVA, F(3 32) = 4.1, p = 0.014; Fig. 1d and Supplementary Fig. 2). We observed a higher density of BrdU-positive cells in enriched groups able to discriminate the two odorants used for enrichment compared to enriched groups unable to do so (T1-T2-T3 versus T4-T5, t-test, p = 0.015, Fig. 1d) and compared to NE groups (enriched T1-T2-T3 vs non enriched T1-T2-T3, t-test, p = 0.028, Fig. 1d and Supplementary Fig. 2). Accordingly, the density of BrdU- positive cells was highly correlated to the index of discrimination (Pearson correlation: R = 0.84; p = 0.038) (Fig. 1e). The density of BrdU-positive cells showed no decline over the time points in NE animals (ANOVA, F(4,11) = 0.8, p = 0.5, Supplementary Fig. 2). y y Here, using the olfactory system in mice, we tackle the issue of how the acquisition of new memories influences the information already stored in the network and we identify the role of adult- born neurons in these processes. We used perceptual olfactory discrimination learning, which depends on newly formed neu- rons in the OB8 to show that (1) adult-born neurons saved by learning are present in the OB as long as the task is remembered; (2) with a second learning task occurring soon after the first, introducing retrograde interference, the new memory overwrites the older one and alters survival of previously recruited adult- born neurons unless (3) the first learned odorants are maintained in the environment. Finally, using sequential labeling of adult- born neurons and selective optogenetic inactivation, we showed (4) that each successive learning is supported by a specific population of adult-born neurons. animals (ANOVA, F(4,11) = 0.8, p = 0.5, Supplementary Fig. 2). ARTICLE To determine whether the involvement of adult-born cell population in processing of the learned odorants relates to the discrimination ability, we assessed the density of cells co- expressing BrdU and Zif268 as an index of adult-born cell activation at the different time points post learning in three experimental groups. More precisely, we assessed the responsive- ness of adult-born cells to +lim (odorant used for the enrichment) in enriched and NE animals. In addition, we assessed the response to dec (odorant not used for enrichment) of adult-born cells in +lim/−lim enriched animals (Fig. 1f). A 2- way ANOVA showed a group effect (F(2,40) = 33.68, p < 0.0001), a time effect (F(2,40) = 14.08, p < 0.0001) and an interaction (F(8,40) = 8.13, p < 0.0001) indicating that the differences in BrdU/Zif268 positive cell density observed between groups depend on the time post learning. Interestingly, the density of Zif268 expression in adult-born neurons was significantly higher in enriched animals in response to +limonene at T1 to T3 compared to the other groups (Tukey tests, T1 versus T4 and T5, p = 0.0003; T2 versus T4 and T5, p = 0.006; T3 versus T4, p = 0.0005 and T3 versus T5, p = 0.0006). This result is in accordance with the performance of discrimination (Fig. 1f). Regarding the NE group, no time effect was observed (ANOVA, F(4,15) = 4.66, p = 0.84). These changes were underlined not only by changes in the density of BrdU positive cells but also by changes in percentage of BrdU/Zif268 positive cells (Supplementary Fig. 3). Notably, a higher percentage of BrdU/Zif268 was found in enriched animals at T1-T3 compared to T4-T5 (t-test, p = 0.00022) in response to +lim but not dec, indicating of a higher involvement of BrdU positive cells in processing the learned Short-term availability of adult-born neurons for memory encoding https://doi.org/10.1038/s41467-019-13521-7 OPEN Jérémy Forest1,2,3, Mélissa Moreno1,2,3, Matthias Cavelius1,2, Laura Chalençon1,2, Anne Ziessel1,2, Joëlle Sacquet1,2, Marion Richard1,2, Anne Didier1,2,4 & Nathalie Mandairon 1,2,4* Jérémy Forest1,2,3, Mélissa Moreno1,2,3, Matthias Cavelius1,2, Laura Chalençon1,2, Anne Ziessel1,2, Joëlle Sacquet1,2, Marion Richard1,2, Anne Didier1,2,4 & Nathalie Mandairon 1,2,4* Adult olfactory neurogenesis provides waves of new neurons involved in memory encoding. However, how the olfactory bulb deals with neuronal renewal to ensure the persistence of pertinent memories and the flexibility to integrate new events remains unanswered. To address this issue, mice performed two successive olfactory discrimination learning tasks with varying times between tasks. We show that with a short time between tasks, adult-born neurons supporting the first learning task appear to be highly sensitive to interference. Furthermore, targeting these neurons using selective light-induced inhibition altered memory of this first task without affecting that of the second, suggesting that neurons in their critical period of integration may only support one memory trace. A longer period between the two tasks allowed for an increased resilience to interference. Hence, newly formed adult-born neurons regulate the transience or persistence of a memory as a function of information relevance and retrograde interference. 1 INSERM, U1028; CNRS, UMR5292; Lyon Neuroscience Research Center, Neuroplasticity and Neuropathology of Olfactory Perception Team, Lyon F-69000, France. 2 Claude Bernard University-Lyon1 and University of Lyon, Lyon F-69000, France. 3These authors contributed equally: Jérémy Forest, Mélissa Moreno. 4These authors jointly supervised this work: Anne Didier, Nathalie Mandairon. *email: nathalie.mandairon@cnrs.fr NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 1 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Non enriched ± lim enrichment ± lim enrichment ± lim enrichment ± lim enrichment Test Test Test Test Test Test T5 T4 T3 T2 T1 ± lim enrichment 0 8 17 Enrichment period 24 34 44 54 64 Days a Non enriched ± lim enrichment ± lim enrichment ± lim enrichment ± lim enrichment Test Test Test Test Test Test T5 T4 T3 T2 ± lim enrichment 0 4 +lim/–lim dec/dodec R = 0.84, p = 0.038 3 2 Investigation time (s) Brdu-positive cell density (cell mm–2) Brdu-positive cell density (cell mm–2) BrdU-Zif268-positive cell density (cell mm–2) Investigation time (s) 1 0 4 3 Hab4 Test Non-enriched and stimulated +lim Enriched and stimulated +lim Enriched and stimulated dec Hab4 Test 2 1 0 NE 70 60 50 40 30 25 20 15 10 5 0 T1 T2 T3 Discrimination index 0.6 0.1 0.2 0.3 0.4 0.5 60 50 40 30 20 10 0 T1 T2 T3 T4 T5 T4 T5 T1 T2 T3 T4 T5 NE T1 T2 T3 T4 T5 8 17 Enrichment period 24 34 44 54 64 Days b c d e f Fig. 1 The presence of adult-born neurons correlates with mnesic performance. a Experimental design. b–f Behavioral results. b Discrimina lim and -lim is assessed at different time points after the enrichment period. Non-enriched control (NE; n = 9) animals do not discriminat −lim at T1. After enrichment, the two odorants are discriminated at T1 (n = 11), T2 (n = 5), and T3 (n = 10) but not at T4 (n = 9) and T5 (n dodec are not discriminated at any time post-learning (NE n = 9; T1 n = 10; T2 n = 7; T3 n = 8; T4 n = 5; T5 n = 10) (for clearer representat belonging to the Test trial of the NE group is not shown (investigation time = 6 s)). T-test, *p < 0.05. d Adult-born neuron density in the experimental groups. Groups that discriminated +lim/−lim (T1–T3) have a higher BrdU-positive cell density compared to groups that do (T4-T5) (n = 4/group), t-test *p < 0.05. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 c Dec and dodec are not discriminated at any time post-learning (NE n = 9; T1 n = 10; T2 n = 7; T3 n = 8; T4 n = 5; T5 n = 10) (for clearer representation, 1 data point belonging to the Test trial of the NE group is not shown (investigation time = 6 s)). T-test, *p < 0.05. d Adult-born neuron density in the OB in enriched experimental groups. Groups that discriminated +lim/−lim (T1–T3) have a higher BrdU-positive cell density compared to groups that do not discriminate (T4-T5) (n = 4/group), t-test *p < 0.05. e Positive correlation between the discrimination index and adult-born neuron density in the OB. f Density of activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/group) and in control non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is observed in enriched groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between enriched animals responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. Fig. 1 The presence of adult-born neurons correlates with mnesic performance. a Experimental design. b–f Behavioral results. b Discrimination between + lim and -lim is assessed at different time points after the enrichment period. Non-enriched control (NE; n = 9) animals do not discriminate the +lim from −lim at T1. After enrichment, the two odorants are discriminated at T1 (n = 11), T2 (n = 5), and T3 (n = 10) but not at T4 (n = 9) and T5 (n = 5). c Dec and dodec are not discriminated at any time post-learning (NE n = 9; T1 n = 10; T2 n = 7; T3 n = 8; T4 n = 5; T5 n = 10) (for clearer representation, 1 data point belonging to the Test trial of the NE group is not shown (investigation time = 6 s)). T-test, *p < 0.05. d Adult-born neuron density in the OB in enriched experimental groups. Results f Performances are linked to adult-born neurons fate. Previous studies have reported increased survival of adult-born granule cells after perceptual discrimination learning8. To better understand the potential role of these neurons in long-term memory, we first asked whether increased bulbar neurogenesis persists for as long as the learned information is remembered. Thus, different groups of mice underwent perceptual discrimination learning consisting in a 10- day enrichment period with (+)limonene and (−)limonene (+lim and −lim), odorants which are not spontaneously discriminated by mice. Their performance in discriminating these two odorants was then analyzed from 1 to 5 weeks post learning (T1, T2, T3, T4, and T5, Fig. 1a) using a habituation/dishabituation test. In this test, mice were exposed to the first odorant of the pair 4 times (Hab1 to Hab4; inter-trial interval 5 min) followed by exposure to the second odorant of the pair (Test, 5 min between Hab4 and Test). The time spent by the mouse investigating the odorant was recorded for all trials. Decreased investigation time across habituation trials reflects habituation to the odorant used and a longer time spent investing Test than Hab4 shows discrimination of the two odorants of the pair. At all time-points analyzed, mice exhibited habituation behavior (Supplementary Table 1, Supplementary Fig. 1). Regarding discrimination, using a two-way ANOVA, we observed significant differences between groups (NE: Non Enriched, T1, T2, T3, T4, T5: F(5,106) = 2.39; p = 0.043) and effect of the trial (Hab4 vs Test, NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 e Positive correlation between the discrimination index and adult-born neuron density in the OB activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/grou non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is obse groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between en responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. dec/dodec Investigation time (s) 4 3 Hab4 Test 2 1 0 NE T1 T2 T3 T4 T5 c 4 +lim/–lim 3 2 Investigation time (s) 1 0 Hab4 Test NE T1 T2 T3 T4 T5 b b c Hab4 Test d Brdu-positive cell density (cell mm–2) 70 60 50 40 30 20 10 0 T1 T2 T3 T4 T5 d R = 0.84, p = 0.038 Brdu-positive cell density (cell mm–2) 60 50 40 30 Discrimination index 0.6 0.1 0.2 0.3 0.4 0.5 e e R = 0.84, p = 0.038 Discrimination index BrdU-Zif268-positive cell density (cell mm–2) Non-enriched and stimulated +lim Enriched and stimulated +lim Enriched and stimulated dec 25 20 15 10 5 0 T1 T2 T3 T4 T5 f BrdU-Zif268-positive cell density (cell mm–2) Non-enriched and stimulated +lim Enriched and stimulated +lim Enriched and stimulated dec 25 20 15 10 5 0 T1 T2 T3 T4 T5 f f Fig. 1 The presence of adult-born neurons correlates with mnesic performance. a Experimental design. b–f Behavioral results. b Discrimination between + lim and -lim is assessed at different time points after the enrichment period. Non-enriched control (NE; n = 9) animals do not discriminate the +lim from −lim at T1. After enrichment, the two odorants are discriminated at T1 (n = 11), T2 (n = 5), and T3 (n = 10) but not at T4 (n = 9) and T5 (n = 5). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Groups that discriminated +lim/−lim (T1–T3) have a higher BrdU-positive cell density compared to groups that do not discriminate (T4-T5) (n = 4/group), t-test *p < 0.05. e Positive correlation between the discrimination index and adult-born neuron density in the OB. f Density of activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/group) and in control non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is observed in enriched groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between enriched animals responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. (T4-T5) (n = 4/group), t-test *p < 0.05. e Positive correlation between the discrimination index and adult-born neuron density in the OB. f Density of activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/group) and in control non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is observed in enriched groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between enriched animals responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 3 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 4 3 2 1 0 30 25 20 15 10 5 0 4 0 BrdU 8 17 24 +lim/–lim +lim/–lim +lim/–lim +lim/–lim +lim/–lim Test Test Test dec/dod dec/dod dec/dod dec/dod Test Test 34 44 54 64 Days +lim/–lim dec/dodec Hab4 Test Hab4 Stimulated with +lim Stimulated with dec Test 3 2 Investigation time (s) Investigation time (s) 1 0 90 80 70 60 50 30 20 BrdU-positive cell density (cell mm–2) BrdU-Zif268-positive cell density(cell mm–2) 40 10 0 T1 T1 T2′ T2′ T3′ T4′ T5′ T3′ T4′ T5′ T1 T2′ T3′ T4′ T5′ T1 T2′ T3′ T4′ T5′ T1 T2′ T3′ T4′ T5′ a b c d e Fig. 2 Time between learning sessions has a critical impact on memory. a Experimental design. The groups differed by the interval between the two enrichment periods: 4, 14, 24 or 34 days separating the two enrichments. b, c Behavioral results. b Discrimination between +lim/−lim. Groups T1 and T3′ discriminate. No discrimination is observed for the other groups (T2′, T4′, T5′) (T1 n = 11; T2’ n = 11; T3′ n = 11; T4′ n = 6; T5′ n = 7) t-test, **p < 0.005; ***p < 0.001. c Dec is discriminated from dodec in Group T2′ to T5′ (T1 n = 10; T2′ n = 11; T3′ n = 11; T4′ n = 10; T5′ n = 11) t-test **p < 0.005; ***p < 0.001. d Adult-born neuron density. Higher density of adult-born neurons is observed in groups that are able to discriminate +lim from −lim (T1 and T3′) (n = 6–7/group), Tukey corrected t-tests *p < 0.05; ***p < 0.001. e Density of activated adult-born neurons (BrdU+/Zif268+ cells) in response to +lim (T1 n = 4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) or dec (T1 n = 4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) after successive enrichments. Density of double- labeled cells is higher in response to +lim at T1 and T3′ compared to T2′, T4′, and T5′. In response to dec, the density of BrdU/Zif268 cells is higher at T3′ compared to the other groups. T-test *p < 0.05 for enriched animals responding to +lim at T1 and T3′ different from all other groups. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. 0 BrdU 8 17 24 +lim/–lim +lim/–lim +lim/–lim +lim/–lim +lim/–lim Test Test Test dec/dod dec/dod dec/dod dec/dod Test Test 34 44 54 64 Days T1 T2′ T3′ T4′ T5′ a 4 3 2 1 0 dec/dodec Hab4 Test Investigation time (s) T1 T2′ T3′ T4′ T5′ c 4 +lim/–lim Hab4 Test 3 2 Investigation time (s) 1 0 T1 T2′ T3′ T4′ T5′ b b c 90 80 70 60 50 30 20 BrdU-positive cell density (cell mm–2) 40 10 0 T1 T2′ T3′ T4′ T5′ d d 30 25 20 15 10 5 0 Stimulated with +lim Stimulated with dec BrdU-Zif268-positive cell density(cell mm–2) T1 T2′ T3′ T4′ T5′ e e e Fig. 2 Time between learning sessions has a critical impact on memory. a Experimental design. The groups differed by the interval between the two enrichment periods: 4, 14, 24 or 34 days separating the two enrichments. b, c Behavioral results. b Discrimination between +lim/−lim. Groups T1 and T3′ discriminate. No discrimination is observed for the other groups (T2′, T4′, T5′) (T1 n = 11; T2’ n = 11; T3′ n = 11; T4′ n = 6; T5′ n = 7) t-test, **p < 0.005; ***p < 0.001. c Dec is discriminated from dodec in Group T2′ to T5′ (T1 n = 10; T2′ n = 11; T3′ n = 11; T4′ n = 10; T5′ n = 11) t-test **p < 0.005; ***p < 0.001. d Adult-born neuron density. Higher density of adult-born neurons is observed in groups that are able to discriminate +lim from −lim (T1 and T3′) (n = 6–7/group), Tukey corrected t-tests *p < 0.05; ***p < 0.001. e Density of activated adult-born neurons (BrdU+/Zif268+ cells) in response to +lim (T1 n = 4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) or dec (T1 n = 4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) after successive enrichments. Density of double- labeled cells is higher in response to +lim at T1 and T3′ compared to T2′, T4′, and T5′. In response to dec, the density of BrdU/Zif268 cells is higher at T3′ compared to the other groups. ARTICLE 3b, c) and that the second enrichment with dec/dodec when performed 4 days after the first one erased the ability to discriminate +lim/−lim (group 2, Fig. 3b, c, Supplementary Table 3). Interestingly, if we maintained the enrichment with +lim/−lim during the second enrichment period (group 3), the animals were able to discriminate both odorant pairs (Fig. 3b, c, Supplementary Table 3). This prevention of forgetting can be due to network reactivation or relearning. Finally, when the retention of the task was tested at T3′, mice were, as previously, able to discriminate both odorant pairs (group 4, Fig. 3b, c, Supplementary Table 3). e o y ete t o (Supp e e ta y ab e , g. b, c). BrdU-positive cell density was then assessed as in the first experiment. BrdU-positive cell density differed between groups (ANOVA, group effect, F(4,26) = 16.1; p < 0.0001) (Fig. 2d). Post hoc Tukey corrected t-tests were then performed and showed that when the second enrichment was performed 4 days after the first one (T2′), the density of labeled adult-born cells was significantly decreased compared to T1 (Tukey corrected t-test p = 0.025). This was accompanied by impaired +lim/−lim discrimination suggest- ing that, at 4 days between two learning, new memory formation altered adult-born neuron survival and already stored informa- tion. However, when the second enrichment was performed 14 days after the first (T3′), both pairs of odorants were discriminated and the density of adult-born cells remained high (Tukey corrected t-test, T1 versus T3′: p = 0.2). Finally, when the second enrichment occurred even later (T4′ or T5′), BrdU density decreased significantly (Tukey corrected t-test, T3′ versus T4′: p < 0.001; T5′ versus T3′: p < 0.001) to a level comparable to T2′ (Tukey corrected t-test, T4′ versus T2′: p = 0.59; T5′ versus T2′: p = 0.99) which is in accordance with the first experiment (Fig. 2d). To study the involvement of adult-born neurons in processing the learned information, we assessed the density of adult-born neurons responding to +lim or to dec (group effect) at the different delays between enrichments (Fig. 2e). Using two-way ANOVA, we found a group effect (F(1,42) = 8.61, p = 0.0054), a time effect (F(1,42) = 42.53, p < 0.0001) and an interaction (F(1,42) = 13.08, p < 0.0001). ARTICLE More precisely, we labeled a first pool of 8-day-old adult- born cells in the OB at the beginning of the first enrichment (with +lim/−lim) with CldU and a second pool of 8-day-old adult- born cells in the OB at the beginning of the second enrichment (with dec/dodec) but not present in the OB at the beginning of the first enrichment (Fig. 3a, d) with IdU. The densities of CldU- and IdU-positive cells were assessed in the OB at T2. Overlapping between CldU and IdU was never observed. Results showed differences between experimental groups for both markers (ANOVA, CldU: F(3,16) = 19.06, p < 0.0001; IdU: F(3,24) = 7.8, p = 0.0008). Post hoc Tukey corrected t-tests revealed that the level of CldU-positive cells was lower in group 2 compared to the other groups, in accordance with +lim/−lim discrimination performance (group 2 versus group 1: p = 0.056, group 2 versus group 3: p = 0.0029, group 2 versus group 4: p < 0.001; Fig. 3e). Interestingly, the density of CldU-positive cells was not significantly higher in group 3 compared to group 1 (p = 0.48) suggesting that the second enrichment recruited a new pool of adult-born neurons. Supporting this hypothesis, all groups that were submitted to dec/dodec enrichment after the +lim/−lim enrichment, including group 2, showed a higher level of IdU- positive cells than group 1 (respectively p = 0.03, p = 0.017 and p < 0.001) (Fig. 3f). This strongly suggests that the second learning recruited a new population of adult-born neurons younger than that recruited during the first learning. We learned from this experiment that reactivating the network of the first learned odorant pair during the second task prevented In all groups, mice exhibited habituation behavior (Supple- mentary Table 3, Supplementary Fig. 8). Using two-way ANOVA, we observed significant effects of trial and group (trial: F(1,164) = 16.88; p < 0.0001, group: F(3,164) = 12.55; p < 0.0001) (Fig. 3b) for +lim/−lim discrimination and a trial effect and a group effect (trial: F(1,164) = 31.33; p < 0.0001, group: F(3,164) = 3.14; p = 0.026) (Fig. 3c) for dec/dodec discrimination. As in the first experiment (Fig. 1a), we observed that +lim/−lim enrichment improved discrimination between these two odorants when tested at T2’ (group 1, see Supplementary Table 3, Fig. NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 ARTICLE would avoid this overwriting of the memory. To do this we maintained exposure to the first pair of odorants during enrich- ment with the second pair (Fig. 3a). in this case) (Supplementary Fig. 6). When the time between the two enrichments increased (14 days, T3′), the animals were able to discriminate the two odorants of each pair (Supplementary Table 2, Fig. 2b, c) suggesting that in this experimental config- uration the formation of a new memory did not impact the memory already stored. At longer times between enrichments (24 and 34 days, respectively T4′ and T5′), +lim and −lim were no longer discriminated while dec and dodec were, which is in accordance with the first experiment reporting the duration of memory retention (Supplementary Table 2, Fig. 2b, c). ment with the second pair (Fig. 3a). In all groups, mice exhibited habituation behavior (Supple- mentary Table 3, Supplementary Fig. 8). Using two-way ANOVA, we observed significant effects of trial and group (trial: F(1,164) = 16.88; p < 0.0001, group: F(3,164) = 12.55; p < 0.0001) (Fig. 3b) for +lim/−lim discrimination and a trial effect and a group effect (trial: F(1,164) = 31.33; p < 0.0001, group: F(3,164) = 3.14; p = 0.026) (Fig. 3c) for dec/dodec discrimination. As in the first experiment (Fig. 1a), we observed that +lim/−lim enrichment improved discrimination between these two odorants when tested at T2’ (group 1, see Supplementary Table 3, Fig. 3b, c) and that the second enrichment with dec/dodec when performed 4 days after the first one erased the ability to discriminate +lim/−lim (group 2, Fig. 3b, c, Supplementary Table 3). Interestingly, if we maintained the enrichment with +lim/−lim during the second enrichment period (group 3), the animals were able to discriminate both odorant pairs (Fig. 3b, c, Supplementary Table 3). This prevention of forgetting can be due to network reactivation or relearning. Finally, when the retention of the task was tested at T3′, mice were, as previously, able to discriminate both odorant pairs (group 4, Fig. 3b, c, Supplementary Table 3). To understand the dynamics of learning-dependent survival of adult-born neurons underlying these effects, we tagged two adult- born neuron populations differing in age using two analogues of thymidine, ChlorodeoxyUridine (CldU) and IododeoxyUridine (IdU). ARTICLE In enriched animals in response to +lim, the density of BrdU/Zif268 cells is higher at T1 and T3’ compared to T2′, T4′, and T5′ (Time effect F(4,21) = 73.09, p < 0.0001; Tukey corrected t-test p < 0.05) which is in accordance with the performance of +lim versus −lim discrimination. In enriched animals in response to dec, the density of BrdU/Zif268 cells is higher at T3′ compared to the other groups (Time effect F(4,21) = 12.08, p < 0.0001; Tukey corrected t-test p < 0.05). These changes in density relied at least partly on changes in percentage of BrdU/Zif268 double-labeled cells (Supplementary Fig. 7). For the T2′, the decrease in the BrdU-Zif268 density is due more to a decrease in BrdU-positive cell density than to a decrease in the percentage of BrdU/Zif268 double-labeled cells. However, overall, the percentage of BrdU/Zif268-positive cells was higher in groups that discriminate +lim from −lim (T1 and T3′) than in groups that do not discriminate (T2′, T4′, and T5′) (p = 0.007). p g p g pp y To understand the dynamics of learning-dependent survival of adult-born neurons underlying these effects, we tagged two adult- born neuron populations differing in age using two analogues of thymidine, ChlorodeoxyUridine (CldU) and IododeoxyUridine (IdU). More precisely, we labeled a first pool of 8-day-old adult- born cells in the OB at the beginning of the first enrichment (with +lim/−lim) with CldU and a second pool of 8-day-old adult- born cells in the OB at the beginning of the second enrichment (with dec/dodec) but not present in the OB at the beginning of the first enrichment (Fig. 3a, d) with IdU. The densities of CldU- and IdU-positive cells were assessed in the OB at T2. Overlapping between CldU and IdU was never observed. Results showed differences between experimental groups for both markers (ANOVA, CldU: F(3,16) = 19.06, p < 0.0001; IdU: F(3,24) = 7.8, p = 0.0008). Post hoc Tukey corrected t-tests revealed that the level of CldU-positive cells was lower in group 2 compared to the other groups, in accordance with +lim/−lim discrimination performance (group 2 versus group 1: p = 0.056, group 2 versus group 3: p = 0.0029, group 2 versus group 4: p < 0.001; Fig. 3e). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 T-test *p < 0.05 for enriched animals responding to +lim at T1 and T3′ different from all other groups. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. odorant in groups showing the ability to discriminate +lim from −lim. discrimination, for +lim/−lim, using two-way ANOVA, we observed a group effect (T1,T2′,T3′,T4′,T5′, F(4,94) = 4.63; p = 0.002) and a trial effect (F(1,94) = 19.99; p < 0.0001). For dec/ dodec, there was no group effect (F(4,108) = 2.19; p = 0.08) but a trial effect (F(1,108) = 45.08; p < 0.0001) and interaction (F(4,108) = 5.49; p = 0.0005). When the second enrichment was performed 4 days after the first one (T2’), animals were only able to dis- criminate the second odorant pair (Supplementary Table 2, Fig. 2b, c) suggesting that the acquisition of the second task erased the memory of the first one. This result was confirmed using another strain of mice (129 mice, Supplementary Fig. 5). As an additional control, we switched the training order, the animals were enriched first with dec/dodec and then with +lim/−lim. Result was similar to that previously obtained: animals were only able to discriminate the second learned odorant pair (+lim/−lim In summary, perceptual memory showed a natural memory decay between T3 and T4. This memory alteration is accom- panied by a decrease in the density of adult-born neurons and in the percentage of adult-born neurons responding to the learned odorants. Time between learning sessions affects stored memory. In total 4, 14, 24 or 34 days after a first enrichment with +lim/−lim, mice were enriched with a new odorant pair, dec/dodec, tested for discrimination of the two pairs of odorants and sacrificed at the same times as in the previous experiment (groups T2′ to T5′) (Fig. 2a). All groups exhibited habituation behavior (Sup- plementary Table 2, Supplementary Fig. 4). Regarding NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 8 0 8 13 17 21 23 30 31 34 40 44 Days Group4 Group3 Group2 Group1 T2′ T3′ +lim/–lim +lim/–lim No odor Test Test Test Test dec/dod dec/dod dec/dod +lim/–lim 7 6 5 Investigation time (s) Investigation time (s) 4 3 2 1 0 Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 8 a b +lim/–lim dec/dodec 7 6 5 Hab4 Test Hab4 Test 4 3 2 1 0 +lim/–lim +lim/–lim c a 8 7 6 5 Investigation time (s) 4 3 2 1 0 Group 1 Group 2 Group 3 Group 4 b +lim/–lim Investigation time (s) Group 1 Group 2 Group 3 Group 4 8 dec/dodec 7 6 5 4 Hab4 Test 4 3 2 1 0 c Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 80 70 60 50 40 CldU-positive cell density (cell/mm2) IdU-positive cell density (cell/mm2) 30 20 10 0 +lim/–lim discrimination dec/dodec discrimination – – + + + + + + 80 70 60 50 40 30 20 10 0 d e f CldU/IdU/DAPI 10 μm d CldU/IdU/DAPI 10 μm d d +lim/–lim discrimination Fig. 3 Reactivation of learned information prevented cell death and maintained memory. a Experimental design. b, c Behavioral results. b +lim and -lim are discriminated in groups 1 (n = 18), 3 (n = 20) and 4 (n = 10) but not in group 2 (n = 18). c Dec/dodec are discriminated in Group 2 (n = 18), 3 (n = 20) and 4 (n = 10) but not in group 1 (n = 18). d Example of CldU/DAPI and IdU/DAPI double labeling. e. Density of CldU-positive cells. A lower density is observed in group 2 compared to groups 1, 3, and 4 and a higher density in group 4 compared to groups 1, 2, and 3 (n = 5/group). f Density of IdU-positive cells is higher in groups 2 (n = 8), 3 (n = 7) and 4 (n = 5) compared to group 1 (n = 8). Tukey corrected t-tests *p ≤0.05, ***p < 0.001. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. but not of dec/dodec (Fig. 4c, Supplementary Table 4). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 d lt b h t f di ti t i W b t t f d /d d (Fi 4 S l t T bl 4) A i 8 0 8 13 17 21 23 30 31 34 40 44 Days Group4 Group3 Group2 Group1 T2′ T3′ +lim/–lim +lim/–lim No odor Test Test Test Test dec/dod dec/dod dec/dod +lim/–lim 7 6 5 Investigation time (s) Investigation time (s) 4 3 2 1 0 Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 8 a b +lim/–lim dec/dodec 7 6 5 Hab4 Test Hab4 Test 4 3 2 80 70 60 50 40 CldU-positive cell density (cell/mm2) IdU-positive cell density (cell/mm2) 30 20 10 0 +lim/–lim discrimination dec/dodec discrimination – – + + + + + + 80 70 60 50 40 30 20 10 0 1 0 +lim/–lim +lim/–lim c d e f CldU/IdU/DAPI 10 μm ctivation of learned information prevented cell death and maintained memory. a Experimental design. b, c Behavioral results. b +lim and minated in groups 1 (n = 18), 3 (n = 20) and 4 (n = 10) but not in group 2 (n = 18). c Dec/dodec are discriminated in Group 2 (n = 18), 3 (n = = 10) but not in group 1 (n = 18). d Example of CldU/DAPI and IdU/DAPI double labeling. e. Density of CldU-positive cells. A lower dens n group 2 compared to groups 1, 3, and 4 and a higher density in group 4 compared to groups 1, 2, and 3 (n = 5/group). f Density of IdU-po her in groups 2 (n = 8), 3 (n = 7) and 4 (n = 5) compared to group 1 (n = 8). Tukey corrected t-tests *p ≤0.05, ***p < 0.001. Data are d as data points and mean ± sem. Source data are provided as a Source Data file. ARTICLE Interestingly, the density of CldU-positive cells was not significantly higher in group 3 compared to group 1 (p = 0.48) suggesting that the second enrichment recruited a new pool of adult-born neurons. Supporting this hypothesis, all groups that were submitted to dec/dodec enrichment after the +lim/−lim enrichment, including group 2, showed a higher level of IdU- positive cells than group 1 (respectively p = 0.03, p = 0.017 and p < 0.001) (Fig. 3f). This strongly suggests that the second learning recruited a new population of adult-born neurons younger than that recruited during the first learning. In summary, the time between the two enrichments is critical to the upkeep of the memory already stored and associated adult- born neurons survival and functional involvement. These data further suggest that the survival of at least some of these adult- born neurons is not assured as, for one week after learning, they go through a fragile state, which is vulnerable to a new learning challenge. g g We learned from this experiment that reactivating the network of the first learned odorant pair during the second task prevented memory erasure and the disappearance of adult-born cells. Thus, for a short time between two successive learning tasks, adult-born neurons can be maintained in the network providing that the relevant sensory inputs remain in the environment. However, despite the presence of these adult-born neurons still in their critical period of integration, a distinct pool of younger adult-born neurons was recruited by the second task. It remains the question of whether the second learning relies on the last recruited adult- born neuron population (IdU-positive cells) or on both adult-born neuron populations (IdU- and CldU-positive cells). In other words, are adult-born neurons allocated to a memory trace able to underlie or contribute to subsequent learning? Sensory reactivation prevents cell and memory loss. With a short period between the two learning sessions (T2′), we found that the new memory altered previously stored information and induced apoptosis of adult-born neurons. Since cell survival is known to be input-dependent, we tested whether reactivating the network that responded to the odorants used for the enrichment 5 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Animals infected with a control virus and submitted to light stimulation behaved as group 3 of the previous experiment, suggesting no direct effect of the light. To test that light-triggered inhibition of adult-born neurons in halorhodopsin animals had no deleterious effect on spontaneous odor discrimination, we assessed the dis- crimination of a pair of dissimilar odorants (+lim/+carvone) and found that it was not impaired by light (Fig. 4d, Supplementary Table 4). We then analyzed the level of viral transfection in control and halorhodopsin groups and found no difference of EYFP-positive cell density (t-test, p = 0.5, Fig. 4e). We also ver- ified the effectiveness of light-mediated inhibition of adult-born granule cells by assessing the expression of Zif268-positive cells in Distinct adult-born neuron cohorts for distinct memories. We used the same learning paradigm as previously (group 3; Figs. 3a and 4a) in which mice underwent a first enrichment period with +lim/−lim and 4 days later a second one with dec/dodec in the presence of +lim/−lim to avoid memory loss. Eight days before the first enrichment period, adult-born neuron progenitors were transduced with halorhodopsin-expressing or control lentiviruses coupled with EYFP expression and animals were implanted with optical fibers in the OB. Light stimulation was used to block adult-born cell activity during the test trial of the habituation/ dishabituation task. Results showed that light-triggered inhibition of neurons born 8 days before the first learning task altered the discrimination of +lim/−lim (Fig. 4b, Supplementary Table 4) Distinct adult-born neuron cohorts for distinct memories. We used the same learning paradigm as previously (group 3; Figs. 3a and 4a) in which mice underwent a first enrichment period with +lim/−lim and 4 days later a second one with dec/dodec in the presence of +lim/−lim to avoid memory loss. Eight days before the first enrichment period, adult-born neuron progenitors were transduced with halorhodopsin-expressing or control lentiviruses coupled with EYFP expression and animals were implanted with optical fibers in the OB. Light stimulation was used to block adult-born cell activity during the test trial of the habituation/ dishabituation task. Results showed that light-triggered inhibition of neurons born 8 days before the first learning task altered the discrimination of +lim/−lim (Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 4b, Supplementary Table 4) NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 7 b a c e f d 0 Halor 8 17 21 30 36 Optogenetic inhibition Test Days T2′ 6 Investigation time (s) Investigation time (s) Investigation time (s) 5 4 3 1 2 0 200 60 40 20 0 100 EYFP-positive cell density (cell mm–2) EYFP-Zif268-positive cells (%) 0 Control Halo Control Halo Hab1 Hab2 Hab3 Hab4 Test Hab1 Hab2 Hab3 Hab4 Test Hab1 Hab2 Hab3 Hab4 Test 7 11 dec/dodec dec/dod +lim/–lim +lim/–lim +lim/–lim +lim/+carvone Cont Halo 10 9 8 7 6 5 4 3 2 1 0 6 5 4 ns 3 1 2 0 Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Experimental design. Adult-born neurons aged 8 days at the beginning of the first enrichment were transduced with a lentivirus expressing halorhodopsin (Halo) or with an empty virus (Cont). Light stimulation was performed during the test trial of the habituation/dishabituation task. b–d Behavioral results. b The learned discrimination of +lim/−lim is abolished by light stimulation in the Halo group (n = 13), but not in the Control group (n = 12). c Discrimination between dec/dodec is not altered by light stimulation (Cont n = 13; Halo n = 14). d Discrimination between a dissimilar odorant pair +lim/ +carvone is not altered by light-triggered inhibition of adult-born neurons (Cont n = 9; Halo n = 14). t-tests *p < 0.05, **p < 0.01, ***p < 0.001 (for difference between Hab4 and Test in b–d). e The density of EYFP-positive transduced cells in the OB is similar between Control (n = 9) and Halo (n = 7) animals. f Percentage of EYFP/Zif268-positive cells after light-triggered inhibition is decreased in Halo (n = 7) compared to Control (n = 8). t-test *p < 0.05 (for difference between Halo and Control groups in f). Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 It is worth noting that in the short time condition, the number of adult-born cells is reduced but a substantial fraction of adult-born neurons (>20%) still respond to the learned odorant even though born neurons present in the OB after the first task are prematurely killed and memory is lost. This conclusion is based on the loss of BrdU-positive neurons and is consistent with previous reports showing that blocking adult-born cell death prevented memory erasure19. In addition to training-dependent changes in BrdU- positive cell density, the functional involvement of these cells in response to the learned odorant increased with performance for neurons aged 8 days at the beginning of learning. We further show that these two learning tasks, despite they are spaced by a short 4- day time period, induced two distinct waves of adult-born neuron survival and that optogenetically inactivating the first cohort altered the first memory, without affecting the second learning. This further reinforces the idea that the 20-day old BrdU-tagged neurons exited the time window during which they are most crucial to enrichment-induced improvement in discrimination. It is worth noting that in the short time condition, the number of adult-born cells is reduced but a substantial fraction of adult-born neurons (>20%) still respond to the learned odorant even though Hence, silencing a cohort of adult-born neurons aged between 8 and 18 days at the time of learning leads to memory erasure demonstrating the crucial role of adult-born neurons in perceptual memory. However, this manipulation did not affect the memory of a new discrimination task learned shortly afterwards, suggesting that these adult-born neurons are allocated to only one memory trace. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Light stimulation was performed during the test trial of the habituation/dishabituation task. b–d Behavioral results. b The learned discrimination of +lim/−lim is abolished by light stimulation in the Halo group (n = 13), but not in the Control group (n = 12). c Discrimination between dec/dodec is not altered by light stimulation (Cont n = 13; Halo n = 14). d Discrimination between a dissimilar odorant pair +lim/ +carvone is not altered by light-triggered inhibition of adult-born neurons (Cont n = 9; Halo n = 14). t-tests *p < 0.05, **p < 0.01, ***p < 0.001 (for difference between Hab4 and Test in b–d). e The density of EYFP-positive transduced cells in the OB is similar between Control (n = 9) and Halo (n = 7) animals. f Percentage of EYFP/Zif268-positive cells after light-triggered inhibition is decreased in Halo (n = 7) compared to Control (n = 8). t-test *p < 0.05 (for difference between Halo and Control groups in f). Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. EYFP-positive neurons after stimulation with light. The expres- sion of Zif268 was significantly lower in the halorhodospin compared to the control group (t-test, p = 0.03, Fig. 4f). born neurons present in the OB after the first task are prematurely killed and memory is lost. This conclusion is based on the loss of BrdU-positive neurons and is consistent with previous reports showing that blocking adult-born cell death prevented memory erasure19. In addition to training-dependent changes in BrdU- positive cell density, the functional involvement of these cells in response to the learned odorant increased with performance for neurons aged 8 days at the beginning of learning. We further show that these two learning tasks, despite they are spaced by a short 4- day time period, induced two distinct waves of adult-born neuron survival and that optogenetically inactivating the first cohort altered the first memory, without affecting the second learning. This further reinforces the idea that the 20-day old BrdU-tagged neurons exited the time window during which they are most crucial to enrichment-induced improvement in discrimination. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 7 b a c d 0 Halor 8 17 21 30 36 Optogenetic inhibition Test Days T2′ 6 Investigation time (s) Investigation time (s) Investigation time (s) 5 4 3 1 2 0 Hab1 Hab2 Hab3 Hab4 Test Hab1 Hab2 Hab3 Hab4 Test Hab1 Hab2 Hab3 Hab4 Test 7 11 dec/dodec dec/dod +lim/–lim +lim/–lim +lim/–lim +lim/+carvone Cont Halo 10 9 8 7 6 5 4 3 2 1 0 6 5 4 ns 3 1 2 0 a 0 8 17 21 30 36 Optogenetic inhibition Test Days T2′ dec/dod +lim/–lim +lim/–lim a d d b e f 200 60 40 20 0 100 EYFP-positive cell density (cell mm–2) EYFP-Zif268-positive cells (%) 0 Control Halo Control Halo f Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Exper Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Experimental design. Adult-born neurons aged 8 days at the beginning of the first enrichment were transduced with a lentivirus expressing halorhodopsin (Halo) or with an empty virus (Cont). Light stimulation was performed during the test trial of the habituation/dishabituation task. b–d Behavioral results. b The learned discrimination of +lim/−lim is abolished by light stimulation in the Halo group (n = 13), but not in the Control group (n = 12). c Discrimination between dec/dodec is not altered by light stimulation (Cont n = 13; Halo n = 14). d Discrimination between a dissimilar odorant pair +lim/ +carvone is not altered by light-triggered inhibition of adult-born neurons (Cont n = 9; Halo n = 14). t-tests *p < 0.05, **p < 0.01, ***p < 0.001 (for difference between Hab4 and Test in b–d). e The density of EYFP-positive transduced cells in the OB is similar between Control (n = 9) and Halo (n = 7) animals. f Percentage of EYFP/Zif268-positive cells after light-triggered inhibition is decreased in Halo (n = 7) compared to Control (n = 8). t-test *p < 0.05 (for difference between Halo and Control groups in f). Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Experimental design. Adult-born neurons aged 8 days at the beginning of the first enrichment were transduced with a lentivirus expressing halorhodopsin (Halo) or with an empty virus (Cont). NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications Discussion h For the olfactory enrichment, 100 μL of pure odorant were placed in two separate tea balls of the home cages for one hour daily over 10 days. For the multiple enrichment (experiment 3, group 3), the two pairs of odorants were presented with an interval of 1 h (SI Methods). We assessed the spontaneous discrimination between two pairs of similar odorants: +lim/−lim and dec/dodec using olfactory habituation/dishabituation. A test session consisted of four 50-s odor presentations of a first odorant (Hab) at 5 min intervals, followed by one 50-s presentation of the second odorant of the pair (Test). Investigation was defined as active sniffing within 1 cm of the tea ball (SI Methods)8,24,25. Exploration assessments were done blind with regard to the experimental group. Perceptual learning. This implicit olfactory learning consisted in passive exposure to odorants (enrichment). For the olfactory enrichment, 100 μL of pure odorant were placed in two separate tea balls of the home cages for one hour daily over 10 days. For the multiple enrichment (experiment 3, group 3), the two pairs of odorants were presented with an interval of 1 h (SI Methods). We assessed the spontaneous discrimination between two pairs of similar odorants: +lim/−lim and dec/dodec using olfactory habituation/dishabituation. A test session consisted of four 50-s odor presentations of a first odorant (Hab) at 5 min intervals, followed by one 50-s presentation of the second odorant of the pair (Test). Investigation was defined as active sniffing within 1 cm of the tea ball (SI Methods)8,24,25. Exploration assessments were done blind with regard to the experimental group. Data analysis. Data analysis was performed using R software (CRAN). Normality was assessed using the Kolmogorov-Smirnov test. Global two-way ANOVAs were performed to evaluate changes in discrimination abilities between groups. Then intra-group one-way RM-ANOVAs and unilateral paired t-tests were performed to determine whether the mice exhibited habituation (trial effect) as well as dis- crimination (by comparing Hab4 and Test). Discrimination was indicated by a significant increase in investigation time during the test trial. Discrimination index was calculated as [1-(Hab4/Test)]26 (SI Methods). Sample sizes were determined based on previous reports8,14,27. Animal assignation to the various experimental groups was randomized. Adult-born cell density assessment. Bromodeoxyuridine (BrdU) (Sigma- Aldrich) was injected 8 days before the enrichment period (50 mg kg-1 in saline, 3x at 2-h intervals; i.p.)14,22. Much of what we experience is ultimately forgotten, but memories for some events persist. Discussion h The OB is continuously receiving waves of adult-born neurons and so contains adult-born neurons of different ages and levels of maturation18. When learning happens successively, which neu- rons take part in underlying behavioral changes? When the time between the two learning tasks is short (4 days), part of the adult- 7 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 discrimination is lost. This state differs from that observed for longer between-task times where both density of adult-born neurons and their responsiveness to the learned odorants are strongly reduced. This suggests only a partial withdrawal of the remaining adult-born neurons from processing the first learned odorants in the short time condition, sufficient however to prevent discrimination. Nevertheless, we conclude that cohorts of neurons of different ages, but still within their critical period, are required to allow the encoding of successive learning experiences. Impor- tantly, when the time between tasks is longer (14 days), no cell loss occurred and performances were maintained. Altogether, these data provide evidence for an unexpectedly early and sharp func- tional transition within the critical period. The critical period of adult-born neurons which, based on their vulnerability to cell death, morphological development and synaptic current devel- opment and plasticity, has been reported to last from 30 days to 60 days post neuronal birth6,10,20–23. Within this critical period, our data suggest that availability of adult-born neurons for encoding new experience lasts no longer than 20 days. tested on their spontaneous discrimination between +lim and −lim and also between another pair of perceptually similar odorants (decanal (dec) and dodecanone (dodec)). Discrimination was assessed using an olfactory habituation/dishabituation task. Animals were sacrificed 24, 34, 44, 54 or 64 days after BrdU injections (Fig. 1a). Regarding the second experiment, eight days after BrdU injection, a different set of mice was similarly enriched with +lim and −lim but this was followed by a second 10-day enrichment period with dec and dodec either 4, 14, 24, 34 or 44 days after the first. At the end of both enrichments, mice were tested on their spontaneous discrimination between the two odorants of each of the pairs using an olfactory habituation/dishabituation task and animals were sacrificed 24, 34, 44, 54 and 64 days post BrdU injections (Fig. 2a) In the third experiment, we used two other DNA markers (analogues of BrdU), 5′- chloro-2′-deoxyuridine (CldU) and 5′-iodo-2′-deoxyuridine (IdU), to label two different populations of adult-born cells. Discussion h Here, we report that modula- tion of the OB circuitry, as a function of the environment and dependent on the fate of maturing adult-born neurons, is responsible for the balance between the transience or persistence of memory. This is made possible thanks to distinct neuronal populations encoding temporally distinct experiences. 24, 34, 44, 54 and 64 days post BrdU injection, five mice were taken randomly from each experimental group for sacrifice and BrdU, IdU and CldU immunohistochemistries were carried on as described in SI Methods14,22. 24, 34, 44, 54 and 64 days post BrdU injection, five mice were taken randomly from each experimental group for sacrifice and BrdU, IdU and CldU immunohistochemistries were carried on as described in SI Methods14,22. The method used for BrdU-, IdU- and CldU-positive cell counting is described in SI Methods14,28. The mean positive cell density of each array was calculated and averaged within each experimental group. Between-groups comparisons of the mean cell density were performed by ANOVA followed by post hoc t-tests with Tukey corrections. Unilateral t-tests were performed for comparisons between two groups. The level of significance was set to 0.05. The method used for BrdU-, IdU- and CldU-positive cell counting is described in SI Methods14,28. The mean positive cell density of each array was calculated and averaged within each experimental group. Between-groups comparisons of the mean cell density were performed by ANOVA followed by post hoc t-tests with Tukey corrections. Unilateral t-tests were performed for comparisons between two groups. The level of significance was set to 0.05. Discussion h CldU was injected 8 days before the first enrichment (again with +lim and -lim) while IdU was injected 8 days before the second enrichment period. The second enrichment period varied among groups: no enrichment (group 1); dec and dodec (group 2 and group 4), +lim and −lim plus dec and dodec (group 3). The time between the two enrichments was either 4 or 14 days. As previously, discrimination was assessed using a habituation/dishabituation task and animals were sacrificed 34 or 44 days post-CldU injection (Fig. 3a). y j g In the fourth experiment using group 3 experimental configuration from the previous experiment, we performed targeted lentiviral-induced halorhodospin channel expression (NpHR3.0) in the subventricular zone, and used optogenetics to specifically inhibit the population of adult-born neurons arriving in the OB at the beginning of the +lim/−lim enrichment (Fig. 4a). g p g y When previously learned information is reintroduced into the environment, we observed no premature cell death and the second learning did no longer erase the first one. To maintain the first learned discrimination, the network could either use adult-born neurons already integrated into this network or recruit a new cohort of adult-born neurons. Optogenetic inactivation of the 8-day old cohort of adult-born neurons present in the OB at the beginning of the first training impaired memory of the first task. This strongly suggests that performance relies on adult-born neurons recruited in the initial task suggesting that the memory is recalled rather than re-encoded. Hence, reactivated memories are not over-written and forgotten. On the contrary, neurons encoding obsolete information (i.e. not present in the environment) could be sentenced to death after about 45 days unless another learning task occurs soon after, causing interference and leading to earlier neuronal death. Our data thus suggest that adult-born neurons are sensitive to interference only during their first 3 weeks and that if further learning occurs when adult-born neurons present in the OB are more than 21 days old, they are resistant to death and memory persists. On the other hand, a second learning task causes neurons aged less than 21 days old to die and erases the encoded memory. This difference could be due to the state of synaptic integration of the adult-born neurons20 which are more or less sensitive to competition from later waves of adult-born neurons. Perceptual learning. This implicit olfactory learning consisted in passive exposure to odorants (enrichment). Additional information y 6. Mouret, A. et al. Learning and survival of newly generated neurons: when time matters. J. Neurosci. 28, 11511–11516 (2008). Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 019-13521-7. 7. Lazarini, F. et al. Cellular and behavioral effects of cranial irradiation of the subventricular zone in adult mice. PLoS ONE 4, e7017 (2009). Correspondence and requests for materials should be addressed to N.M. 8. Moreno, M. M. et al. Olfactory perceptual learning requires adult neurogenesis. Proc. Natl Acad. Sci. USA 106, 17980–17985 (2009). Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. g 9. Moreno, M. M. et al. Action of the noradrenergic system on adult-born cells is required for olfactory learning in mice. J. Neurosci. 32, 3748–3758 (2012). 10. Nissant, A., Bardy, C., Katagiri, H., Murray, K. & Lledo, P.-M. Adult neurogenesis promotes synaptic plasticity in the olfactory bulb. Nat. Neurosci. 12, 728–730 (2009). Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 11. Sultan, S. et al. Learning-dependent neurogenesis in the olfactory bulb determines long-term olfactory memory. FASEB J. 24, 2355–2363 (2010). 12. Sultan, S., Lefort, J. M., Sacquet, J., Mandairon, N. & Didier, A. Acquisition of an olfactory associative task triggers a regionalized down-regulation of adult born neuron cell death. Front. Neurosci. 5, 52 (2011). 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. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 19. Sultan, S., Rey, N., Sacquet, J., Mandairon, N. & Didier, A. Newborn neurons in the olfactory bulb selected for long-term survival through olfactory learning are prematurely suppressed when the olfactory memory is erased. J. Neurosci. 31, 14893–14898 (2011). implanted with bilateral optic fibers (200 nm core diameter, 0.22 N.A., Doric Lenses) in the OB, with the following coordinates respective to bregma: antero-posterior +4.6 mm, medio-lateral ±0.75 mm, dorso-ventral −2 mm. Mice were injected with a ketoprophen solution (2 mg kg−1) after the surgery as well as during the following days and allowed to recover with food and water ad libidum. 20. Kelsch, W., Lin, C.-W., Mosley, C. P. & Lois, C. A critical period for activity- dependent synaptic development during olfactory bulb adult neurogenesis. J. Neurosci. 29, 11852–11858 (2009). During the habituation/dishabituation mice were automatically stimulated (crystal laser, 561 nm, 10–15 mW, continuous stimulation) only during the test trial (Test) when they entered a 2.5 cm diameter zone around the tea ball. 21. Yamaguchi, M. & Mori, K. Critical period for sensory experience-dependent survival of newly generated granule cells in the adult mouse olfactory bulb. PNAS 102, 9697–9702 (2005). In order to control of light-triggered inhibition, 36 days post-surgery and lentiviral infusion, mice were stimulated during the hour before sacrifice with light patterns mimicking the average light stimulating pattern during the test trial (0.75 s light ON, 5 s light OFF for 1 h). After brain sectioning (see above), EYFP and Zif268 double immunohistochemistry was performed: incubation with rabbit Zif268 antibody (1:1.000, Santa Cruz, ref: Sc-189), chicken GFP antibody (1:1.000, Anaspec TEBU, ref. 29, 55423). The density of EYFP, Zif268+ and double-stained cells were counted on 1–2 slices under the fiber implantation to allow assessment of the inhibition. Statistical significance was assessed using a unilateral t-test. 22. Mandairon, N. et al. Neurogenic correlates of an olfactory discrimination task in the adult olfactory bulb. Eur. J. Neurosci. 24, 3578–3588 (2006). y 23. Ge, S., Yang, C.-H., Hsu, K.-S., Ming, G.-L. & Song, H. A critical period for enhanced synaptic plasticity in newly generated neurons of the adult brain. Neuron 54, 559–566 (2007). 24. Mandairon, N., Stack, C., Kiselycznyk, C. & Linster, C. Broad activation of the olfactory bulb produces long-lasting changes in odor perception. Proc. Natl Acad. Sci. USA 103, 13543–13548 (2006). Reporting summary. References 1. Altman, J. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137, 433–457 (1969). 1. Altman, J. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137, 433–457 (1969). Code availability y The codes are provided in supplementary file entitled Supplementary Software 1. 28. Mandairon, N., Sacquet, J., Jourdan, F. & Didier, A. Long-term fate and distribution of newborn cells in the adult mouse olfactory bulb: influences of olfactory deprivation. Neuroscience 141, 443–451 (2006). The codes are provided in supplementary file entitled Supplementary Software 1. olfactory deprivation. Neuroscience 141, 443–451 (2006). Received: 8 January 2019; Accepted: 12 November 2019; Received: 8 January 2019; Accepted: 12 November 2019; Received: 8 January 2019; Accepted: 12 November 2019; 29. Kermen, F. et al. Topographical representation of odor hedonics in the olfactory bulb. Nat. Neurosci. https://doi.org/10.1038/nn.4317 (2016). ARTICLE Further information on research design is available in the Nature Research Reporting Summary linked to this article. 25. Cleland, T. A., Morse, A., Yue, E. L. & Linster, C. Behavioral models of odor similarity. Behav. Neurosci. 116, 222–231 (2002). 26. Rey, N. L., Sacquet, J., Veyrac, A., Jourdan, F. & Didier, A. Behavioral and cellular markers of olfactory aging and their response to enrichment. Neurobiol. Aging 33, 626.e9–626.e23 (2012). Data availability y The data associated to all main figures are provided as a Source Data file. The data associated to all main figures are provided as a Source Data file. 27. Forest, J. et al. Role of adult-born versus preexisting neurons born at p0 in olfactory perception in a complex olfactory environment in mice. Cereb. Cortex https://doi.org/10.1093/cercor/bhz105 (2019). Author contributions 2. Lois, C. & Alvarez-Buylla, A. Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145–1148 (1994). J.F., M.M., A.D., and N.M. designed the research. J.F., M.M., A.Z, M.C., J.S., and L.C. performed the research. J.F., M.M., M.R., A.D., and N.M. wrote the paper. 3. Ming, G. & Song, H. Adult neurogenesis in the mammalian central nervous system. Annu. Rev. Neurosci. 28, 223–250 (2005). Acknowledgements This work was supported by CNRS, Inserm, Lyon 1 University and Ecole Normale Supér- ieure de Lyon. We would like to thank C. Benetollo from the Neurogenetic and Optogenetic Platform of the CRNL for lentiviral production and G. Froment, D. Nègre and C. Costa from the lentivector production facility /SFR BioSciences de Lyon (UMS3444/US8). This work was supported by CNRS, Inserm, Lyon 1 University and Ecole Normale Supér- ieure de Lyon. We would like to thank C. Benetollo from the Neurogenetic and Optogenetic Platform of the CRNL for lentiviral production and G. Froment, D. Nègre and C. Costa from the lentivector production facility /SFR BioSciences de Lyon (UMS3444/US8). The authors declare no competing interests. 5. Alonso, M. et al. Olfactory discrimination learning increases the survival of adult-born neurons in the olfactory bulb. J. Neurosci. 26, 10508–10513 (2006). adult-born neurons in the olfactory bulb. J. Neurosci. 26, 10508–10513 (2006). 6. Mouret, A. et al. Learning and survival of newly generated neurons: when time matters. J. Neurosci. 28, 11511–11516 (2008). Methods Mi Ad l Mice. Adult C57BL/6J mice (8 weeks old, male, Charles River, L’arbresles, France) and 129 mice (8 weeks old, male, Charles River, L’arbresles, France) were used in this study. They were housed in standard laboratory cages with water and food ad libitum and were kept on a 12-hr light/dark cycle at a constant temperature of 22 °C. All behavioral training was conducted in the afternoon (12:00–18:00). Experiments were done following procedures in accordance with the European Community Council Directive of 22 September 2010 (2010/63/UE) and approved by the National Ethics Committee (Agreement DR2013-48(vM)). Every effort was made to minimize animal suffering. BrdU/Zif268 experiment. To investigate immediate early gene expression in response to odorant exposure, mice were presented with a tea ball containing 100 µl of pure +lim or dec for 1 h, 1 h before sacrifice. A rabbit anti-Zif268 (1:1000, Santa Cruz Biotechnology) and a rat anti-BrdU (1:100, Harlan Sera-Lab) were used. The appropriate secondary antibodies, coupled to Alexa Fluor 633 and 488 (Invitrogen) were used for revelation of the different markers. BrdU/Zif268 density was cal- culated by combining the mean percentage of BrdU/Zif268 positive cells per group to individual density of BrdU positive cells. All cell counts were conducted blind with regard to the experimental group. Different animals were used in different experimental groups. Histological data were obtained from a sample of animals used for behavioral tasks. Optogenetics in freely behaving mice. Hundred and fifty nanoliters of pLenti- hSyn-eNpHR3.0-EYFP lentivirus (9.22 × 106IU ml−1) or 300 nL of control pLenti- hSyn-EYFP (1.1 × 106 IU ml−1, expressing only the reporter gene EYFP29) injections were done bilaterally in the subventricular zone, with the following coordinates respective to bregma: antero-posterior + 1 mm, medio-lateral ± 1 mm, dorso-ventral −2.3 mm and at a rate of 150 nL min−1. Just after virus infusions, mice were Experimental designs. Regarding the first experiment, mice were injected with a DNA marker, BrdU, in order to label a cohort of adult-born neurons. Eight days later, they began the enrichment procedure with (+)limonene (+lim) and (−)limonene (−lim) for one hour daily over 10 days. At the end of the enrichment, mice were NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 8 Competing interests 4. Ming, G. & Song, H. Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron 70, 687–702 (2011). p g The authors declare no competing interests. Additional information 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/. 13. Grelat, A. et al. Adult-born neurons boost odor-reward association. Proc. Natl Acad. Sci. USA 115, 2514–2519 (2018). 14. Mandairon, N. et al. Opposite regulation of inhibition by adult-born granule cells during implicit versus explicit olfactory learning. eLife 7, e34976 (2018). 15. Akers, K. G. et al. Hippocampal neurogenesis regulates forgetting during adulthood and infancy. Science 344, 598–602 (2014). y 16. Frankland, P. W. & Josselyn, S. A. Hippocampal neurogenesis and memory clearance. Neuropsychopharmacology 41, 382–383 (2016). 17. Richards, B. A. & Frankland, P. W. The persistence and transience of memory. Neuron 94, 1071–1084 (2017). 18. Carleton, A. et al. Making scents of olfactory neurogenesis. J. Physiol.-Paris 96, 115–122 (2002). © The Author(s) 2019 9 NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunicatio
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Serine/Threonine Kinase Inhibitor XL418
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Serine/Threonine Kinase Inhibitor XL418 National Cancer Institute National Cancer Institute Qeios ID: 3TS65E · https://doi.org/10.32388/3TS65E Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Source National Cancer Institute. Serine/Threonine Kinase Inhibitor XL418. NCI Thesaurus. Code C68963. A selective, orally active small molecule, targeting protein kinase B (PKB or AKT) and ribosomal protein S6 Kinase (p70S6K), with potential antineoplastic activity. XL418 inhibits the activities of PKB and p70S6K, both acting downstream of phosphoinosotide-3 kinase (PI3K). These kinases are often upregulated in a variety of cancers. Inhibition of PKB by this agent will induce apoptosis, while inhibition of p70S6K will result in the inhibition of translation within tumor cells. Qeios ID: 3TS65E · https://doi.org/10.32388/3TS65E 1/1
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Topography evolution of germanium thin films synthesized by pulsed laser deposition
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RESEARCH ARTICLE | APRIL 17 2017 Topography evolution of germanium thin films synthesized by pulsed laser deposition RESEARCH ARTICLE | APRIL 17 2017 P. Schumacher; S. G. Mayr; B. Rauschenbach AIP Advances 7, 045115 (2017) https://doi.org/10.1063/1.4981800 Articles You May Be Interested In Initial perturbation matters: Implications of geometry-dependent universal Kardar–Parisi–Zhang statistics for spatiotemporal chaos for spatiotemporal chaos Chaos (November 2021) Effects of grains’ features in surface roughness scaling Variational approach to KPZ: Fluctuation theorems and large deviation function for entropy production Chaos (July 2020) 24 October 2024 05:20:36 AIP ADVANCES 7, 045115 (2017) Topography evolution of germanium thin films synthesized by pulsed laser deposition P. Schumacher,1,a S. G. Mayr,1,2 and B. Rauschenbach1,2 1Leibniz-Instiut f¨ur Oberfl¨achenmodifizierung (IOM), Permoserstr. 15, 04318 Leipzig, Germany 2Fakult¨at f¨ur Physik und Geowissenschaften, Universit¨at Leipzig, 04103 Leipzig, Germany (Received 1 February 2017; accepted 9 April 2017; published online 17 April 2017) P. Schumacher,1,a S. G. Mayr,1,2 and B. Rauschenbach1,2 1Leibniz-Instiut f¨ur Oberfl¨achenmodifizierung (IOM), Permoserstr. 15, 04318 Leipzig, Germany 2Fakult¨at f¨ur Physik und Geowissenschaften, Universit¨at Leipzig, 04103 Leipzig, Germany (Received 1 February 2017; accepted 9 April 2017; published online 17 April 2017) (Received 1 February 2017; accepted 9 April 2017; published online 17 April 2017) Germanium thin films were deposited by Pulsed Laser Deposition (PLD) onto single crystal Ge (100) and Si (100) substrates with a native oxide film on the surface. The topography of the surface was investigated by Atomic Force Microscopy (AFM) to evaluate the scaling behavior of the surface roughness of amorphous and polycrystalline Ge films grown on substrates with different rough- nesses. Roughness evolution was interpreted within the framework of stochastic rate equations for thin film growth. Here the Kardar-Parisi-Zhang equation was used to describe the smoothening process. Additionally, a roughening regime was observed in which 3-dimensional growth occurred. Diffusion of the deposited Ge adatoms controlled the growth of the amorphous Ge thin films. The growth of poly- crystalline thin Ge films was dominated by diffusion processes only in the initial stage of the growth. © 2017 Author(s). All article content, except where other- wise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). [http://dx.doi.org/10.1063/1.4981800] 24 October 2024 05:20:36 aElectronic mail: philipp.schumacher@iom-leipzig.de I. INTRODUCTION Since the first report of Pulsed Laser Deposition (PLD) in 1965,1 it has become an established technique for coating surfaces and producing thin films. This technique has several advantages with respect to molecular beam epitaxy, among which are the high versatility, applicability and possi- bility to perform stoichiometric transfer, even with complicated compounds.2,3 For applications of the PLD technique, it is important to understand the physical principles that govern the growth process. Recent studies performed with Ge have yielded results about the roughness evolution, parameter dependence of the epitaxial breakdown and coarsening of mound-like structures on the surface.4–6 PLD has also been used to grow Ge nanostructures that have potential importance in device fabrication.7 This work contributes to the understanding of basic, physical mechanisms in thin film growth by PLD by taking into account the crystalline phase, film thickness, as well as substrate material for the growth of Ge as an exemplary material. Particularly, diffusion rates of adatoms in PLD can be increased and thereby change the surface topography significantly in comparison to other deposition techniques.8 Consequently, the question arises, for which conditions diffusion processes dominate the growth. With this objective in mind, the scaling behavior of the surface roughness was investigated. By applying the power spectral density (PSD) function to the surface topography as obtained by an Atomic Force Microscope (AFM), physical mechanisms that govern the growth processes on certain length scales can be identified. The basic feasibility of such an approach has been demonstrated within different scopes previously.9,10 In this context, the following continuum models that describe the surface evolution were considered. In a simplest case, random deposition is assumed, which means that particles are deposited on random positions and remain there. I. INTRODUCTION The growth exponent β, which characterizes the scaling behavior of the roughness over the time, can be calculated as β=0.5 for random deposition.11 If surface diffusion is a dominant factor (in addition to random deposition) aElectronic mail: philipp.schumacher@iom-leipzig.de © Author(s) 2017 7, 045115-1 7, 045115-1 7, 045115-1 2158-3226/2017/7(4)/045115/7 045115-2 Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) AIP Advances 7, 045115 (2017) the Wolf-Villain equation12 that combines Mullins-Herring curvature-induced surface diffusion13,14 with stochastic noise has been demonstrated to yield results in good accordance with experimental data: the Wolf-Villain equation12 that combines Mullins-Herring curvature-induced surface diffusion13,14 with stochastic noise has been demonstrated to yield results in good accordance with experimental data: ∂h(x, y, t) ∂t = -K ∇4h (x, y, t) + η(x, y, t). (1) (1) Here, h(x, y, t) is the height of the surface at position (x, y) for time t. K is a temperature dependent constant and η(x, y, t) a noise term. For the first term, diffusion of particles on the surface is assumed to occur according to the local chemical potential, dependent on the local curvature of the surface. The second term is a noise term that takes into account the randomness of the process. For this process the growth exponent is β=0.25, assuming a two-dimensional system.15 The roughness exponent α characterizes the scaling behavior of the roughness, depending on the length scale. For processes dominated by the Mullins-Herring equation, the roughness exponent is α=1 for two dimensions.15 Another growth mechanism is represented by the Kardar-Parisi-Zhang equation16 Here, h(x, y, t) is the height of the surface at position (x, y) for time t. K is a temperature dependent constant and η(x, y, t) a noise term. For the first term, diffusion of particles on the surface is assumed to occur according to the local chemical potential, dependent on the local curvature of the surface. The second term is a noise term that takes into account the randomness of the process. For this process the growth exponent is β=0.25, assuming a two-dimensional system.15 The roughness exponent α characterizes the scaling behavior of the roughness, depending on the length scale. I. INTRODUCTION For processes dominated by the Mullins-Herring equation, the roughness exponent is α=1 for two dimensions.15 Another growth mechanism is represented by the Kardar-Parisi-Zhang equation16 ∂h(x, y, t) ∂t = v∇2h (x, y, t) + λ 2 ∇h(x, y, t)2 + η (x, y, t) , (2) (2) (2) where h(x, y, t) is again the height of the surface at the position (x, y) and for time t. λ and ν are constants. The first term reflects a deposition-desorption phenomenon. Depending on the chemical potential of the vapor and surface, which depends on the local surface curvature, either desorption from ordepositionontothesurfacedominates.Thesecondtermreflectstheexistenceoflateralgrowth, while the third term takes into account noise since random deposition is assumed. Growth which is governed by this equation shows a scaling behavior characterized by a roughness exponent α=0.39 and a growth exponent β=0.25.17 These exponents are numerical results of the Kardar-Parisi-Zhang equation. 24 October 2024 05:20:36 If three-dimensional growth occurs, the coarsening exponent γ describes the relationship between the film thickness and separation of mound-structures on the surface. For Ge films produced by PLD, Shin et al. experimentally found a coarsening exponent γ=0.40±0.05.4 II. EXPERIMENTAL CONDITIONS In the present investigations, Ge films were deposited from a high purity Ge target by means of PLD. As substrates, Si (100) and Ge (100) were used. The native oxide layers on the substrates were not removed. A KrF-excimer laser with a pulse length of 20 ns and wavelength of 248 nm was used for PLD. The pulse frequency was 10 Hz and the pulse energy was fixed at 220 mJ, with a focal spot size of roughly 0.05 cm2 that resulted in a fluence of 4.4 J/cm2. For all depositions, the background pressure was <8×10 8 mbar. The average deposition rate was on the order of 0.001 nm per pulse. The thickness of the Ge films was controlled via the number of laser pulses and monitored by a quartz crystal microbalance, which was calibrated regularly by means of X-ray reflectometry. The substrates could be heated using a resistive heater and the temperature was corrected using a thermocouple. The topography of the films was investigated by an AFM in tapping mode with a scan size of 2x2 µm2. The Si tips had a nominal radius of 7 nm. From the statistical point of view, the height fluctuations of the surface could be described by the root mean square roughness (RMS) Rq, which is given by Rq = r 1 NM XN i=1 XM j=1 [h(xi, yj) −¯h]2, (3) (3) where h(xi, yj) is the surface height at a given point (xi, yj), N and M are the number of points in x- and in y-direction, respectively and ¯h = 1 NM PN i=1 PM j=1 h(xi, yj) is the average height of the surface. The RMS value of each film was measured on a square area of 500 x 500 nm2. During film growth, different points on the surface are not independent and the height at each point is related to the height at other points nearby. The information about these correlations and about characteristic distances of structures is provided by the height-height correlation function H  δx, δy, t  = D [h  x + δx, y + δy, t  −h(x, y, t)]2E , (4) (4) Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) AIP Advances 7, 045115 (2017) 045115-3 where δx and δy are distances in x- and in y-direction respectively. The angular brackets denote averaging over the plane. II. EXPERIMENTAL CONDITIONS Additionally, the PSD function was calculated in order to analyze the height profiles in reciprocal space. The scaling behavior of this function is related to the aforementioned roughness exponents.18 It is therefore possible to deduce the physical principles governing the growth of the material. By performing the discrete two dimensional fast Fourier transformation (FFT) of the height profile of the AFM images, the dominating spatial frequencies on the surface and amplitude of the roughness can be determined. For the discrete case, a square image of N x N points, the two-dimensional fast Fourier transformation is given by FFT  fx, fy  = 1 N2 XN i=1 XN j=1 h  xi, yj  exp " −2iπ N  xifx + yjfy # , (5) (5) where f x and f y are the spatial frequency coordinates along the x-axis and y-axis, respectively. By calculation of the magnitude square of FFT(f x, f y), the two-dimensional power spectral density function PSD(f x, f y) can be obtained.19 The structures on the surface were expected to be isotropic, so the angular averaged PSD(f ) was used to evaluate the experimental roughness data. The averaging was performed over all spatial frequencies with constant magnitude f = q f 2x + f 2y . For the PSD function, an expression can be derived from which the contributing processes can be deduced10 PSD (f , t) = D(f )1 −e−2t P4 i=1 aif i P4 i=1 aif i (6) (6) where D(f ) is the strength of the white noise, t is the time and ai ≥0 are constants whose relations contain information about the growth process and the dominating mechanisms. 24 October 2024 05:20:36 III. RESULTS AND DISCUSSION Epitaxial growth of Ge on Si (100) by physical vapor deposition has been previously observed (see e.g. Ref. 20). However, in this study, epitaxial growth was not expected due to the presence of native oxide on the substrates and the comparably low temperatures during the deposition process. FIG. 1. (a,b) AFM images of polycrystalline Ge films deposited on Ge at 175 ◦C. The RMS value and the thickness d are given below each image. (c) The roughness evolution of polycrystalline Ge films deposited on Ge at 175 ◦C. (d) The evolution of the roughness in the roughening regime with a rescaled film thickness (see text), shown with a power law fit. The same data is also plotted with respect to the number of laser pulses, indicated by circular data points. FIG. 1. (a,b) AFM images of polycrystalline Ge films deposited on Ge at 175 ◦C. The RMS value and the thickness d are given below each image. (c) The roughness evolution of polycrystalline Ge films deposited on Ge at 175 ◦C. (d) The evolution of the roughness in the roughening regime with a rescaled film thickness (see text), shown with a power law fit. The same data is also plotted with respect to the number of laser pulses, indicated by circular data points. 045115-4 Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) FIG. 2. The mound separation determined by the height-height correlation function with respect to the film thickness of polycrystalline Ge films deposited at 175 ◦C. IG. 2. The mound separation determined by the height-height correlation function with respect to the film thickness of olycrystalline Ge films deposited at 175 ◦C. The evolution of the surface topography was studied by variation of both the substrate material and substrate temperature. The Ge deposition was either carried out at room temperature to produce amorphous films or at (175±30) ◦C to produce polycrystalline films. The evolution of the surface topography was studied by variation of both the substrate material and substrate temperature. The Ge deposition was either carried out at room temperature to produce amorphous films or at (175±30) ◦C to produce polycrystalline films. In Figs. 1 (a) and (b), AFM images of polycrystalline Ge films deposited on Ge substrates and with two different film thicknesses are compared. Additionally, Fig. 1 presents the roughness evolution with respect to the film thickness. III. RESULTS AND DISCUSSION The AFM images and roughness evolution revealed that the deposited Ge film was not initially closed. The Ge film was smoothened until it closed above a critical layer thickness of approximately 45 nm. Consequently, the roughness rose with the layer thickness, which was caused by a coarsening process during the deposition. In agreement to the study of Shin et al.,4 the formation of irregularly shaped mounds was observed. During the pulsed laser ablation process, the deposition rate was not constant; therefore, the film thickness was not a time-equivalent quantity. For this reason, the film thickness d was rescaled in order to determine the growth exponent. The rescaled and time-equivalent film thickness was calculated according to d′ = (¯r/r) (d −45nm). Here, d is divided by the time-averaged deposition rate r of each sample and multiplied by a constant ¯r. This rescaling generated the film thickness that would exist if all the samples were deposited at a constant deposition rate of ¯r. The result of this rescaling is depicted in Fig. 1 (d). The growth exponent β could be determined for the roughening regime above a film 24 October 2024 05:20:36 FIG. 3. The roughness evolution of Ge films on Si substrates at two different temperatures. The filled points represent the data for the polycrystalline films (deposited at 175 ◦C), while the hollow points represent the data for the amorphous films (deposited at room temperature). The circular points (red) show the roughness evolution with respect to the number of laser pulses, and the square points (black) show the roughness evolution with respect to the film thickness. FIG. 3. The roughness evolution of Ge films on Si substrates at two different temperatures. The filled points represent the data for the polycrystalline films (deposited at 175 ◦C), while the hollow points represent the data for the amorphous films (deposited at room temperature). The circular points (red) show the roughness evolution with respect to the number of laser pulses, and the square points (black) show the roughness evolution with respect to the film thickness. 045115-5 Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) FIG. 4. The PSD function calculated from AFM measurements of Ge films with different thicknesses on various substrates: (a) amorphous films, deposited on Si at room temperature, (b) polycrystalline films, deposited on Si at 175 ◦C and (c) polycrystalline films, deposited on Ge at 175 ◦C. thickness of 45 nm. III. RESULTS AND DISCUSSION It was calculated from the slope of the log-log representation of the roughness as FIG. 4. The PSD function calculated from AFM measurements of Ge films with different thicknesses on various substrates: (a) amorphous films, deposited on Si at room temperature, (b) polycrystalline films, deposited on Si at 175 ◦C and (c) polycrystalline films, deposited on Ge at 175 ◦C. thickness of 45 nm. It was calculated from the slope of the log-log representation of the roughness as a function of thickness. By fitting the roughness data, β=0.45±0.17 was obtained. Compared to the aforementioned growth exponents, random deposition gave the most adequate representation of this thickness of 45 nm. It was calculated from the slope of the log-log representation of the roughness as a function of thickness. By fitting the roughness data, β=0.45±0.17 was obtained. Compared to the aforementioned growth exponents, random deposition gave the most adequate representation of this Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) 045115-6 roughening regime. Nevertheless, the values for growth dominated by diffusion and growth along the local surface were within the margins of error. The roughening regime was analyzed by means of the height-height correlation function (see eq. 4). The separation of mounds was found by determining the distance of the first maximum to the center peak in the height-height correlation. After rescaling,7 it could be shown, that the mound separation increased with the film thickness (see Fig. 2). The coarsening exponent was determined to be 0.48±0.29. This corresponded to a high coarsening rate, which was also found by Shin et al.4 In that context, the high coarsening exponent was justified by defect-mediated filling of gaps and increased effective corner-diffusion, which was confirmed by the observation of irregularly shaped mounds (Fig. 1 (b)). The roughness evolution of Ge films deposited on Si substrates at room temperature and at 175 ◦C is depicted in Fig. 3. For small film thicknesses the RMS roughness increased slightly up to 0.2 nm corresponding to a polycrystalline film thickness of about 10 nm. After that, a smoothening regime occurred. For large film thicknesses (>50 nm) the roughness was rather constant. For all films, only small differences between the roughness evolution of the amorphous and polycrystalline films were observed. In Fig. 4, the evolution of the PSD function for different crystalline phases of the film and different substrate materials are presented. III. RESULTS AND DISCUSSION For the deposition of amorphous Ge on Si, the fits revealed that a4 (see eq. 6) was large compared to the other constants, while a3 was not negligible either. Mullins- Herring diffusion was therefore identified as the presumably dominating process. On small length scales, surface diffusion was important for the evolution of the surface. From the PSD curves at intermediate spatial frequencies (f ≈0.03 nm-1), the evolution of the topography could be described by the Kardar-Parisi-Zhang equation. This behavior was similar for all the film thicknesses shown here. For the deposition of polycrystalline Ge on Si, the fits revealed a comparably high a4-constant for small film thickness (4 nm), while a4=0 and a3 dominated for medium film thickness (8 nm and 30 nm). In the beginning of the growth, surface diffusion as described by Mullins and Herring governed the process. After a certain film thickness, surface diffusion was suppressed and the quadratic term of the Kardar-Parisi-Zhang equation became more prominent. Traditionally the interpretation of this term has been desorption, however due to the relatively small deposition temperatures, desorption was assumed to be negligible here. Different, more complex mechanisms might also account for such a functional dependency. For high film thickness (112 nm), ‘bumps’ and irregularities in the PSD function appeared due to the formation of mound structures with characteristic separations. 24 October 2024 05:20:36 pp p For the deposition of polycrystalline Ge on Ge, the fits revealed domination of surface diffusion up to a film thickness of 57 nm. In these cases the PSD curves show an f −4-behavior for large f and an f 1-behavior for smaller f. Traditionally the f 1-dependency has been interpreted as viscous flow.21 It is to be noted however, that noise might have also contributed here. It is not possible to distinguish between noise and the real f 1-dependency. For larger film thicknesses, again ‘bumps’ evolved, leading to relatively large deviations between the fits and measured data. The ‘bumps’ moved to lower spatial frequencies from film thicknesses of 74 nm to 96 nm. This shift indicated, that the average separation of mound-structures increased, as was also shown above by means of the height-height correlation. Kinetic Monte-Carlo simulations of PLD of Si on Si have already been performed.22 For thermal deposition, the occurrence of mound-like structures and existence of a second maximum in the height-height correlation were observed. III. RESULTS AND DISCUSSION Additionally the roughness exponent was determined to be α≈0.95±0.1 for a deposition temperature of 400 ◦C. Comparable roughness exponents were found in this study at lower temperatures depending on the exact deposition conditions. IV. SUMMARY For the deposition of polycrystalline Ge on Ge at 175 ◦C the roughness of the films initially decreased. This regime was dominated by a Mullins-Herring diffusion process. Beginning at a certain film thickness (approx. 45 nm), a roughening occurred. This roughening was accompanied by the occurrence of mound-structures, which were observed in the height-height correlation function as well as in the PSD spectra. These changes were also accompanied by a comparably high coarsening rate. 045115-7 Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) AIP Advances 7, 045115 (2017) For the deposition of amorphous Ge on Si at room temperature, the surfaces were smoothened after reaching film thicknesses of approx. 10 nm. Here, Mullins-Herring diffusion was found to be the governing mechanism on small length scales, independent of the film thickness. For the deposition of polycrystalline Ge at 175 ◦C, the surfaces were also smoothened after reaching film thicknesses of several nm. However, the Mullins-Herring diffusion was identified as present only for small film thickness (4 nm). For larger film thicknesses, surface diffusion was suppressed and the Kardar-Parisi-Zhang equation was a suitable description of the process already on small length scales. ACKNOWLEDGMENTS We would like to thank Dr. Erik Thelander for the technical assistance and for several useful discussions and Emilia Wisotzki for proofreading the manuscript. 1 H. M. Smith and A. F. Turner, Appl. Optics 4, 147 (1965). 2 2 P. R. Willmott and J. R. Huber, Rev. Mod. Phys. 72, 315 (2000). 3 3 M. Stafe, A. Marcu, and N. Puscas, Pulsed Laser Ablation of Solids, Springer Series in Surface Sciences 53, Springer, 201 4 B. Shin, J. P. Leonard, J. W. McCamy, and M. J. Aziz, Appl. Phys. Lett. 87, 181916 (2005). 3 M. Stafe, A. Marcu, and N. Puscas, Pulsed Laser Ablation of Solids, Springer Series in Surfac 4 3 M. Stafe, A. Marcu, and N. Puscas, Pulsed Laser Ablation of Solids, Springer Series in Surface Sciences 53, Springer, 2014. 4 B. Shin, J. P. Leonard, J. W. McCamy, and M. J. Aziz, Appl. Phys. Lett. 87, 181916 (2005). 3 M. Stafe, A. Marcu, and N. Puscas, Pulsed Laser Ablation of Solids, Springer Series in Surface Sciences 53, Springer, 2014. 4 B. Shin, J. P. Leonard, J. W. McCamy, and M. J. Aziz, Appl. Phys. Lett. 87, 181916 (2005). f p g p g 4 B. Shin, J. P. Leonard, J. W. McCamy, and M. J. Aziz, Appl. Phys. Lett. 87, 181916 (2005). 5 5 M. J. Aziz, Appl. Phys. A 93, 579 (2008). 5 M. J. Aziz, Appl. Phys. A 93, 579 (2008). 6 6 B. Shin and M. J. Aziz, Phys. Rev. B 76, 085431 (2008). 6 B. Shin and M. J. Aziz, Phys. Rev. B 76, 085431 (2008). 7 7 C. V. Cojocaru, A. Bernardi, J. S. Reparaz, M. I. Alonso, J. M. MacLeod, C. Harnagea, and F. Rosei, Appl. Phys. Lett. 91, 113112 (2007). 7 C. V. Cojocaru, A. Bernardi, J. S. Reparaz, M. I. Alonso, J. M. MacLeod, C. Harnagea, and F. Rosei, Appl. Phys. Lett. 9 113112 (2007). 8 P.-O. Jubert, O. Fruchart, and C. Meyer, Surf. Sci. 522, 8 (2003). 8 P.-O. Jubert, O. Fruchart, and C. Meyer, Surf. Sci. 522, 8 (2003). 9 y 9 J. R¨oder, T. Liese, and H.-U. Krebs, J. Appl. Phys. 107, 103515 (2010). 9 J. R¨oder, T. Liese, and H.-U. Krebs, J. Appl. Phys. 107, 103515 (2010). 10 0 S. G. Mayr and R. S. Averback, Phys. Rev. Lett. 87, 196106 ( y , y , ( ) 11 J. T. Drotar, Y.-P. Zhao, T.-M. ACKNOWLEDGMENTS Lu, and G.-C. Wang, Phys. Rev. B 62, 2118 (2000). 12 11 J. T. Drotar, Y.-P. Zhao, T.-M. Lu, and G.-C. Wang, Phys. Rev. B 62, 2118 (2000). 12 1 J. T. Drotar, Y.-P. Zhao, T.-M. Lu, and G.-C. Wang, Phys. Re 12 D. E. Wolf and J. Villain, Europhys. Lett. 13, 389 (1990). 12 D. E. Wolf and J. Villain, Europhys. Lett. 13, 389 (1990). 24 October 2024 05:20:36 13 W. Mullins, J. Appl. Phys. 28, 333 (1957). 13 W. Mullins, J. Appl. Phys. 28, 333 (1957). 14 14 C. Herring, J. Appl. Phys. 21, 301 (1950). 14 C. Herring, J. Appl. Phys. 21, 301 (1950). 5 15 A. L. Barabasi and H. E. Stanley, Fractal Concepts of Surface Growth (Cambridge University Press, 1995). 16 5 A. L. Barabasi and H. E. Stanley, Fractal Concepts of Surfac 6 15 A. L. Barabasi and H. E. Stanley, Fractal Concepts of Surface Growth (Cambridge University P 16 16 M. Kardar, G. Parisi, and Y. C. Zhang, Phys. Rev. Lett. 56, 889 (1986). 16 M. Kardar, G. Parisi, and Y. C. Zhang, Phys. Rev. Lett. 56 17 17 J. G. Amar and F. Family, Phys. Rev. A 41, 3399 (1990). 18 17 J. G. Amar and F. Family, Phys. Rev. A 41, 3399 (1990). 18 Amar and F. Family, Phys. Rev. A 41, 3399 (1990). 7 J. G. Amar and F. Family, Phys. Rev. A 41, 3399 (1990). y y 18 W. M. Tong and R. S. Williams, Annu. Rev. Phys. Chem. 45, 401 (1994). 19 18 W. M. Tong and R. S. Williams, Annu. Rev. Phys. Chem. 45, 40 19 Y. Zhao, G.-C. Wang, and T.-M. Lu, Characterization of amorphous and Crystalline Rough Surfaces: Principles and Applications (Academic Press, San Diego, 2001). 20 19 Y. Zhao, G.-C. Wang, and T.-M. Lu, Characterization of amorphous and Crystalline Rough Surfaces: Principles and Applications (Academic Press, San Diego, 2001). Applications (Academic Press, San Diego, 2001). 20 A. N. Larsen, Mat. Sci. Semicon. Proc. 9, 454 (2006). 21 20 A. N. Larsen, Mat. Sci. Semicon. Proc. 9, 454 (2006). 21 21 C. C. Umbach, R. H. Headrick, and K.-C. Chang, Phys. Rev. Lett. 87, 246104 (2001). 22 21 C. C. Umbach, R. H. Headrick, and K.-C. Chang, Phys. Rev. Lett. 87, 246104 (2001). 22 22 S. G. Mayr, M. Moske, K. Samwer, M. E. Taylor, and H. A. Atwater, Appl. Phys. Lett. 75, 4091 (1999). ACKNOWLEDGMENTS 22 S. G. Mayr, M. Moske, K. Samwer, M. E. Taylor, and H. A. Atwater, Appl. Phys. Lett. 75, 4091 (1999).
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Agile logistics challenges of petroleum product distribution in Nigeria
International Journal of Science and Research Archive
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Agile logistics challenges of petroleum product distribution in Nigeria national Journal of Science and Research Archive, 2024, 11(02), 531–542 Publication history: Received on 04 February 2024; revised on 12 March 2024; accepted on 15 March 2024 Article DOI: https://doi.org/10.30574/ijsra.2024.11.2.0436 Abstract Agile distribution of petroleum product is a strategy towards uninterrupted supply of quality products at affordable prices. Industry and economic productivity as well as transportation in Nigeria is largely driven by petroleum energy due to low investments in other sources of energy, yet the petroleum energy supply and distribution is marred by lingering history of scarcity, high prices, and distribution bottlenecks to end users all over the nation. The study through a descriptive survey following literature view, primary and secondary data collection sought to determine at granular level the limiting factors of agile petroleum product distribution in Nigeria. Petroleum supply, consumption and pricing are significant aspects of the study survey. Study findings shows limiting factors of distribution agility cut across pricing, supply, technology, infrastructure, accessibility and lead time, storage, management, and Human Resource issues. Significantly lack of local refining capacity and poor distribution network are notorious for supply limitations and rising price of products. Addressing the challenges would require strong political will, driven by genuine commitment and focused attention towards economic growth and improvement in the welfare of the citizens. Keywords: Petroleum; Distribution; Agility; Supply Keywords: Petroleum; Distribution; Agility; Supply Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons * Corresponding author: Goodluck Tochukwu Njoku Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4 2.1. Distribution Enterprise Task A significant aspect of the enterprise logistics function is distribution. Specifically referring to the delivery of the finished products to the customer. Schewe and Smith (1980) define distribution as the physical movement of products to the ultimate consumers. It consists of order processing, warehousing, and transportation. Distribution Straka (2017) identify is responsible for storage and transportation of goods to consumers as well as related information, management, and control activities. It ensures the most appropriate way, selection, and analysis of transport, which is most suitable for transfer of products manufactured by enterprises to achieve failure-free performance of the market. The distribution network comprise of specific nodes which include; factories where products are manufactured or assembled; a depot or deposit relating to standard type of warehouse for storing of merchandise (high level of inventory); distribution centres for order processing and fulfillment (lower level of inventory) and also for receiving returning items from clients, transit points built for cross docking activities, which consist in reassembling cargo units based on deliveries scheduled (only moving merchandise). 2. Literature review Energy is key to economic productivity and industrial growth and is central to the operation of any modern economy. Energy is one of the most important inputs for economic development and from a physical viewpoint, the use of energy drives economic productivity, consumption, and growth. Many production and consumption activities involve energy as a basic input (Zahid, 2008). In Nigeria the petroleum products and the derivatives constitute the main source of energy for production, manufacturing, distribution and general logistics of goods, passengers, and services. A logistics system which meets the need for timely availability, quality, and affordable products is necessary to drive economic productivity and growth. The components of petroleum and its by-products traded include the following: Premium Motor Spirit (PMS), Automotive Gas Oil (AGO), Household Kerosene (HHK), Dual Purpose Kerosene (DPK), Aviation Turbine Kerosene (ATK), Low Pour Fuel Oil (LPFO), Liquefied Petroleum Gas (LPG), Bitumen, Lubricants and Base Oil. Fuels are extracted from crude petroleum and pass through various processing units by thermal cracking in the refineries. International Journal of Science and Research Archive, 2024, 11(02), 531–542 reflecting rapid growth in the number of automobiles, industries, households, intensified rural-urban migration, economic and political developments. The bulk of products consumption has been the Premium Motor Spirit (PMS) or petrol, Automotive Gas Oil (AGO) or diesel, dual purpose kerosene and bitumen/asphalt. Together, they account for more than 60.0 per cent of the total consumption of petroleum products. The petroleum product distribution in Nigeria is challenged by short deliveries, high lead time, increased cost of distribution, scarcity of products and high pump prices. Nigeria is an emerging and fast developing market and the demand for petroleum product continues to icrease. As such, the need for sufficient and uninterrupted supplies is a major task for national development and economic growth. A sustainable economic development cannot be successfully achieved without an appreciable vibrant supply chain which involves the even distribution of petroleum products to keep alive economic activities such as manufacturing, production distribution and general logistics involving the movement of materials and human resources from one place to the other. In this regard, the study seeks to:  Examine the limiting factors to agile distribution of petroleum products in Nigeria.  Evaluate petroleum supply and consumption.  Evaluate petroleum supply and  And analyse product pricing. 1. Introduction The petroleum industry is involved in a global supply-chain that includes domestic and international transportation, ordering and inventory visibility and control, materials handling, and information technology. Logistics is a supply chain function that plays an essential role to ensure a safe, timely and cost-effective delivery. Oil and gas logistics includes typically transportation of crude oil from the production sites to refineries as well as transportation and distribution of oil products to markets and customers. The demand for oil and gas has increased in tandem with the economy of the world as it continued to prosper. From the reference point of 1960, Organization of the Petroleum Exporting Countries (OPEC) projects that oil demand would increase up to five times by 2035 (OPEC, 2014). The demand for petroleum product is affected by myriad of challenges. Alongside the need to replace depleted wells, old plant, and equipment, and to accommodate ever higher standards and tighter regulations is the need to adopt logistics strategies that ensure consumers continue to receive products in a timely and orderly manner (OPEC, 2014). An effective and efficient logistics process is an industry key for reducing lead times and costs, increase the company's profits and in managing supply chains; demand management, efficient distribution of petroleum products among customers, better transportation scheduling, warehouse management, and quality and timeliness of information (Lisitsa, Levina and Lepekhin, 2019). In Nigeria, the consumption of petroleum products Onwioduokit and Adenuga (2000) recount has continually been on the increase with most of the demand for products fulfilled over time by importation due to the poor condition of the refineries. Starting from the 1980s the demand for petroleum products they acknowledge have increased tremendously * Corresponding author: Goodluck Tochukwu Njoku International Journal of Science and Research Archive, 2024, 11(02), 531–542 The agile supply chain is a more recent concept which focuses on leveraging responsiveness to customer demand. Here, logistics plays a crucial role in aligning material flow across the supply network with customer demand, and in ensuring execution in a flexible and customized manner (Harrison & Hoek, 2008). The origins of the agility paradigm in logistics and supply chain can be traced back to the theory of lean enterprises, which comprises concepts and methods which aim to minimize waste and consequently maximize efficiency and cost-effectiveness as the company uses fewer resources (capital, financial, human, organisational) and less time to achieve the same goal (Sajdak, 2015). Agile logistics strategy of petroleum product distribution represents a distribution process which ensures the availability right products to the right customer at the right place, in the right condition and right quantity at the right time, and right (lowest possible) costs. Logistics is the task of managing two key supply chain flows; material flow of the physical goods from suppliers through the distribution centers, stores and flow of Information; demand data from the end-customer back to purchasing and to suppliers, and supply data from suppliers to the retailer, so that material flow can be accurately planned and controlled (Harrison & Hoek, 2008). The agile strategy ensures the adequacy of place (moving refined products to places where there is a demand for them) and time (maintaining the right stocks levels) and proper distribution of the products through procedural alignment, end-to-end accountability, social proximity, and deep integration of suppliers. 2.2. Agility of Petroleum Product Distribution The petroleum industry is frequently classified into three main sections: upstream, midstream, and downstream operation. Upstream activities refer to the exploration and production of crude oil. This includes searching for potential underground or underwater oil and gas fields, drilling of exploratory wells, and subsequently operating the wells that recover and bring the petroleum crude oil and/or raw natural gas to the surface. Midstream activities refer to the refining, transportation (by pipelines, rail, tankers), storage and wholesale marketing of crude or refined petroleum products. Downstream activities refer to the transportation and marketing of end-user products, e.g. petrol, diesel, and liquefied petroleum gas (LPG). The agility of petroleum product distribution is a strategy to building an agile supply chain of the same. Agility Tiefenbacher (2020) denotes is a strategy to thrive, improve performance and be competitive in VUCA environments, that is environments that are volatile, uncertain, chaotic, and ambiguous characterized by being unpredictable and thereby necessitating adaptive, flexible approaches and interventions to muster them. Logistics and Supply chains are one of the same ultimately concerned with delivering the right thing to the right place at the right time (LaBotz, 2022). 532 International Journal of Science and Research Archive, 2024, 11(02), 531–542 2.4.1. Lack Local Refinery Capacity 2.4.1. Lack Local Refinery Capacity Nigeria is blessed with the abundance of petroleum resource and natural gas, but the country is seriously challenged by local refining capacity which meet the soaring energy need. World Energy 2023 report according to Akintayo (2023) reveal local refineries output of Nigeria has dropped to near zero output in 10 years. According to data the statistical review, output from the nation’s four refineries fell from 92,000 barrels per day in 2012 to just 6,000 barrels per day in 2022 representing a 92 per cent drop in production capacity. Similarly, the Organisation of the Petroleum Exporting Countries’ Annual Statistical Bulletin (2023), also revealed the country’s crude oil refining capacity fell from 33,000 bpd in 2018 to 6,000 bpd in 2022, representing about 81 per cent drop in production output. The drop in refining capacity has made the country to rely solely on the importation of refined petroleum products to meet the local energy consumption needs. NBS report spanning from 1997 up to 2014 presented in table 1 below shows a continued decline in capacity utilization of the refineries and year on year growth in crude oil refining percentage respectively. Adeosun and Oluleye (2017) acknowledge Nigeria despite having a nameplate refining capacity, 445,000 bpd which exceeds demand has over the last four decades consistently struggled to keep its refineries functioning optimally and is ranked as the 3rd highest importer of petroleum products in Africa, importing over 80% of products consumed. 2.3. Petroleum Distribution Chain in Nigeria The actors in the Nigerian industry consist of both private and public organizations. The public actors are the government agents and functionaries such as the Nigerian National Petroleum Corporation (NNPC) and its subsidiaries, the Department of Petroleum Resources (DPR), the petroleum products pricing regulatory authority (PPPRA), the Pipeline and Product Marketing Company (PPMC) among others. The private segment consists of both indigenous and foreign actors. The PPMC is charged with the task of large quantities supply, distribution, and marketing of petroleum products in Nigeria. There exists network of four thousand (4,000) kilometers of pipelines which are interconnected with twenty-one (21) highly dispersed depots. The products may be obtained from the four local refineries or in the event of a supply short-fall from off-shore refineries by way of imports. Imported refined products are received at PPMC depots at Atlas Cove. From here, products are pumped to nearby depots at Mosimi in Shagamu, from which products are pumped into various other depots through the pipelines; Booster pump stations are provided along the route and between two adjoining depots. This arrangement is necessary to boost the flow of products in the pipelines along the routes. Movement of products from the depots is the responsibility of major oil marketers. Source: NNPC/PPMC Bulletin, 2010 Figure 1 Chanel of Petroleum Product Distribution in Nigeria Source: NNPC/PPMC Bulletin, 2010 Source: NNPC/PPMC Bulletin, 2010 Figure 1 Chanel of Petroleum Product Distribution in Nigeria 533 International Journal of Science and Research Archive, 2024, 11(02), 531–542 International Journal of Science and Research Archive, 2024, 11(02), 531–542 2.4.1. Lack Local Refinery Capacity Table 1 Yearly Domestic Crude Oil Refining 1997-2014 Year Crude oil received Crude oil processed CAPACITY UTILIZATION (%) Year on Year Growth in Crude Oil Refining % 1997 80,473,488.91 78,001,995.34 140.8 1998 52,422,070.13 54,304,185.98 93.16 -30.38 1999 65,979,134.80 66,409,253.60 116.18 22.29 2000 36,189,139.39 36,282,789.51 58.64 -45.36 2001 82,578,546.57 81,512,099.57 140.41 124.66 2002 78,160,576.55 79,579,031.26 142.65 -2.37 2003 42,754,815.49 44,811,925.54 72.11 -43.69 2004 38,019,906.64 38,026,965.93 66.14 -15.14 2005 72,360,780.00 70,637,019.00 129.44 85.76 2006 42,471,739.00 43,445,397.00 62.45 -38.49 2007 18,191,136.45 19,059,670.28 24.87 -56.13 2008 45,533,304.84 39,264,519.46 75.92 106.01 2009 19,392,571.25 17,745,656.61 78 -54.80 2010 33,633,907.94 34,871,693.84 167.87 96.51 2011 40,405,605.00 39,408,108.00 61.95 13.01 2012 33,595,260.00 33,628,602.00 68.95 -14.67 2013 36,193,237.20 35,233,125.59 74.5 4.77 2014 25,839,373.09 23,360,372.27 44.42 -33.70 Source: National Bureau of Statistics e-Library, 2014 Table 1 Yearly Domestic Crude Oil Refining 1997-2014 The scarcity of petroleum products and the rising cost of the petroleum products is an indication that refined product importation is not sustainable for agile distribution of petroleum products. Okereke, Emodi and Diemuodeke, (2022) lament the shortage of refining capacity at existing oil refineries is the main driver of Nigeria’s fuel crisis, which hampers the socio-economic development of the country, placing a high subsidy burden on the government and has long made Nigeria dependent on imported petroleum products. Similarly, Ehinomen and Adeleke (2012) decry the low-capacity utilization of government owned refineries and petrochemical plants in Kaduna, Warri and Port Harcourt and inadequate crude oil allocation to the refineries for domestic consumption as well as long turnaround time for the 534 International Journal of Science and Research Archive, 2024, 11(02), 531–542 maintenance of the refineries poses a lot of challenge to NNPC and result into petroleum product shortage and eventual importation of the products a situation that puts a drain on the scarce foreign exchange of the nation. An attempt by the government at the removal of subsidy without addressing the issues of local refining capacity has compounded the issue of product scarcity and high prices with PMS and AGO pricing reaching 600 NGN and 1300 NGN respectively, the highest prices ever recorded in the history refined product distribution in Nigeria. 2.4.1. Lack Local Refinery Capacity Because industrial production activity and business operation in Nigeria largely depend on petroleum energy following the epileptic electric power supply and lack of technology to harness the solar power, the increase in the price of the petroleum products have resulted to hyperinflation which has affected the cost of production, local cost of transportation and prices of consumer commodities. Sales and business struggle due to high cost of doing business, low demand, and patronage while many shut down and workers lose their jobs. Source: Author’s Analysis Figure 2 Capacity Utilization and Year on Year % Growth in Oil Refining Figure 2 Capacity Utilization and Year on Year % Growth in Oil Refining Figure 2 above presents a graphical analysis of the capacity utilization and year on year percentage growth in crude refining with reference table 1. The line graph shows a staggered progression in the refinery capacity utilization and year on year growth in oil refining for the years referenced depicting a very hopeless situation for the industry in Nigeria. There has not been a serious approach in addressing the issues of local refinery capacity in the country since time and the challenges have compounded till date. With growth in population and increasing demand for development which put pressure on energy supply to drive industrial and economic productivity, the reliance on importation of refined products has become highly unsustainable. A significant aspect of agile product distribution is delivering the products available to the end users at affordable cost. This cannot be realized by dependance on importation to feed the local market. No doubt pump price of petroleum products in Nigeria is due to importation is determined by world crude prices, and inflation in the import countries. Interruption in production due political instability, social unrest and natural disaster in the import countries would translate to scarcity of product in Nigeria. 2.4.4. Dependency on Road Haulage The supply of refined petroleum products to end users is faced with many challenges. Efficient transportation of petroleum product requires a modal integration of shipping via vessels, pipeline, road trucking and rail tankers. While the pipeline is acknowledged the most efficient single mode due to the benefit of large volume delivery at high speed and constant flow resulting to cheaper cost relative to other modes, zero accidents and limited environmental concerns the case is different in Nigeria where on the contrary road haulage is the dominant mode of petroleum distribution. This is because the pipeline mode is underdeveloped and fraught by the combined force of vandalism by oil bunkering, industrial sabotage, militancy, poor maintenance, and neglect. According to Bataiya, (2018) 98% petroleum products that include fuel components like premium motor spirit, domestic pure kerosene, and automotive gas oil’s lifting from supply sources to all parts of the country is done by road which of itself is in gross disrepair and affecting the distribution of the products. 70% of the federal road which constitute a 70% of the vehicular freight movement is overstretched with 40% and 27% in poor and fair condition respectively requiring rehabilitation and maintenance work and a 3% unpaved. 78% and 87% of state and local government roads respectively are in poor condition. The road transport research acknowledge is not efficient for petroleum distribution over long distances due to lower speed, interrupted flow, limited capacity leading to high cost of transportation per unit volume. Nwoloziri et al. (2021) acknowledge the inefficiency of railways and pipeline transportation heavily contribute to total dependency on road transportation in the distribution of petroleum products to various depots and stations located all over the country. Ehinomen and Adeleke (2012) recounts the despair, neglect and repeated vandalization of the state-run petroleum products pipelines and oil movement infrastructure nationwide, coupled with frequent accidents of haulage trucks on the nations heavily used highways pose complex problems managers, and operators in the oil industry. Road haulage of petroleum products is marred by road traffic accidents, theft, and robbery incidence, bad and poor road networks as well as various hindrances such as delays at police, customs check points and many other bottlenecks (Thomas et al., 2014, Nwachukwu & Chike, 2015, Bataiya, 2018, Nwoloziri et al., 2021, Ehinomen & Adeleke, 2012). International Journal of Science and Research Archive, 2024, 11(02), 531–542 Nigeria recorded over over 16,083 cases of “pipeline tampering” and vandalism amounting to a loss of 174.57 billion (or approximately $484 million) and between 2016 and 2017, a total of 992 cases of pipeline vandalism were recorded across pipelines and depot lines, amounting to a loss of 167 billion (approximately $464 million). Nigeria Extractive Industries Transparency Initiative (NEITI) records between 2017 and 2021 (in 5 years) Nigeria recorded a total of 7,143 cases of pipeline vandalism. The country recorded in 11 years (2009-2020) a loss of 619.7 million barrels of oil valued at $46.16 billion to theft, pipeline tampering and vandalism. This is higher than the size of the country’s foreign reserves. In addition, Nigeria lost 4.2 billion litres of petroleum products from refineries, valued at $1.84 billion at the rate of 1440,000 barrels per day, from 2009 to 2018 (Aduloju, 2023). 2.4.3. Corruption and Mismanagement Unscrupulous and corrupt practices of government agencies, oil companies and marketers as well as mismanagement of petroleum revenue largely fraught the agility of petroleum product distribution in Nigeria. Subsidy of petroleum products leads to a situation where the pump price of petroleum product is higher in neighbouring countries, Benin, Cameroon, and Chad. The aim of the petroleum subsidy was undermined following large-scale smuggling of petroleum meant for Nigerian consumption and subsidized by the government to the neighbouring countries where they are sold at higher prices making superfluous profits short-changing the government and causing scarcity in Nigeria (Terkimbi, 2015 & Ayanruoh, 2023). Oshunkeye, (2012) alleges the oil subsidy is bedeviled by criminal tendencies of oil importers and sharp practices in the distribution of import allocation, approval of subsidy payments and actual release of subsidy cheques. While the significance of petroleum resources as a substantial source of economic advantages became increasingly obvious, susceptibility of the resource regions to military attacks, regional pressure, and oil-related problems, such as oil spills, crude oil theft, illegal refining of crude oil, accidents involving oil trucks, pipeline vandalism, and explosions will continued to exacerbate and requiring strategic management innovations by government to bring under control (Francis et al., 2022). Mismanagement, lack of proper guidelines and standards for the design, construction, and operation of major oil pipelines result in corrosion and aging pipelines, ruptures, operational error, and mechanical failure which lead to leaks and explosion that disrupt product flow (Akinpelu 2021, Achebe, Nneke, & Anisiji, 2012,). 2.4.2. Insecurity, Terrorism and Acts of Sabotage Agile supply and distribution of petroleum products will remain a mirage in the presence of insecurity and safety concerns which disrupt operation and investment in oil production, refining and distribution. Industrial sabotage, crude oil theft, illegal refining operations, pipeline vandalism and piracy present significant challenges which adversely impact onshore oil and gas production and fraught delivery of products to the market. The disruptive activities of the various militant group and instability in the Niger Delta region is acknowledged a major threat to the security of the oil & gas industry. Oturugbum (2021) acknowledge pipeline vandalism and militancy represents a means which the inhabitants of the oil producing areas adopt in pressing their demands to the government and oil companies. Unfortunately, this is due to the prevalence of corruption, poverty, and governments unfulfilled promises, among others. The Petroleum Production Act and other legislation which criminalize the act of tampering and vandalism of crude oil and natural gas pipelines with the death penalty and life imprisonment have not deterred the commission of relevant offences as the challenge continues to escalate. Adeosun and Oluleye (2017) lament Nigeria lost over NGN 50 billion to pipeline vandalism between January and April 2016. Okumagba (2019) reports within a space of 10 years between 2006 – 2016, 535 International Journal of Science and Research Archive, 2024, 11(02), 531–542 International Journal of Science and Research Archive, 2024, 11(02), 531–542 3.1. Limiting factors of Agile Distribution The qualitative data is coded with the capability of the SPSS and analysed for data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology, infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 = undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology, infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 = undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology, infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 = undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology, infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. Table 3 Descriptive Statistics of limiting factors to agile distribution Table 3 Descriptive Statistics of limiting factors to agile distribution N Minimum Maximum Mean Std. 3.1. Limiting factors of Agile Distribution At a very granular level, the study sought to survey the factors which limit the agile distribution of refined petroleum products to end users in Nigeria. Distribution agility based on the review of literature hinges on continuous product availability at affordable prices without quality compromise. Research observation and findings shows the distribution of refined petroleum products in Nigeria is fraught with the challenges of scarcity, product adulteration, and high prices. And efforts by the government to address this have not yielded the desired results. Perceived factors which limit distribution agility were presented to selected staff of six major independent petroleum marketers and NNPC retail as presented in Table 2 below. 30 questionnaires were distributed to the executive staff of each of the study organizations drawn from sales, marketing, admin, HR, imports, finance, business analyst, logistics and transport units via one-on-one interview making a total of 210 questionnaires. 187 questionnaires, which represent 89% of the total questionnaires distributed, were returned valid for the research. Table 2 Instrument Analysis Company Questionnaires Distributed Returned and Valid % Returned and Valid Total Energies 30 28 13 OVH Energy (Oando) 30 27 13 MRS 30 26 12 Conoil Plc 30 27 13 Ardova Plc 30 27 13 11 Plc (Mobil) 30 28 13 NNPC Retail 30 24 11 Total Energies 210 187 89 Source: Authors Field Survey Table 2 Instrument Analysis y Company Questionnaires Distributed Returned and Valid % Returned and Valid Total Energies 30 28 13 OVH Energy (Oando) 30 27 13 MRS 30 26 12 Conoil Plc 30 27 13 Ardova Plc 30 27 13 11 Plc (Mobil) 30 28 13 NNPC Retail 30 24 11 Total Energies 210 187 89 Source: Authors Field Survey The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 = undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology, infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 = undecided, 4 = agree, 5 = strongly agree. 3. Study Framework and Data The agile logistics of petroleum product distribution is a concept representing a distribution strategy which ensures uninterrupted supply of right products to the right customer at the right place, in the right condition and right quantity at the right time, and right (lowest possible) costs. The study follows a descriptive survey design based on quantitative and qualitative approach relying on primary and secondary data. While Primary data is gotten by the distribution 536 International Journal of Science and Research Archive, 2024, 11(02), 531–542 structures questionnaire, secondary data is ensued from e-library of National Bureau of Statistics (NBS) and literatures reviews. 3.1. Limiting factors of Agile Distribution Deviation Fluctuation in crude prices and scarce foreign exchange (F1) 187 2.00 5.00 4.4813 0.76441 Insecurity, fire incidence, theft, and acts of terrorism ((F2) 187 1.00 5.00 4.2193 0.82960 Lack of technology and integrated ICT system (F3) 187 3.00 5.00 4.4652 0.67411 Lack of local refinery capacity (F4) 187 3.00 5.00 4.5134 0.71360 Access limitations to retail delivery locations (F5) 187 3.00 5.00 4.3422 0.74088 Long delivery cycle, transit time and high cost (F6) 187 2.00 5.00 4.4118 0.69288 Lack of integrated management system and process (F7) 187 3.00 5.00 4.3636 0.76648 Poor transport infrastructure and networks (F8) 187 3.00 5.00 4.4332 0.64722 Expertise limitations (F9) 187 2.00 5.00 4.1551 0.76377 Valid N (listwise) 187 Source: Author’s SPSS Data Computation Output Source: Author’s SPSS Data Computation Output 537 International Journal of Science and Research Archive, 2024, 11(02), 531–542 Table 3 above presents the analysis of the response of the respondents as per their level of agreement or disagreement to the perceived limiting factors of petroleum distribution agility in Nigeria. The sampled factors on a five-point likert scale shows a mean response ranged 4 – 4.5 higher than the likert mean value 3 and standard deviation values less the likert scale mean value indicating the respondents largely agree the sampled variables are limiting factors to petroleum product distribution in Nigeria. The sample factor of lack of local refinery capacity has the highest mean response above 4.5 indicating the respondents are very highly concerned about the limitations of product importation to meet local consumption demand. The data is further subjected to K-S Test for more information about the sample and to test the significance of the sample mean to the liket mean value and that the data result is not by random chance. Table 4 One-Sample Kolmogorov-Smirnov Test F1 F2 F3 F4 F5 F6 F7 F8 F9 N 187 187 187 187 187 187 187 187 187 Normal Parametersa,b Mean 4.4813 4.2193 4.4652 4.5134 4.3422 4.4118 4.3636 4.4332 4.1551 Std. Deviation 0.76441 0.82960 0.67411 0.71360 0.74088 0.69288 0.76648 0.64722 .76377 Most Extreme Differences Absolute 0.377 0.276 0.353 0.383 0.315 0.326 0.337 0.328 .240 Positive 0.249 0.173 0.214 0.242 0.187 0.200 0.203 0.230 .206 Negative -0.377 -0.276 -0.353 -0.383 -0.315 -0.326 -0.337 -0.328 -.240 Test Statistic .377 0.276 0.353 0.383 0.315 0.326 0.337 0.328 0.240 Asymp. Sig. (2-tailed) .000c 0.000c 0.000c 0.000c 0.000c 0.000c 0.000c 0.000c 0.000c a. Test distribution is Normal. b. 3.1. Limiting factors of Agile Distribution Calculated from data. c. Lilliefors Significance Correction. Table 4 One-Sample Kolmogorov-Smirnov Test The K-S Test is presented in table 4. The variables F1-F9 represent the perceived limiting factors of agile product distribution presented in table 9. The K-S Test mean values and standard deviation as in descriptive statistics of table 9 are greater and less than the liker mean values respectively. The P value < 0.05, and negative and positive upper and lower bound mean interval for the data parameters are indication that the mean of the data parameters is significantly different from the likert mean value 3. This validates the descriptive result of table 3 and leads to accept that the sample variables are limiting factors to agile distribution of petroleum products. 3.2. Supply Consumption Analysis 3.2. Supply Consumption Analysis Table 5 Product Supply and Consumption Product Category 2017 2018 2019 PMS Import 17,313,512,018 20,147,419,127 20,892,934,107 Consumption 18,335,739,889 19,467,446,039 20,584,964,536 Local Refinery Output 1,022,227,871 -679,973,088 -307,969,571 5.58% -3.49% -1.50% AGO Import 4,277,630,139 4,236,496,957 5,148,860,318 Consumption 4,748,102,354 4,583,290,750 5,158,861,275 Local Refinery Output 470,472,215 346,793,793 10,000,957 9.91% 7.57% 0.19% 538 International Journal of Science and Research Archive, 2024, 11(02), 531–542 International Journal of Science and Research Archive, 2024, 11(02), 531–542 J , , ( ), HHK Import 340,333,700 537,605,965 128,110,313 Consumption 944,390,851 619,859,750 270,215,589 Local Refinery Output 604,057,151 82,253,785 142,105,276 63.96% 13.27% 52.59% Source: NBS E-library Two sources of product supply import and local refinery are identified. Data is sourced from the NBS e-library and products considered are Premium Motor Spirit (PMS), Automated Gass Oil (AGO) and Household Kerosene (HHK). Data parameter of product category examined are import volume, local consumption, and local refinery output in liters for the period of 2017 – 2019 as available on the NBS e-library data base. With regard to table 5 above, data analysis shows PMS import has increased steadily from 17.3 billion liters in 2017 to 20.8 billion liters in 2019 representing a 17.6% increase. The consumption volume also experienced a steady increase form 18.3 billion liters in 2017 to 20.5 billion liters in 2019 representing a 11% increase. The local refinery capacity decreased from 1 billion liters in 2017 to 307 million liters in 2019 representing a 70% decline in output. The local refinery contribution to PMS consumption nose- dived from 5.58% in 2017 to 0% in 2018 and 2019. There is a gap of 679 and 307 million liters of PMS in the 2018 and 2019 respectively which infers smuggled volume PMS. This is also validated by the recorded supply excess against local consumption for the same years. The imports and local consumption of AGO increased from 4 billion liters in 2017 to 5 billion liters in 2019 representing an increase of 25% respectively. Local refining capacity decreased from 470 million in 2017 to 10 million liters in 2019 which is a 97% decline. Local refinery contribution to total supply decrease from 9.9% in 2017 to 0% in 2019. HHK imports decreased from 340 million litres in 2017 to 128 million litres in 2019 representing a 62% decline. Consumption decreased from 944 million litres in 2017 to 270 million litres in 2019 which is a 73.4% decline in consumption. 4. Study Findings The agility of petroleum product distribution is a strategy towards uninterrupted supply of refined petroleum products to consumers at affordable prices and best quality. Research observation and finding shows the petroleum supply and distribution in Nigeria have had history of challenges ranging from product scarcity, inconsistent pricing, and quality compromise. At the root of the challenge is festering issues of corruption, mismanagement petroleum resource and infrastructure, lack of local refinery capacity, dependency on importation, poor distribution networks and technology constraints. Lack of local refinery capacity is fuel by corruption, sabotage, and lack of political will. There is no identified radical approach by the government to resuscitate the hitherto moribund refineries and to build new ones. The local refineries currently operate at near zero out levels. Corrupt gains of oil subsidy and importation has engineered the sabotage of any government effort at building local refinery capacity. Insecurity, pipeline vandalism, theft, and terrorism which disrupt supply are acts of sabotage and militancy that the government must address. Product importation is the reason for increasing cost of petroleum products following rising foreign exchange, devaluation of Naira, rising global oil prices and inflation in the country of import. The product importation itself places foreign exchange burden on the Naira leading to further devaluation of the currency. Also supply disruption in the exporting countries will translate to scarcity in the country of import. Storage capacity constraints, access limitations to retail delivery locations, poor transport infrastructure and networks are transport and distribution challenges which lead to long delivery cycle, high transit time and delivery cost per unit volume. Furthermore, expertise limitations, lack of integrated management system and technology driven process have led to poorly coordinated distribution system. 3.3. Product Pricing The price of the products has increased over the years with YOY percentage increase nearing a 200% for PMS in the year 2023. This is occasioned by the sudden removal of subsidy on the product without plans of local supply. The AGO and HHK reached highest price increase of 167% and 96% respectively in the year 2022. This is also following the removal of subsidy on the products. Table 6 above presents the yearly average price of AGO, PMS and HHK for the period of 2017 to 2023 alongside with the Year on Year (YOY) percentage increase. The price of the products has increased over the years with YOY percentage increase nearing a 200% for PMS in the year 2023. This is occasioned by the sudden removal of subsidy on the product without plans of local supply. The AGO and HHK reached highest price increase of 167% and 96% respectively in the year 2022. This is also following the removal of subsidy on the products. 3.2. Supply Consumption Analysis Contribution of local refinery to consumption decreased from 63% to 52%. Figure 3 Product Supply and Consumption Analysis Figure 3 Product Supply and Consumption Analysis Figure 3 Product Supply and Consumption Analysis Figure 3 shows local consumption for the years under review increasingly depends on foreign supply of products. Local supply capacity for all product group have declined over the years with PMS reaching negative levels in 2018 and 2019. This explains the reason for low levels of agility in product supply and distribution. There is a limit to which a country will effectively meet her local consumption requirements depending solely on importation. Figure 3 shows local consumption for the years under review increasingly depends on foreign supply of products. Local supply capacity for all product group have declined over the years with PMS reaching negative levels in 2018 and 2019. This explains the reason for low levels of agility in product supply and distribution. There is a limit to which a country will effectively meet her local consumption requirements depending solely on importation. Dependence on imported refined products is notorious for the high cost of products in the hands of the consumers following rising cost of dollar, cost of production in the exporting countries port clearance and landing costs. It also means Nigeria will not be in control of its supply. Any challenge in production, supply shortage, political unrest or economic concern in the exporting countries will translate to supply shortages in Nigeria. The supply country would 539 International Journal of Science and Research Archive, 2024, 11(02), 531–542 prioritize local consumption and cut short export in the event of production constraints. This will translate to severe shortage to Nigeria, scarcity of product and hike in price as few distributors with products would hoard them to sell at exploitive prices. prioritize local consumption and cut short export in the event of production constraints. This will translate to severe shortage to Nigeria, scarcity of product and hike in price as few distributors with products would hoard them to sell at exploitive prices. 3.3. Product Pricing The study surveys the pump price of AGO, PMS and HHK for the period of 2017 to 2023 to examine how the price of the products have fared over the years. I significant aspect of product agility is continual availability of best quality at affordable prices. But it is unfortunate that the price of the refined petroleum products has continued to increase over the years becoming unaffordable and making life difficult for the people following the attendant hyperinflation bearing in mind that industrial and economic productivity in the country is driven largely by the petroleum energy. Table 6 Petroleum Pump Price for the period of 2017 to 2023 Product 2017 2018 2019 2020 2021 2022 2023 AGO Yearly Average 212.73 210.79 227.19 223.69 248.95 664.81 1,152.00 YOY Difference 0.00 -1% 8% -2% 11% 167% 73% PMS Yearly Average 146.40 154.82 145.45 147.77 166.47 181.01 483.02 YOY Difference 0.00 6% -6% 2% 13% 9% 167% HHK Yearly Average 297.57 287.70 315.35 340.31 394.02 771.78 1,211.65 YOY Difference 0.00 -3% 10% 8% 16% 96% 57% Source: NBS E-Library Table 6 Petroleum Pump Price for the period of 2017 to 2023 Product 2017 2018 2019 2020 2021 2022 2023 AGO Yearly Average 212.73 210.79 227.19 223.69 248.95 664.81 1,152.00 YOY Difference 0.00 -1% 8% -2% 11% 167% 73% PMS Yearly Average 146.40 154.82 145.45 147.77 166.47 181.01 483.02 YOY Difference 0.00 6% -6% 2% 13% 9% 167% HHK Yearly Average 297.57 287.70 315.35 340.31 394.02 771.78 1,211.65 YOY Difference 0.00 -3% 10% 8% 16% 96% 57% Source: NBS E-Library Table 6 above presents the yearly average price of AGO, PMS and HHK for the period of 2017 to 2023 alongside with the Year on Year (YOY) percentage increase. The price of the products has increased over the years with YOY percentage increase nearing a 200% for PMS in the year 2023. This is occasioned by the sudden removal of subsidy on the product without plans of local supply. The AGO and HHK reached highest price increase of 167% and 96% respectively in the year 2022. This is also following the removal of subsidy on the products. Table 6 Petroleum Pump Price for the period of 2017 to 2023 Table 6 above presents the yearly average price of AGO, PMS and HHK for the period of 2017 to 2023 alongside with the Year on Year (YOY) percentage increase. References [1] Acta Logistica 4(2):23-26 [2] Adeosun, O. and Oluleye, A. (2017). Nigerian’s refining revolution. https://www.pwc.com/ng/en/assets/pdf/nigerias-refining-revolution.pdf [3] Akintayo, O. (2023, Dec. 23). Domestic refining output drops by 92%. The Punch Nigeria. [4] Bataiya, K. I. (2018, May 10th). Road infrastructure in Nigeria: Impact on petroleum product distribution [4th quadrennial delegates conference of the petroleum tanker drivers (ptd) branch of NUPENG Abuja]. https://narto.org/road-infrastructure-in-nigeria-impact-on-petroleum-products-distribution-by-alhaji-dr- kassim-ibrahim-bataiya-national-president-nigerian-association-of-road-transport-owners-narto-deliv/ [5] Nwachukwu, M. U., and Chike. H. (2015). Energy supply and road transport challenges in Nigeria. Energy Sci. & Tech, 3. [5] Nwachukwu, M. U., and Chike. H. (2015). Energy supply and road transport challenges in Nigeria. Energy Sci. & Tech, 3. [6] Okumagba, E. O. (2019). Oil and gas pipeline "vandalism" in nigeria: analysing alternative options beyond the traditional legal approach. International Energy Law Review, 37(7),181-189. [6] Okumagba, E. O. (2019). Oil and gas pipeline "vandalism" in nigeria: analysing alternative options beyond the traditional legal approach. International Energy Law Review, 37(7),181-189. [7] Straka, M. (2017). The position of distribution logistics in the logistic system of an enterprise. [7] Straka, M. (2017). The position of distribution logistics in the logistic system of an enterprise. [8] Thompson, O. O., Martina, F. and James J. W. (2014). Road haulage constraints in the transportation of petroleum products in northern Nigeria. Journal of Environmental Science, Toxicology and Food Technology, 8(3) 1-8. [8] Thompson, O. O., Martina, F. and James J. W. (2014). Road haulage constraints in the transportation of petroleum products in northern Nigeria. Journal of Environmental Science, Toxicology and Food Technology, 8(3) 1-8. [9] Zahid, A. (2008). Energy–gdp relationship: a causal analysis for the five countries of South Asia. Journal of Applied Econometrics and International Development, 8 (1). [9] Zahid, A. (2008). Energy–gdp relationship: a causal analysis for the five countries of South Asia. Journal of Applied Econometrics and International Development, 8 (1). [10] Oshunkeye, S. (2012, Jan. 2015). At Last, People’s Power. Sun Nigeria. [10] Oshunkeye, S. (2012, Jan. 2015). At Last, People’s Power. Sun Nigeria. [11] Ayanruoh, F. (2023, June 1st) Fuel Subsidy Removal is Good for Nigeria Vanguard, Thursda [12] Johnson, F. I., Laing, R., Bjeirmi, B & Leon, M. (2022). Examining the causes and impacts of pipeline disasters in Nigeria. AIMS Environmental Science, 9(5): 636–657 [13] Nwoloziri, C. N, Dakoru, E and Nwabueze, E. and Elias, Eleyi (2021). Risk assessment of long-distance road haulage of petroleum products in Nigeria. International Journal of Science and Research Archive, 2024, 11(02), 531–542 Savings from removal of petroleum subsidy as implemented by the current government should be invested in local refinery capacity, revitalization of the pipeline for efficient distribution and development of other sources of energy especially electricity supply to reduce over dependence on petroleum energy. International Journal of Science and Research Archive, 2024, 11(02), 531–542 and other sources of energy. Over time, the demand for petroleum products has soared in the following rising population, increasing demand for economic productivity, general logistics and distribution, as well as quest for improved standard of living. While the consumption demand for petroleum product has continued to increase, the issues of scarcity, high prices and corrupt practices which fraught the distribution of petroleum in Nigeria has compounded and successive governments have not shown a political will in addressing them. Significantly lack of local refinery capacity and poor distribution network the study identifies are notorious for supply limitations and rising price of products. Nigeria is blessed with abundance of natural petroleum resource yet depends on importation of refined petroleum products to satisfy her local consumption demand. The reliance on road haulage in distribution is inefficient and possess significant bottleneck and risk to product supply. To ensure the availability of products which meet local demand at affordable prices and even distribution to all regions, the government is tasked to show genuine commitment in addressing the issues identified. Savings from removal of petroleum subsidy as implemented by the current government should be invested in local refinery capacity, revitalization of the pipeline for efficient distribution and development of other sources of energy especially electricity supply to reduce over dependence on petroleum energy. and other sources of energy. Over time, the demand for petroleum products has soared in the following rising population, increasing demand for economic productivity, general logistics and distribution, as well as quest for improved standard of living. While the consumption demand for petroleum product has continued to increase, the issues of scarcity, high prices and corrupt practices which fraught the distribution of petroleum in Nigeria has compounded and successive governments have not shown a political will in addressing them. Significantly lack of local refinery capacity and poor distribution network the study identifies are notorious for supply limitations and rising price of products. Nigeria is blessed with abundance of natural petroleum resource yet depends on importation of refined petroleum products to satisfy her local consumption demand. The reliance on road haulage in distribution is inefficient and possess significant bottleneck and risk to product supply. To ensure the availability of products which meet local demand at affordable prices and even distribution to all regions, the government is tasked to show genuine commitment in addressing the issues identified. Disclosure of conflict of interest The author declares that there is no conflict of interest in this manuscript. 5. Conclusion More than 70% of energy requirement in Nigerian for transportation, domestic consumption, economic and industrial productivity depend on petroleum and its derivatives. This is chiefly because of the underdevelopment of electric power 540 References International Research Journal of Modernization in Engineering Technology and Science, 3(10). [14] Achebe, C.H., Nneke, U.C. and O.E. Anisiji (2012). Analysis of oil pipeline failures in the oil and gas industries in the Niger delta area of Nigeria Proceeding of the International Multiconference of Engineers and Computer Scientist, 2. [14] Achebe, C.H., Nneke, U.C. and O.E. Anisiji (2012). Analysis of oil pipeline failures in the oil and gas industries in the Niger delta area of Nigeria Proceeding of the International Multiconference of Engineers and Computer Scientist, 2. [15] Akinpelu, Y. (2021, April 11th). Analysis: Nigeria spends billions on pipeline maintenance as hundreds are vandalised annually. Premium Times Nigeria. [15] Akinpelu, Y. (2021, April 11th). Analysis: Nigeria spends billions on pipeline maintenance as hundreds are vandalised annually. Premium Times Nigeria. 541 International Journal of Science and Research Archive, 2024, 11(02), 531–542 [16] Ehinomen, C. and Adeleke, A. (2012). An assessment of the distribution of Petroleum products in Nigeria. Journal of Business Management and Economics, 3(6) 232-241. [17] Oturugbum, D. (2021). Sabotage in the Nigerian petroleum industry and the attitude of courts. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3841077. [18] Adology, B. (2023, Nov. 10). NEITI: Oil theft, pipeline vandalism have become national emergency. The Cable: Nigeria. https://www.thecable.ng/neiti-oil-theft-pipeline-vandalism-have-become-national-emergency. [19] Schewe, D. C. & Smith, R. M. (1980). Marketing Concepts and Application, New York. [20] Tiefenbacher, W. (2020), Agile for all: The principles of agility, agile leadership, and how to be an agile leader. https://www.ckju.net/en/dossier/agile-all-principles-agility-agile-leadership-and-how-be-agile-leader. [21] LaBotz, K. (2022). Agile logistics for superb profits and sustainability. https://blog.flexis.com/agile-logistics-for- superb-profits-and-sustainability-0-0-0-0 [22] van Hoek R., Harrison A. and Christopher M. (2001). Measuring agile capabilities in the supply chain. International Journal of Operations and Production Management, 21(1–2) 126–47 [23] Harrison, A. and Hoek (2008). Logistics Management and Strategy; competing through the supply chain, 3rd edition. Pearson Education Limited Edinburgh, Gate Harlow Essex CM20 2JE, England. 542
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Clinical Activity and Tolerability of SL-401 (Tagraxofusp): Recombinant Diphtheria Toxin and Interleukin-3 in Hematologic Malignancies
Biomedicines
2,019
cc-by
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biomedicines Review Clinical Activity and Tolerability of SL-401 (Tagraxofusp): Recombinant Diphtheria Toxin and Interleukin-3 in Hematologic Malignancies Omar Alkharabsheh * and Arthur E. Frankel Division of Medical Oncology, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA; afrankel@health.southalabama.edu * Correspondence: oalkharabsheh@health.southalabama.edu; Tel.: +1-251-665-8000 Received: 20 November 2018; Accepted: 3 January 2019; Published: 5 January 2019   Abstract: Overcoming the leukemia stem cell resistance to intensive chemotherapy has been an area of extensive research over the last two decades. Advances and greater understanding of the molecular biology of leukemia stem cells are in rapid progress. Targeted therapies are currently being used in clinical practice with reasonable response rates, but a cure is being achieved in only a small percentage of patients, most likely due to tumor mutational heterogeneity. A genetically engineered diphtheria toxin fused with interleukin-3 (SL-401 or tagraxofusp) has shown robust activity in blastic plasmacytoid dendritic cell neoplasm and promising response rates in different myeloid malignancies, including eradication of minimal residual disease. Multiple clinical trials are being conducted using this drug and the preliminary results are encouraging. This article reviews the clinical trials for SL-401, its mechanism of action, clinical activity, and the adverse event profile. Keywords: SL-401 (tagraxofusp); diphtheria immunotoxin; adverse events; Myeloid neoplasms 1. Introduction The current treatment approach for myeloid malignancies is heavily dependent on intensive chemotherapy regimens that follow the old strategy of “one size fits all”. Novel targeted agents are emerging in clinical practice and clinical trials. Data on their efficacy and tolerability are promising, although no treatment has produced a cure thus far [1]. Intensive chemotherapy treatments are designed to induce hypocellular marrow and allow hematopoietic stem cells to recover and resume normal production of blood cell components. The durability and duration of remission by this kind of therapy can vary and will depend primarily on the mutational signature of the disease and epigenetic aberration. In addition, the planned consolidation strategy, whether it is allogeneic stem cell transplantation or further consolidative chemotherapy, has an effect on leukemia-free survival and how deeply we can eradicate leukemic stem cells. New agents that target actionable mutations, such as FLT3 and IDH, have been approved for clinical practice [2,3]. However, they have not had a significant enough effect on overall survival rates to replace chemotherapy, partly because of the diversity of cytogenetic abnormalities and the clonal evolution of new mutations. Nevertheless, it is important to assess the performance status and comorbidities of these treatments before exploring which treatment approach is the most appropriate in terms of toxicity and survival. Unfortunately, even with the most intensive treatment, the risk of relapse is high in the first two years, especially in patients with adverse cytogenetic profiles [4]. Extensive time and effort were spent in clinical trials optimizing the sequence of well-known acute leukemia chemotherapy protocols. There is a need to revolutionize the treatment of acute leukemia. Unlike traditional chemotherapy, the specificity of the target on the tumor cells is the focus of the new era in cancer treatment. Various examples have been investigated in clinical trials, and Biomedicines 2019, 7, 6; doi:10.3390/biomedicines7010006 www.mdpi.com/journal/biomedicines Biomedicines 2019, 7, 6 2 of 9 many agents have been approved and are available in clinical practice. These include, for example, tyrosine kinase inhibitors to target the FLT3 mutation, monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor T cell therapies. The ideal target, for example, in acute myeloid and lymphoid leukemia, is highly expressed in leukemic blasts, with minimal or no expression on normal hematopoietic cells, to induce deeper remission. Targets that have been studied in acute myeloid leukemia include CD33, CD135, FLT3, CXCR4, and vascular endothelial growth factor [5]. This article focuses on the diphtheria toxin-interleukin 3 fusion protein as a targeted therapy to leukemic blasts. Hematopoiesis is a complex process with multiple factors, undertaken to avoid the production of clones and leukemic stem cells. Interleukin 3 (IL-3) is one of the growth factors and cytokines that participate in this process at the level of granulocytic and monocytic lineage [6]. Expression of the interleukin 3 receptor (IL-3R) starts at the CD34+ hematopoietic cell and is maintained during all stages of development for granulocytes and monocyte precursors. Multiple investigators have targeted this receptor for the treatment of different types of myeloid leukemia [7]. Most recently, experiments have used chimeric antigen receptor T cell technology to target CD123 in Hodgkin lymphoma in an effort to overcome the immunosuppressed microenvironment and allow the patient’s T lymphocytes to attack tumor cells [8]. The structure of IL-3R consists of an α subunit, which is the site of ligand attachment and represents the specificity of the receptor, and a β subunit, which is shared with the granulocyte-macrophage colony-stimulating factor and is important for signal transduction, internalization of the ligand–receptor complexes, and activation through phosphorylation of the Ras pathway [9]. The development of monoclonal antibodies and cytokines was a major advance in cancer treatment because of their ability to deliver cancer therapies, such as cytotoxic drugs, isotopes, and toxins. Toxins are powerful pro-apoptosis agents, and they achieve their cellular effect by various pathways. One of them is by shutting down protein synthesis [10]. After removal of the natural binding domain of the toxins, they are conjugated to a monoclonal antibody or cytokine as their ligand [11]. One toxin that has been used in cancer therapy is the diphtheria toxin, which is an exotoxin secreted by Corynebacterium diphtheriae [12]. This toxin inhibits protein synthesis in the host, which is the underlying pathophysiology of diphtheria infection [8]. Previously, the diphtheria toxin was engineered to target interleukin-2 and was approved by the Food and Drug Administration (FDA) in 2002 for the treatment of cutaneous T cell lymphoma [13]. However, because of the side effects, marketing of this drug was discontinued by the manufacturer. Diphtheria toxin consists of three domains and several linkers elements: The C terminus binding domain, the N terminus catalytic domain, and the translocation domain [14]. IL-3 is a cytokine that promotes the differentiation of hematopoietic stem cells into various myeloid cells [12]. IL-3R is composed of two subunits (α and β). The α subunit (CD123) directly binds to IL-3, whereas the β subunit (CDw131) functions as the signaling subunit (see Figure 1) [10]. The idea is to remove the diphtheria toxin binding domain and replace it with IL-3 to target leukemic stem cells that express CD123 and CDw131 by inhibiting intracellular protein synthesis and therefore inducing cell death. This combination (diphtheria toxin and IL-3) is named SL-401 (tagraxofusp) and has reached phase II clinical trials, with robust activity in the blastic plasmacytoid dendritic cell neoplasm (BPDCN) and other hematological malignancies [15–20]. Subsequently, the FDA approved this drug in December 2018 for adult and pediatric BPDCN. In this review, we report the preclinical data and describe the clinical trials for SL-401 and its activity in various myeloid malignancies. Biomedicines 2019, 7, 6 Biomedicines 2019, 7, x FOR PEER REVIEW 3 of 9 3 of 9 Figure Structure andand Mechanism of Action (provided by Stemline Therapeutics, Inc., New Figure1.1.SL-401 SL-401 Structure Mechanism of Action (provided by Stemline Therapeutics, Inc., New NY York, NYUSA). 10022, USA). Abbreviations: IL-3, 3; interleukin 3; IL-3R, interleukin 3 receptor; York, 10022, Abbreviations: IL-3, interleukin IL-3R, interleukin 3 receptor; DT, diphtheria DT, diphtheria toxin; EF2, elongation factor ribonucleic 2; mRNA,acid; messenger ribonucleic acid; ADP, toxin; EF2, elongation factor 2; mRNA, messenger ADP, adenosine diphosphate. adenosine diphosphate. This combination (diphtheria toxin and IL-3) is named SL-401 (tagraxofusp) and has reached 2. Preclinical Data and Experiments phase II clinical trials, with robust activity in the blastic plasmacytoid dendritic cell neoplasm (BPDCN) and other hematological malignancies [15–20]. Subsequently, theGM-CSF FDA approved drug Previously, investigators fused the truncated diphtheria toxin with using athis His-Met in December 2018 for adult and pediatric BPDCN. In this review, we report the preclinical data anda linker. However, due to the toxicity profile and the hepatic side effects, they decided to explore describe clinical trials[21]. for SL-401 andof itsIL-3R activity in various myeloid malignancies. differentthe growth factor The level expression was examined by Testa and colleagues and they demonstrated a higher level of IL-3R α chain in leukemic stem cells (LSC) and a low 2.expression PreclinicalonData and hematopoietic Experiments stem cells, which makes it a marker for LSC and a target for normal treatment [2]. Using genetic engineering, the first 388 amino acid residues of the diphtheria are Previously, investigators fused the truncated diphtheria toxin with GM-CSF using a toxin His-Met fused with interleukin 3 via H-M linker (DT IL3). The mechanism of action starts when the ligand linker. However, due to the toxicity profile388 and the hepatic side effects, they decided to explore ais attachedgrowth to the IL-3R subunit Along with binding to the IL-3R subunit, will trigger different factorα[21]. The (CD123). level of IL-3R expression was examined byβTesta and this colleagues and receptor-mediated endocytosis into a vesicle which will deliver the diphtheria toxin to the endosomes they demonstrated a higher level of IL-3R α chain in leukemic stem cells (LSC) and a low expression on where ithematopoietic is cleaved by stem furincells, to generate the A and B fragments of the The environment of normal which makes it a marker for LSC andtoxin. a target foracidic treatment [2]. Using the endosomes will partially unfold all three toxin domains to facilitate the escape of the A fragment genetic engineering, the first 388 amino acid residues of the diphtheria toxin are fused with interleukin the(DT catalytic domain into the cytosol. Finally, the catalytic domain will ADP-ribosylate 3that via contains H-M linker 388IL3). The mechanism of action starts when the ligand is attached to the IL-3R α the elongation factor 2, which lead to synthesis inhibition deathreceptor-mediated [22] (see Figure 1). subunit (CD123). Along with will binding toprotein the IL-3R β subunit, this and willcell trigger Additionally, several animal studies confirmed the in vivo efficacy of the engineered diphtheria toxin endocytosis into a vesicle which will deliver the diphtheria toxin to the endosomes where it is cleaved and provided more information about the appropriate dose and its readiness for the first human clinical by furin to generate the A and B fragments of the toxin. The acidic environment of the endosomes will trials [14,15]. To all examine the level of expression on different myeloid researchers from Mayo partially unfold three toxin domains to facilitate the escape of thelineage, A fragment that contains the Clinic performed a study of CD123 expression on myeloproliferative neoplasm subtypes with flow catalytic domain into the cytosol. Finally, the catalytic domain will ADP-ribosylate the elongation factor expression on systemic mastocytosis, and aseveral minor 2,cytometry. which willThey leadreported to protein synthesismostly inhibition and cell death [22] (see clonal Figureeosinophilia, 1). Additionally, population of myelofibrosis [16]. This broadened the potential activity on several myeloid disorders, animal studies confirmed the in vivo efficacy of the engineered diphtheria toxin and provided more along with the previously described leukemic stem cellsfor in acute myeloid To understand information about the appropriate dose and its readiness the first humanleukemia. clinical trials [14,15]. To more about the bone marrow microenvironment and its effect on disease pathogenesis, along with examine the level of expression on different myeloid lineage, researchers from Mayo Clinic performed the possible use of this agent as an investigational drug in clinical trials, researchers at Dana-Farber a study of CD123 expression on myeloproliferative neoplasm subtypes with flow cytometry. They Cancer Institute demonstrated plasmacytoid dendritic cells (pDCs) play a role in protecting clonal reported expression mostly on that systemic mastocytosis, clonal eosinophilia, and a minor population of plasma cells in multiple myeloma from treatment. Additionally, they reported that SL-401 induces myelofibrosis [16]. This broadened the potential activity on several myeloid disorders, along with the pDC apoptosis, which resultedstem in overcoming treatment resistance inTo multiple myeloma previously described leukemic cells in acute myeloid leukemia. understand more[17]. about the bone marrow microenvironment and its effect on disease pathogenesis, along with the possible use of 3. Clinical Trials this agent as an investigational drug in clinical trials, researchers at Dana-Farber Cancer Institute The first that in human trial was presented the American Society of Clinical Oncology demonstrated plasmacytoid dendritic cellsat(pDCs) play a role in protecting clonal plasma (ASCO) cells in annual meeting in 2006. The preliminary findings showed manageable toxicity in elderly patients with multiple myeloma from treatment. Additionally, they reported that SL-401 induces pDC apoptosis, acute myeloid (AML) or in those with relapsed or refractory disease which resulted leukemia in overcoming treatment resistance in multiple myeloma [17]. [18]. The study included Biomedicines 2019, 7, 6 4 of 9 31 patients who received one cycle of SL-401. The findings showed mild toxicity and promising biologic activity. At the 2007 ASCO annual meeting, investigators reported a patient with AML who achieved complete remission (CR) for eight months as well as two cases of partial response (PR) [19]. These patients were heavily pretreated, including bone marrow transplantation, and the cohort included patients with secondary AML either from a previous myelodysplastic syndrome or therapy-related AML. The major side effects reported in this abstract were mild to moderate and included transient fever, chills, hypotension, and hypoalbuminemia [19]. Table 1 highlights the clinical trials for SL-401, adverse events, and response rates. Phase I data were first published in 2008 [20] and highlighted the following points. First, the maximum tolerated dose is 12.5 µg/kg/day, which can be infused safely over 15 min, twice daily every 48 h with a total of six doses as one cycle. Second, toxicity and side effects were manageable, and no grade IV/V adverse events were reported. Grade II/III adverse events included transaminitis, fever, hypoalbuminemia, hypotension, and hypocalcemia. This cohort included elderly patients, most of whom had a disease that was refractory to standard therapy. The median age was 67 years, and only 11% of patients had previously untreated AML. In addition, 96% of patients had an intermediate cytogenetic profile. The trial reported one case of CR and two cases of PR (Both AML and MDS). All of the patients who showed some form of response had relapsed or refractory disease and the patients with previously untreated AML showed no response to the experimental treatment [20]. The high level of IL-3R expression on BPDCN [23] provided the basis for investigation of SL-401 activity in this type of myeloid disease. Because BPDCN is an aggressive tumor, unfortunately, patients have a short survival, irrespective of the intensity of chemotherapy [24]. This accelerated the pathway to move SL-401 from in vitro studies to more early phase clinical trials [13,25]. The first prospective trial in BPDCN using SL-401 published its results and outcomes in 2014. Frankel et al. reported the detailed toxicity profile and response rates for the phase I/II trial [26]. The maximum tolerated dose was 12.5 µg/kg/day for five days in 11 patients with BPDCN. The reported adverse events were grade II/III, including edema, hypoalbuminemia, fever, chills, and transaminitis as well as one case of grade IV thrombocytopenia that was not related to an underlying thrombotic microangiopathy. Another adverse event that the investigators described in their outcomes data is vascular leak syndrome (VLS). This was reported as mild to moderate, and it manifested clinically with edema, with laboratory findings showing hypoalbuminemia. The VLS phenomenon had been reported in other fusion proteins with toxins [27]. Of the patients who underwent evaluation for response, 78% had an objective response, including 55% CR after the first cycle. Because of the nature of the drug, there is a question about whether the antibody developed because of previous diphtheria vaccination during childhood or whether the infused drug itself may trigger antibody development that might inactivate SL-401. The investigators examined this theory and reported that pretreatment titers were positive as a result of previous vaccination. A repeat evaluation after therapy showed an increased titer; however, this did not affect toxicity events, pharmacokinetics, or the clinical response rate [26]. Biomedicines 2019, 7, 6 5 of 9 Table 1. Summary of SL-401 clinical trials in various hematologic neoplasms, baseline characteristics, adverse events and clinical outcomes (See page 14). Abbreviations: NCT, National Clinical Trial; R/R, Relapsed or refractory; AML, Acute myeloid leukemia; MDS, Myelodysplastic syndrome; CMML, Chronic myelomonocytic leukemia; BPDCN, Blastic plasmacytoid dendritic cell neoplasm; MM, Multiple myeloma; ORR, Overall response rate; CR, Complete response; PR, Partial response; LFT, Liver function tests; ASCO, American Society of Clinical Oncology; ASH, American Society of Hematology; CLS, Capillary leak syndrome; NA, Not available. SL-401 Clinical Trial Registry Numbers Primary Malignancy Status Study Start Date Phase Published Data Number of Patients Number of Cycles Age Male/Female Adverse Cytogenetics Intermediate Cytogenetics Relapsed (any) and Refractory NA R/R or elderly AML and high risk MDS Completed NA I Leukemia and Lymphoma 2007 45 1 67 (32–81) 23/22 17 25 35 NCT00397579 BPDCN Completed May 2013 I/II Blood 2014 for the BPDCN 11 1 70 (40–77) 11/0 NA NA 7 NA RR/AML and BPDCN NA NA NA ASH 2015 17 multiple 63 NA NA NA NA Recruiting February 2015 I/II NA NA NA NA NA NA NA NA Recruiting December 2014 I/II ASH 2016 19 multiple 69 (42–81) NA NA NA Recruiting June 2017 I NA NA NA NA NA NA NCT02270463 NCT02268253 NCT03113643 Consolidation Rx in adverse risk AML CR1 Advanced high risk MNP (SM, PED, MF, CMML) With AZA for Rx naïve AML/high risk MDS not eligible for standard Rx Adverse Events ORR CR PR NA 1 3 NA 5 0 NA 4 NA NA NA NA NA 19 Grade III thrombocytopenia and anemia NA 1 NA NA NA NA NA NA NA Grade III LFTs, Grade II fever, chills, low albumin and hypotension Grade IV thrombocytopenia, Grade III LFTs and neutropenia Grade V CLS, Grade IV CLS, Grade III LFTs NCT02661022 R/R MM Recruiting January 2016 I/II ASH 2016 2 multiple 65 (63–67) NA NA NA 2 Grade II thrombocytopenia and hypoalbuminemia NA 0 2 NCT02113982 BPDCN and R/R AML Recruiting September 2014 I/II ASCO 2016 (only BPDCN) 18 multiple 70 (45–82) NA NA NA 10 CLS and thrombocytopenia 87% 8 NA Biomedicines 2019, 7, 6 6 of 9 The current advances in measuring minimal residual disease (MRD) have shown that conventional chemotherapy may not provide the deepest response or subsequently a better outcome. For example, the use of traditional consolidation chemotherapy after achieving CR in AML might not be enough to reduce the risk of relapse. For that, new treatment strategies are needed. Currently, a phase I/II study (NCT02270463) is underway in patients with AML who are at risk for relapse based on intermediate-high risk cytogenetics and are not candidates for allogeneic stem cell transplant. The following initial results were reported at the 2016 ASH annual meeting. First, the adverse event profile was similar to that of other clinical trials of SL-401, with a maximum tolerated dose of 12 µg/kg/day [28]. Second, the concept of targeting MRD-positive AML with SL-401 is promising, and the trial will continue to recruit patients until December 2018 (www.clinicaltrials.gov). For multiple myeloma, studies are still in the early phase, with evidence showing that SL-401 targets myeloma stem cells [29]. As with pDC in the marrow microenvironment, those cells express CD123 and have a role in the proliferation of myeloma, which makes SL-401 a promising agent for myeloma backbone therapy. Currently, a phase I/II trial (NCT02661022) is recruiting patients with relapsed or refractory myeloma to be treated with pomalidomide, dexamethasone, and SL-401 [30]. The current evidence from early clinical trials indicates that side effects are tolerable and manageable. Additionally, clinical activity is seen in tumors that express CD123, especially BPDCN. Recently, investigators showed robust data at the American Society of Hematology annual meeting in December 2017 for a phase II trial of single-agent SL-401 in BPDCN, with an 84% overall response rate. Additionally, two abstracts were presented at the European Society of Hematology annual meeting in June 2018. The first one described chronic myelomonocytic leukemia, and all patients showed a spleen reduction response, while 12.5% showed marrow CR. The second abstract described primary myelofibrosis, and 50% of patients showed a spleen response. In summary, the results of trials of SL-401 indicate no serious adverse events, unlike other forms of immunotherapy, and a convening clinical activity in various hematologic malignancies, most effectively in BPDCN. In the future, we will likely see more positive results from phase III trials. 4. Side Effects and Tolerability The currently reported side effects and clinical activity from early-phase trials are shown in Table 1. To review those in detail, this is a recombinant protein from diphtheria toxin conjugated with IL-3 to internalize the toxin inside the tumor cells. Based on the nature of this agent, some form of immune or infusion reaction would be expected. The most serious adverse event that was reported in human clinical trials was capillary leak syndrome (CLS), previously called VLS, which is manifested by edema, hypoalbuminemia, hypotension, and fatigue. This was reported as grade ≤3; however, three cases of fatal CLS were reported in the BPDCN trial (www.ashclinicalnews.org). Other significant side effects were reported up to grade 4, including thrombocytopenia. However, it was reversible and was not associated with a major bleeding event. In addition, less than grade 3 neutropenia was reported. Patients with marrow-involved malignancies are susceptible to cytopenia because of disease expansion and the side effects of treatment. As shown in Table 1, the rest of the side effects were tolerable and reversible, so one could argue that these patients have refractory disease and are heavily pretreated, and this by itself can explain the cytopenia. Additionally, transaminitis has been reported across trials, but it was reversible and less than or equal to grade 3. 5. Pediatric Experience Although BPDCN is a disease of the elderly, a few patients who were younger than 18 years old were included for compassionate use because their disease was refractory to standard therapy. Sun et al. from the City of Hope reported the first three cases of pediatric patients with this type of malignancy. Overall, the treatment was very well tolerated, with side effects similar to those of CLS and infusion reaction [31]. The number of patients in this report was very small, so a conclusion about the efficacy of this treatment cannot be made, although all patients showed some response. Biomedicines 2019, 7, 6 7 of 9 6. Conclusions The concept of using diphtheria toxin in cancer treatment is not new but evolving. Alternative therapies to traditional chemotherapy are needed to improve outcomes, especially in patients with primary refractory or relapsing hematologic malignancies. Using IL-3 as a conjugate offers an advantage because multiple hematologic malignancies express CD123 and CDw131. Therefore, having a target that is highly expressed on tumor cells will increase the likelihood that the drug will exhibit anti-tumor activity by internalizing the diphtheria toxin and inducing apoptosis. The most striking activity was noted in BPDCN, with excellent responses in the affected organs, including bone marrow, skin, and lymph nodes. Outcomes in other hematologic malignancies, such as multiple myeloma and chronic myelomonocytic leukemia, are still in early clinical trials, and these studies are providing a better understanding of the bone marrow microenvironment and the role of pDC in tumor progression and treatment resistance. It has been reported that pDCs have anti-tumor activity through the production of interferon alfa [32]. Funding: This research received no external funding. Acknowledgments: All sources of funding of the study should be disclosed. Please clearly indicate grants that you have received in support of your research work. Clearly state if you received funds for covering the costs to publish in open access. Conflicts of Interest: Omar Alkharabsheh has no conflict of interest. Arthur E. Frankel has an intellectual property interest in Stemline Therapeutics, Inc. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Kayser, S.; Levis, M.J. Advances in targeted therapy for acute myeloid leukaemia. Br. J. Haematol. 2018, 180, 484–500. [CrossRef] [PubMed] Testa, U.; Riccioni, R.; Diverio, D.; Rossini, A.; Lo Coco, F.; Peschle, C. Interleukin-3 receptor in acute leukemia. Leukemia 2004, 18, 219. [CrossRef] [PubMed] Chang, F.; Steelman, L.S.; Lee, J.T.; Shelton, J.G.; Navolanic, P.M.; Blalock, W.L.; Franklin, R.A.; McCubrey, J.A. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention. Leukemia 2003, 17, 1263–1293. 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Results from ongoing phase 2 trial of SL-401 as consolidation therapy in patients with acute myeloid leukemia (AML) in remission with high relapse risk including minimal residual disease (MRD). Blood 2016, 128, 215. Ray, A.; Song, Y.; Das, D.S.; Macri, V.; Chen, J.; Paba-Praba, C.; Richardson, P.G.; Brooks, C.L.; Chauhan, D.; Anderson, K.C. SL-401, a novel IL-3Rα/CD123-directed agent targets stem-like cells in multiple myeloma. Blood 2016, 128, 4463. Biomedicines 2019, 7, 6 30. 31. 32. 9 of 9 Htut, M.; Gasparetto, C.; Zonder, J.; Martin, T.G.; Scott, E.C.; Chen, J.; Shemesh, S.; Brooks, C.L.; Chauhan, D.; Anderson, K.C.; et al. Results from ongoing phase 1/2 trial of SL-401 in combination with pomalidomide and dexamethasone in Relapsed or refractory multiple myeloma. Blood 2016, 128, 5696. Sun, W.; Liu, H.; Kim, Y.; Karras, N.; Pawlowska, A.; Toomey, D.; Kyono, W.; Gaynon, P.; Rosenthal, J.; Stein, A. First pediatric experience of SL-401, a CD123-targeted therapy, in patients with blastic plasmacytoid dendritic cell neoplasm: report of three cases. J. Hematol. Oncol. 2018, 11, 61. [CrossRef] Candolfi, M.; King, G.D.; Yagiz, K.; Curtin, J.F.; Mineharu, Y.; Muhammad, A.K.M.G.; Foulad, D.; Kroeger, K.M.; Barnett, N.; Josien, R.; et al. Plasmacytoid dendritic cells in the tumor microenvironment: immune targets for glioma therapeutics. Neoplasia 2012, 14, 757–770. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine
Nanoscale research letters
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© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine Scarlet Xiaoyan Wang1*, Johan Michiels2, Kevin K. Ariën2, Roger New3, Guido Vanham2 and Ivan Roitt1 Scarlet Xiaoyan Wang1*, Johan Michiels2, Kevin K. Ariën2, Roger New3, Guido Vanham2 and Iva Wang et al. Nanoscale Research Letters (2016) 11:350 DOI 10.1186/s11671-016-1558-7 Open Access Open Access Abstract Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations. Keywords: HIV/AIDS, Liposome, Neutralizing antibody, Drug delivery, Prophylaxis * Correspondence: x.wang@mdx.ac.uk 1Department of Natural Sciences, Middlesex University, London, UK Full list of author information is available at the end of the article Background HIV-1 is more pathogenic and transmissible and is mainly responsible for the global AIDS pandemic [4]. HIV infec- tion has continued spreading by the transfer of body fluids due to exposure to blood or blood products, by homo- or heterosexual contact, or prenatally from mother-to-child. Since the start of the epidemic, around 78 million people have become infected with HIV and 35 million people have died of AIDS-related illnesses according to the global statis- tical report from UNAIDS in 2016. In addition, 2.1 million people became newly infected with HIV and 36.7 million people were living with HIV globally in 2015. Human immunodeficiency virus (HIV), responsible for the acquired immunodeficiency syndrome (AIDS), is a lentivirus belonging to the retrovirus family. Infection causes extensive destruction of T-helper cells, macro- phages, dendritic cells and other cellular components asso- ciated with cell-mediated immunity, eventually leading to AIDS [1, 2]. As a consequence, HIV-infected patients are more susceptible to opportunistic infections [3]. At present, HIV-1 and HIV-2 are the two known types of HIV [4]. Currently, the major means of efficient management of HIV and prevention of its progression towards AIDS * Correspondence: x.wang@mdx.ac.uk 1Department of Natural Sciences, Middlesex University, London, UK Full list of author information is available at the end of the article * Correspondence: x.wang@mdx.ac.uk 1Department of Natural Sciences, Middlesex University, London, UK Full list of author information is available at the end of the article Wang et al. Nanoscale Research Letters (2016) 11:350 Page 2 of 10 Page 2 of 10 Page 2 of 10 is highly active antiretroviral therapy (HAART), which combines a minimum of three antiretroviral drugs from at least two classes. These include nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitor, integrase inhibitors and/or entry inhibitors [5]. Although the therapy has improved long- term survival rate, there are associated toxic side effects and emergence of multi-drug resistance. Furthermore, there is a limited access to anti-HIV drugs in the devel- oping countries due to their high costs [6]. Clearly, there remains an urgent demand for an effective and low-cost system for prophylaxis and treatment of HIV infection. liposome platform due to their specificity, high stability and low production cost [31, 32]. Moreover, these anti- body fragments showed very efficient ability to penetrate human tissue by diffusing into the mucosal layer when placed in a vaginal ring [31]. Background In this study, multivalent liposomal antibody constructs were developed by grafting Vhh (J3), which achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 env [33], onto the liposome membrane via either non-covalent metal chelation or a co- valent linkage. The neutralization potency of multivalent presentation of these fragments by liposomes was analyzed by antiviral assays with TZM-bl cells. The results indicated that the avidity of the Vhh was significantly increased while the neutralization potency was compromised when the Vhh was covalently conjugated to the lipid. The additional ability of the liposome construct to effectively deliver dapivirine, a potent non-nucleoside reverse transcriptase inhibitor presently being developed as a vaginal HIV microbicide, was further investigated. y y The challenge of developing an effective vaccine or entry-inhibitor drug for HIV infection lies in the rapid mutation of the glycoproteins comprising gp120 and gp41 and the structural features that facilitate antibody evasion [7–13]. Another recent explanation is the spatial arrangement of the envelope spikes on HIV virus, which contributes to its ability to escape the neutralization by antibodies [14]. As is known, most neutralization activities can be attributed to antibody-antigen interactions and the avidity resulting from bivalent binding between two Fabs and two physically linked antigens greatly increase the neutralization potency of the antibody [15–17]. The pub- lished studies indicated that there are 4 to 35 spikes per HIV virus particle [18–22] with very restricted mobility [23, 24]. This makes the antibody cross-linking very un- likely because the hinge (10–15 nm) between the two Fabs is not long enough to reach the two spikes randomly dis- tributed on the surface of the virus. When the anti-HIV antibody b12 was tested as an IgA, IgM or IgG, equivalent neutralization potency was observed [14]. Actually, a number of studies have demonstrated that neutralizing antibodies against HIV cannot take the advantage of avid- ity effects resulting from the IgG cross-linking epitopes on the surface of virus [15–17], while antibodies have been shown to bind bivalently to other viruses comprising closely spaced epitopes [25, 26]. Materials Soy phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3- [(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (18:1 DGS-NTA(Ni)) and 1,2-distearoyl-sn-glycero-3-phos- phoethanolamine-N-[maleimide(polyethylene glycol)-2000] (malDSPE-PEG2000) were purchased from Avanti Polar Lipids. Methoxypolyethyleneglycol-di-stearoyl-phosphatidyl- ethanolamine (DSPE-PEG2000, with mPEG MW2000Da) was obtained from Genzyme. Cholesterol (Chol), PBS and Sephadex G25 and G50 were purchased from Sigma. Acetone, methanol and dichloromethane were from Fisher Scientific. RPMI1640, L-glutamine, penicillin-streptomycin and foetal bovine serum (FBS) were from Invitrogen Life Technologies. Dulbecco’s minimum essential medium (DMEM) was purchased from Lonza. Vhhs, the variable region fragment of single heavy-chain llama antibodies, including J3 and lab5, were purchased from Quality in Antibodies (Netherlands). Dapivirine, a non-nucleoside re- verse transcriptase inhibitor (NNRTI), was kindly provided by the authors Prof. Guido Vanham and Dr. Kevin Arien. CN54 gp140 was kindly provided by Prof. Robin Weiss from University College London. Liposomes are artificially prepared vesicles composed of a lipid bilayer and have been used extensively for drug delivery due to their unique properties [27]. Liposomes are also platforms for the development of multivalent antibody constructs by coupling antibody molecules onto liposome membranes. Chiu et al. have demonstrated that the antibody potency was increased 25-fold by employing the multivalent liposome formulation [28], which even induced cascade effects by cross-linking the antibody complex [29]. Cells, Virus and Reagents Cells, Virus and Reagents TZM-bl cells (obtained through the NIH AIDS Research and Reference Reagent Program, Germantown, MD) were used for the evaluation of the anti-HIV activity of free and liposome conjugated Vhh with and without dapivirine. TZM-bl cells express high densities of CD4, CCR5 and CXCR4. They contain a luciferase reporter gene under control of HIV LTR, which will be tran- scribed and translated into luciferase protein, if the cells Recently, llama heavy-chain antibody fragments (Vhh), which compete with surface CD4 for binding to the HIV-1envelope glycoprotein, have been described as po- tent cross-clade HIV-1 candidate microbicides [30, 31]. Such Vhh would be excellent candidates for multivalent Wang et al. Nanoscale Research Letters (2016) 11:350 Page 3 of 10 Page 3 of 10 Page 3 of 10 get infected with HIV and start producing Tat. TZM-bl cells were cultured in DMEM supplemented with 10 % heat-inactivated FBS and 50 μg gentamicin/ml medium, at 37 °C in a humidified 7 % CO2 environment. Cells were split twice a week and plated at 106 cells in tissue culture flasks and at 104 cells/well in 96-well plates. 10 mM DTT for 30 min at room temperature. Covalent immunoliposomes were prepared via interaction be- tween J3-cys and maleimide-derivatized lipid inserted in lipo3. The mixture of J3-cys and liposomes with the maleimide group was allowed to conjugate at room temperature overnight. Unreacted maleimide groups were subsequently saturated by adding cysteine. The immunoliposomes were purified by running through a Sephadex G50 column to remove excess cysteine or/ and unbound protein. The subtype B CCR-5 using reference HIV-1 strain Ba-L was obtained from the NIH AIDS Research and Reference Reagent Program, Germantown, MD. It was grown and titered in human activated peripheral blood mononuclear cells (PBMC). PBMC were isolated from buffy coats from HIV-seronegative blood donors (provided by the Antwerp Blood Transfusion Centre) using Ficoll density gradient centrifugation. PBMC were stimulated for 48 h with 2 μg/ml of phytohemagglutinin (PHA) (Remel, Kent, UK) in RPMI 1640 medium containing 10 % FBS, 50 μg/ml gentamicin and 2 μg/ml polybrene (Sigma- Aldrich, Bornem, Belgium). Subsequently, PBMC were activated for 24 h with 1 ng/ml of interleukin-2 (IL-2) (Gentaur, Brussels, Belgium) in RPMI 1640 medium supplemented with 10 % FBS, 50 μg/ml gentamicin, 2 μg/ ml polybrene and 5 μg/ml hydrocortisone (Calbiochem, Leuven, Belgium). PHA, IL-2 activated PBMC. Characterization of Liposomes The phospholipid, mainly PC, in the liposome was quan- tified by the Stewart assay [34]. The number of lipo- somes per ml was estimated by assuming there were 80,000 phospholipid molecules per liposome of 100 nm or 320,000 phospholipid molecules per liposome of 200- nm diameter [35]. The mean particle size distribution and polydispersity index of liposome suspensions were determined by dynamic light scattering in a Malvern Zetasizer Nano-2S (Malvern Instruments, Malvern, UK). The buffer for zeta potential is MilliQ water pH 7. The stability of liposomes was evaluated by storing the lipo- somes at 4 °C for 1 month, and samples were taken for size measurement. Liposome Preparation Liposome compositions used in this study with diame- ters in brackets were as follows: (a) lipo1 (200 nm), PC/ Chol/DSPE-PEG2000/18:1 DGS-NTA in proportions 65/33/1/1 mol%; (b) lipo2 (100 nm), PC/Chol/DSPE- PEG2000/18:1 DGS-NTA in proportions 65/33/1/1 mol% and (c) lipo3 (100 nm), PC/Chol/DSPE-PEG2000/mal- DSPE-PEG2000 in proportions 65/33.5/0/1.5 mol%. Briefly, the lipids with or without dapivirine were dissolved in methanol: dichloromethane 1:2 (v/v) at room temperature. The lipid mixtures were deposited on the side wall of a rotary glass vial by removing the solvent with nitrogen. The resulting dried lipid films were hydrated in 10-mM sodium phosphate buffer pH 7.4. This process led to the spontaneous formation of pegylated liposomes. The lipo- somes were then extruded through 0.2 or 0.1 μm Anotop 10 filters (Whatman). Excess dapivirine was removed on a Sephadex G25 column [34]. SDS-PAGE was employed to confirm the conjugation of Vhh to the liposomes. Fluorescamine was used to quantify the protein conjugated to liposomes [36]. Fluor- escamine is a non-fluorescent compound, which reacts rapidly with primary amines in proteins, such as the ter- minal amino group of peptides and the ε-amino group of lysine, to form highly fluorescent moieties. Basically, the liposome suspension was diluted 1:20 in PBS pH 7. An aliquot of 25 μl of 2 mg/ml fluorescamine was added to a total assay volume of 100-μl diluted liposome suspen- sion. The fluorescence was measured at ex 390 nm/em 475 nm with a cut-off at 455 nm, and the background was subtracted. The concentration of conjugated protein was calculated by comparison with standard samples of the same protein. The concentration of dapivirine in lipo- somes was determined by measuring the absorbance at 310 nm after incubating the liposomes in ethanol [37, 38]. J3 is a neutralizing Vhh, while lab5 is an irrelevant Vhh (irr-Vhh). J3 and lab5 were polyhistidinylated (J3- His and lab5-His) for the non-covalent attachment to liposomes via nitrilotriacetic acid (NTA) linkage. Mean- while another form of J3 (J3-cys) with extra cysteine in the C-terminus was generated for covalent conjugation to liposomes via interaction with a maleimide group. Vhhs were produced as described before [31]. Non- covalent immunoliposomes were produced by mixing Vhh-His with the liposome suspension in a 1:10 M ra- tio at room temperature for 1 h on a bench roller. Free thiol groups were generated by incubating J3-cys with Enzyme-Linked Immunosorbent Assay An enzyme-linked immunosorbent assay (ELISA) was carried out to determine the interaction between liposome- linked Vhh and gp140 which is a derivative of gp160 that can be cleaved into gp120 and gp41. Briefly, 1 μg/ml CN54 gp140 was coated at 4 °C overnight on 96-well Maxisorp plates (NUNC, Denmark). After blocking with 200 μl/well Microwell Blocking Buffer with Stabilizer (Rockland), serial dilutions of immunoliposomes in PBS pH 7, free Vhh and negative control liposomes coated with irr-Vhh (lab5) were added to the plates for 1 h. After three washes with PBS, Wang et al. Nanoscale Research Letters (2016) 11:350 Page 4 of 10 Page 4 of 10 Page 4 of 10 Vhh density of the liposomes will contribute to the neutralization efficiency of the liposomes for HIV virus. Lipo1 was of 200-nm diameter while lipo2 and lipo3 were of 100-nm diameter. His-tagged Vhh (J3-His and lab5-His) were conjugated to lipo1 or lipo2 by inter- action with the nickel-chelating lipid 18:1 DGS-NTA-Ni which was incorporated into the liposome bilayer (Fig. 1a). The sulphydryl-reactive lipid (malDSPE-PEG2000) in lipo3 allowed covalent attachment of the reduced J3-cys to the liposome (Fig. 1b). Immunoliposomes were then purified by size exclusion chromatography to remove the unbound Vhh and impurities. The conjugation and purification were successful in all cases, and the resulting products were analyzed by SDS-PAGE (Fig. 2). The conjugated Vhhs gave bands of higher molecular weight than the free Vhhs, and this was due to the contribution of the conju- gated lipids including 18:1 DGS-NTA-Ni or malDSPE- PEG2000. More than 95 % of the Vhh was shown to be conjugated to the liposomes by the fluorescamine method described above [34, 36]. The density of Vhh on liposome surfaces was shown in Table 1. anti-myc-horse radish peroxidase (HRP) (Roche), which targets the myc-tag in Vhh, was diluted 1:1000 in HRP Conjugate Stabilizer (Rockland) and added to detect the bound Vhh. 3,3′,5,5′-Tetramethylbenzidine liquid (Sigma) substrate was added and left to develop for 30 min at room temperature; after which, the reaction was stopped with 2 M H2SO4. Absorbance of the product was measured at 450 nm. Antiviral Assays for Antibody-Mediated Neutralization and Effectiveness of Liposomal Dapivirine Drug Delivery Antiviral Assays for Antibody-Mediated Neutralization and Effectiveness of Liposomal Dapivirine Drug Delivery The antiviral activity of the compounds was determined by pre-incubating 104 TZMbl cells/well in a 96-well plate for 2 h at 37 °C and 7 % CO2 to maintain the opti- mal pH with or without a serial dilution of compounds. Next, 200 TCID50 of HIV-1 BaL virus was added to each well, and cultures were incubated for 48 h before luciferase activity was quantified. To this end, 120 μL of supernatant was removed, 75 μl of the luciferase substrate Steadylite (Perkin Elmer, Life Sciences, Zaventem, Belgium) was added to the wells, and the plates were incubated at room temperature on an orbital shaker for 10 min. Next, the luciferase activity was measured using a TriStar LB941 luminometer (Berthold Technologies GmbH & Co. KG., Bad Wildbad, Germany) and expressed in relative light units (RLU). The final immunoliposomes were characterized, and the data was shown in Table 1. Phospholipid (PC) was detected in the liposomes by Stewart assay. It was esti- mated to result in approximately 160 Vhh molecules per 200-nm liposome or 95–125 Vhh molecules per 100-nm liposome on the basis that one 100-nm liposome contains 80,000 phospholipid molecules and one 200-nm liposome contains 320,000 phospholipid molecules [29, 35]. This resulted in similar Vhh density on the surface of the liposomes. PEGylated liposome suspensions were prepared by the lipid film rehydration method [34] and extrusion tech- nique, with a final mean diameter of 200–230 nm or 120–140 nm (polydispersity index of 0.1–0.2). The nega- tive charge of the liposome formulations were attributed to the DSPE-PEG2000 or malDSPE-PEG2000which ac- counts for 1–1.5 % mol of the lipid. The liposomes were stable for at least 1 month, with no significant change in size or charge (Fig. 3). Each compound was tested in triplicate and each ex- periment was repeated in three independent runs. Anti- viral activity was expressed as the percentage of viral growth compared to the control and plotted against the compound concentration. Next, non-linear regression analysis was used to calculate the EC50, using GraphPad Prism 5.03 using non-linear regression (GraphPad Software, San Diego, CA, USA). In Vitro Cytotoxicity Human cancer cells, Hela cells (Sigma-Aldrich, UK), were cultured in 96-well plates overnight and treated with differ- ent concentrations of liposomes for 48 h. Medium was then removed from the plates, and the cells washed twice with PBS. Fifty microlitres of 3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyltetrazolium bromide (MTT) was added per well. Cells were incubated for 30 min at 37 °C with 95 % O2 and 5 % CO2. MTT was removed carefully before 100-μl pro- panol was added per well to dissolve crystals and incubated for at least 30 min. The absorbance of this coloured solu- tion was quantified by measuring at a wavelength of 570 nm by FLUOstar Omega (BMG Labtech, Aylesbury, UK). Each treatment was conducted in triplicate. Immunoliposomes with or without NNRTI dapivirine were produced to investigate whether conjugated Vhh could show higher avidity to antigen and improve the ef- ficacy of Vhh in HIV viral inhibition, as compared to free Vhh. Dapivirine was dissolved in the solvent with lipid mixture when formulating the liposomes. Unincor- porated dapivirine was removed by size exclusion chro- matography, and the concentration of dapivirine was determined by UV spectrometry. Liposome Preparation and Characterization Potential cytotoxicity of the liposomes was estimated from cell viability relative to control Hela cells treated with medium only. MTT assay demonstrated that cell Three groups of liposome suspensions: lipo1, lipo2 and lipo3, were prepared to investigate whether the size or Wang et al. Nanoscale Research Letters (2016) 11:350 Page 5 of 10 Fig. 1 Structural basis of conjugation. a Non-covalent ligation between Vhh-His and nickel-chelating lipid 18:1 DGS-NTA-Ni. b Covalent ligation between Vhh-cys and malDSPE-PEG2000 lipid Fig. 1 Structural basis of conjugation. a Non-covalent ligation between Vhh-His and nickel-chelating lipid 18:1 DGS-NTA-Ni. b Covalent ligation between Vhh-cys and malDSPE-PEG2000 lipid viability was between 80 and 120 % in relation to the control samples (Fig. 4). One-way ANOVA statistical analysis showed there is no evidence of liposome toxicity at concentrations up to 1 mg/ml (p > 0.05). the free J3 at the same concentrations, together with the irrelevant Vhh (lab5-His) and liposome-coated the same irrelevant Vhh as negative controls. J3-His and J3-cys revealed similar binding affinities for gp140 as expected (Fig. 5). No improved or compromised affinity was ob- served for lipo1-J3 and lipo2-J3 compared to free J3-His, implying size differences are not influencing the avidity despite similar Vhh densities on the liposome surfaces. Meanwhile, one-way ANOVA statistical analysis showed that J3 conjugated to lipo3 demonstrated much higher avidity to gp140 than free J3-cys (p < 0.001), indicating covalent attachment greatly facilitated J3 interacting with Characterization of Neutralizing Antibody by Antiviral y The neutralization activity of liposomal Vhhs and free Vhhs for HIV virus was evaluated by subsequent incuba- tion with TZM-bl cells. Lipo1-J3 and lipo2-J3 revealed similar potencies for neutralization compared to free J3. Surprisingly, lipo3-J3 only provided 60 % of viral inhib- ition at J3 concentration of 286 ng/ml, whereas lipo1-J3 and lipo2-J3 inhibited approximately 90 % of viral growth at the same concentration (Fig. 6a). No viral growth reduc- tion was observed for control liposomes, liposomes coated with irrelevant Vhhs or free Vhhs alone. There was no significant difference in neutralizing activities between Analysis of Binding of Liposomal Vhhs to Recombinant HIV gp140 by ELISA As Vhh competed with soluble CD4 (sCD4) for binding to gp120 [32], an ELISA assay was carried out to deter- mine whether the Vhh conjugated to liposomes showed compromised binding affinity or increased avidity to gp140 (CN54). Liposome-linked J3 was compared with Fig. 2 Vhh and conjugated Vhh in liposomes analyzed by SDS-PAGE. Lanes: 1, molecular weight marker; 2, J3-His; 3, lipo1-J3; 4, lipo2-J3; 5, lab5; 6, lipo1-lab5; 7, lipo2-lab5; 8, J3-cys and 9, lipo3-J3 Fig. 2 Vhh and conjugated Vhh in liposomes analyzed by SDS-PAGE. Lanes: 1, molecular weight marker; 2, J3-His; 3, lipo1-J3; 4, lipo2-J3; 5, lab5; 6, lipo1-lab5; 7, lipo2-lab5; 8, J3-cys and 9, lipo3-J3 Wang et al. Nanoscale Research Letters (2016) 11:350 Page 6 of 10 Table 1 Characterisation of the immunoliposomes Liposome PC conc. (mg/ml)a Dapivirine (μM) No. of Vhh molecules per liposome Vhh (μg/ml) Diameter (nm)a Zeta potential (mV)a Lipo1-lab5 11.937 ± 0.001 – 160 126.3 228.0 ± 1.0 −45.0 ± 0.9 Lipo1-J3 11.708 ± 0.008 – 160 118.4 223.4 ± 4.5 −37.4 ± 0.5 Lipo2-lab5 5.974 ± 0.005 – 95 150.0 130.9 ± 1.4 −38.7 ± 0.7 Lipo2-J3 6.149 ± 0.003 – 108 159.9 131.2 ± 1.9 −38.5 ± 0.2 Lipo2-dapi 6.478 ± 0.001 6.5 – – 122.9 ± 1.8 −46.0 ± 0.9 Lipo2-dapi-J3 5.548 ± 0.002 5.5 125 169.6 123.4 ± 1.8 −38.5 ± 0.4 Lipo3 control 7.573 ± 0.003 – – – 130.9 ± 1.7 −42.5 ± 0.4 Lipo3-J3 6.456 ± 0.004 – 121 168.2 142.2 ± 3.2 −36.4 ± 0.4 Lipo3-dapi 6.675 ± 0.003 6.7 – – 128.2 ± 2.6 −41.7 ± 0.5 Lipo3-dapi-J3 6.806 ± 0.001 6.8 108 157.0 139.1 ± 1.8 −37.8 ± 0.6 Dapi: dapivirine aData are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments gp140. In contrast, irrelevant negative controls demon- strated no binding to gp140. lipo1-J3 and lipo2-J3 compared to free J3-His (Fig. 6b), revealing no contribution from the size differences. However, covalently conjugated J3 (lipo3-J3) had a sig- nificantly higher EC 50 (Fig. 6b), indicating less potency for neutralization than non-covalently attached J3 or free J3. The result is unexpected because the ELISA data revealed significantly increased binding affinity of lipo3-J3 to gp140 which should contribute to the neutralization activity of conjugated J3. Discussion The treatment of HIV remains a serious challenge due to the high genetic variation of the virus and associated toxic side effects of the antiviral drugs. HIV-1 neutraliz- ing antibodies have been considered to be critical for vaccine development and prevention of HIV infection. Liposomes provided another approach shown to have potential for HIV prophylaxis [39]. In the present study, a combined platform with both liposome and antibody fragments was developed to investigate the antiviral effi- ciency of the multivalent liposomal formulation. y p Previous studies have demonstrated that Vhhs produced by llamas (lama glama) exhibited comparable affinity and specificity for gp120 to conventional immunoglobulins despite their much smaller size. This smaller size of Vhhs is an advantage over conventional immunoglobulins as it has been shown to reduce their immunogenicity [33]. In particular, J3 Vhh, which achieved broad and potent neutralization of HIV-1 via interaction with the CD4- binding site, neutralized 96 of 100 tested HIV-1 strains and chimeric simian-HIV strains with HIV subtypes B and C env [33]. Unlike most of the enveloped viruses which express large numbers of closely spaced spikes on the sur- face, HIV virus has a low spike surface density (4 to 35 spikes) and the arrangement of these spikes is likely to be static over the time periods relevant to neutralization [14]. Therefore, the location and arrangement of the neutraliz- ing Vhhs on the liposome surfaces is critical for their interaction with the virus particles. Fig. 4 Cell viability of Hela cells after 48 h incubation with control liposomes or immunoliposomes. No significant difference in cell viability was observed against the media only treatment from one-way ANOVA (p > 0.05). Data are means ± standard deviations of three replicate measurements in one representative experiment of at least two independent experiments. Dapi: dapivirine Fig. 4 Cell viability of Hela cells after 48 h incubation with control liposomes or immunoliposomes. No significant difference in cell viability was observed against the media only treatment from one-way ANOVA (p > 0.05). Data are means ± standard deviations of three replicate measurements in one representative experiment of at least two independent experiments. Dapi: dapivirine although no significant difference was observed between lipo2 and lipo3 with or without neutralizing Vhhs (Fig. 7a). Comparison of Antiviral Effects of Liposomal Dapivirine with Free Dapivirine We compared the antiviral effects of different formula- tions of liposomal dapivirine with free dapivirine. The dose-response curves indicated increased antiviral effect of liposomal dapivirine compared with free dapivirine, Fig. 3 Diameter and zeta potential of the liposome formulations over 1-month period. Data are means ± standard deviations of three replicate measurements in one representative experiment of at least two independent experiments. Using unpaired t test (p > 0.05), no significant change in diameter or zeta potential of liposomes was observed after 1-month storage at 4 °C. Dapi: dapivirine Fig. 3 Diameter and zeta potential of the liposome formulations over 1-month period. Data are means ± standard deviations of three replicate measurements in one representative experiment of at least two independent experiments. Using unpaired t test (p > 0.05), no significant change in diameter or zeta potential of liposomes was observed after 1-month storage at 4 °C. Dapi: dapivirine Wang et al. Nanoscale Research Letters (2016) 11:350 Page 7 of 10 Page 7 of 10 Fig. 4 Cell viability of Hela cells after 48 h incubation with control liposomes or immunoliposomes. No significant difference in cell viability was observed against the media only treatment from one-way ANOVA (p > 0.05). Data are means ± standard deviations of three replicate measurements in one representative experiment of at least two independent experiments. Dapi: dapivirine Discussion Furthermore, there was approximately 76 % of viral growth at a concentration of 1-nM free dapivirine, whereas only 50–56 % of viral growth was detected with liposomal dapivirine at this concentration. Calculated EC50 (Fig. 7b) confirmed that liposomal dapivirine is more effective than free dapivirine in inhibiting HIV virus. However, the anti- viral effects were dominated by dapivirine since no differ- ence was observed between the liposome preparations with or without neutralizing J3. To optimize the size of the liposomal platform, J3 was non-covalently conjugated to the surface of two lipo- some formulations with diameters of 100 or 200 nm, each with approximately the same Vhh densities. Metal chelation via NTA has been employed to attach peptide and protein antigens to liposomes to compare with cova- lent linkage for their ability to elicit antibody responses [40]. Pegylated liposomes were employed to ensure the stability and minimal immunogenicity of the platform. To check that J3 was available and not masked by the PEG coating on the liposome surface, appropriate access of J3 to gp140 was verified by ELISA (Fig. 5). The identi- cal binding curves demonstrated that lipo1-J3 and lipo2- J3 bound to gp140 with the same avidity as free J3, showing that the different geometrical location of J3 on the surface of spherical liposomes did not compromise its interaction with gp140. Surprisingly, no enhanced avidity was observed for these liposomal J3 constructs compared to free J3. This may be due to the charge on the nickel-histidine complex which would inhibit close association of the J3 antibody fragments to generate multivalent attachment to the gp140 on ELISA surface. This presumption is confirmed by the neutralization results indicating lipo1-J3 and lipo2-J3 inhibited HIV infection in a dose-response manner similar to free J3 Fig. 5 The binding of free J3/J3 linked to liposomes to recombinant gp140 (CN54) was compared in ELISA. Gp140 was immobilized on the plate, and Vhh was detected with anti-myc-HRP. Irrelevant Vhh (lab5) and liposome-irrelevant Vhh were included as negative controls. Values are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences were observed (p < 0.001) between lipo3-J3 and the other liposome formulations or free Vhhs Fig. 5 The binding of free J3/J3 linked to liposomes to recombinant gp140 (CN54) was compared in ELISA. Gp140 was immobilized on the plate, and Vhh was detected with anti-myc-HRP. Discussion Irrelevant Vhh (lab5) and liposome-irrelevant Vhh were included as negative controls. Values are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences were observed (p < 0.001) between lipo3-J3 and the other liposome formulations or free Vhhs Wang et al. Nanoscale Research Letters (2016) 11:350 Page 8 of 10 a b Fig. 6 Antiviral effect of different liposomal formulations and free Vhhs. a Dose-response curves for the antiviral effect of different liposomal formulations and free Vhhs. b Comparison of calculated EC50 of each liposomal formulation and free J3. Liposome preparations were pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in EC50 were observed between lipo3-J3 and the other liposome formulations or free Vhhs b a Fig. and f for 2 expe othe a b a Fig. 6 Antiviral effect of different liposomal formulations and free Vhhs. a Dose-response curves for the antiviral effect of different liposomal formulations and free Vhhs. b Comparison of calculated EC50 of each liposomal formulation and free J3. Liposome preparations were pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in EC50 were observed between lipo3-J3 and the other liposome formulations or free Vhhs J3 were distinct from each other, indicating that their inter- actions with gp140 were qualitatively different with superior avidity for lipo3-J3. This implied that a multivalent platform had been formed with cross-linking or multimerization of Vhhs. This is not surprising since the absence of the nickel complex allows the free movement and geometrical ar- rangement of J3-lipid complex within the lipid liposome membrane so permitting the formation of multivalent lipo- somal Vhh constructs through cross-linking the envelope in antiviral assay (Fig. 6). There was no significant differ- ence in the EC50 between lipo1-J3, lipo2-J3 and free J3, suggesting no improved antiviral potency for the non- covalently anchored Vhh independent of liposome sizes. J3-cys was then covalently attached to liposomes to produce lipo3-J3 (Fig. 1). Discussion One hundred-nanometre diam- eter liposomes were used for economic reasons and since liposomes of this size are broadly applied as drug delivery system [41, 42]. The binding curves for lipo2-J3 and lipo3- a b Fig. 7 Antiviral effect of different liposomal formulations and free dapivirine. a Dose-response curves for the antiviral effect of different liposomal formulations and free dapivirine. b Comparison of calculated EC50 of each liposomal formulation and free dapivirine. Liposome preparations were pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in EC50 were observed between free dapivirine and the liposomal dapivirine. Dapi: dapivirine a Fig. form pre-i meas EC50 a b a b Fig. 7 Antiviral effect of different liposomal formulations and free dapivirine. a Dose-response curves for the antiviral effect of different liposomal formulations and free dapivirine. b Comparison of calculated EC50 of each liposomal formulation and free dapivirine. Liposome preparations were pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in EC50 were observed between free dapivirine and the liposomal dapivirine. Dapi: dapivirine Fig. 7 Antiviral effect of different liposomal formulations and free dapivirine. a Dose-response curves for the antiviral effect of different liposomal formulations and free dapivirine. b Comparison of calculated EC50 of each liposomal formulation and free dapivirine. Liposome preparations were pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in EC50 were observed between free dapivirine and the liposomal dapivirine. Dapi: dapivirine Wang et al. Nanoscale Research Letters (2016) 11:350 Page 9 of 10 Page 9 of 10 spikes which are sparsely distributed on the virion surface and remained static over the time periods. Unexpectedly, the antiviral potency of lipo3-J3 was remarkably reduced as evaluated in the antiviral assay compared to lipo1-J3, lipo2- J3 or free J3, the EC50 for lipo3-J3 being approximately four times higher than that for lipo2-J3 or free J3. Competing Interests Th h d l h The authors declare that they have no competing interests. Authors’ Contributions SXW and IR are working in Middlesex University. SXW has designed the study, prepared the liposome formulations for all the experiments in this study and wrote the manuscript. IR provided consultation on the whole study including the design and use of the liposome and Vhh antibody fragments. JM, KKA and GV are working in Institute of Tropical Medicine, Belgium. JM has carried out the antiviral assay in this study. KKA has designed the antiviral assay. GV provided consultation on HIV infection and treatment. RN is the chief scientist in Proxima Concepts Ltd. He has provided consultation on the liposome preparation. All authors read and approved the final manuscript. References 1. Wyatt R, Sodroski J (1998) The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280:1884–8 2. Chan DC, Kim PS (1998) HIV entry and its inhibition. Cell 93:681–4 3. Wong HL, Chattopadhyay N, Wu XY, Bendayan R (2010) Nanotechnology applications for improved delivery of antiretroviral drugs to the brain. Adv Drug Deliv Rev 62:503–17 4. Freed EO (2001) HIV-1 replication. Somat Cell Mol Genet 26:13–33 5. Da Cunha J (2015) Impact of antiretroviral therapy on lipid metabolism of human immunodeficiency virus-infected patients: old and new drugs. World J Virol 4:56 6. Rao KV1, Chitturi RT2, Kattappagari KK2, Kantheti LP2, Poosarla C2, Baddam V (2015) Impact of highly active antiretroviral therapy on oral manifestations of patients with human immunodeficiency virus_acquired immuno deficiency synd. Indian J Sex Transm Dis 36(1):35–39 Acknowledgements Thi k t This work was supported by the CHAARM EU consortium to develop vaginal HIV microbicides. We would like to thank Dr. Lei Wang for his assistance in statistical analysis. Author details 1 f 1Department of Natural Sciences, Middlesex University, London, UK. 2Institute of Tropical Medicine, Antwerp, Belgium. 3Proxima Concepts. Ltd, London, UK. In addition to the neutralization capacity of antibody- coated liposomes, we investigated whether the liposomes could have the extra-function of delivering antiviral drugs intracellularly, in particular using high doses of otherwise poorly soluble hydrophobic drugs whose encap- sulation in the liposome would limit its general toxicity. All of these dapivirine containing liposomes revealed higher efficacy in reducing viral replication than free dapi- virine, although no significant differences were observed between them i.e. irrespective of the presence of irrelevant or J3 Vhh on the surface. This suggested that the antiviral activities of J3 were masked by the high dose of dapivirine and that the liposome platform per se is an efficient deliv- ery system for dapivirine. Received: 3 February 2016 Accepted: 16 July 2016 Received: 3 February 2016 Accepted: 16 July 2016 Discussion We hypothesize that in the case of non-covalent linkage, J3 was dissociated from liposome after it bound to gp140 on the virion surface because the interaction between J3 and gp140 was so strong (K ≅10−9 M ~ 10−12 M compared to K ≅10−5 M ~ 10−6 M for attachment to the nickel-chelating lipid) [32, 40, 43], that J3 was pulled apart from the nickel complex inserted in the liposome membrane. The remaining surface antibody molecules were now free to react with further virus, the net effect being that lipo2-J3 neutralized the virus as efficiently as free J3. Although the covalent attachment of the J3 in lipo3 enhanced lipo3-J3-gp140 interaction, this fixed the viruses to the surface of the liposome and due to the comparability in size of the interacting particles, the number bound to the liposome surface will be restricted and the small number of viral spikes involved will leave a large fraction of the antibodies remaining uncombined which were otherwise available in the non-covalently linked constructs. liposome platform encapsulating dapivirine with non- covalently attached J3 and covalently linked anti-CD4 Vhh could be a powerful potential candidate for HIV prevention where J3 neutralizes free virus and dissoci- ates from liposome while the anti-CD4 Vhh coated lipo- some efficiently delivers the antiviral drug to HIV target cells overexpressing CD4. 10. 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Gaddy DF, Lee H, Zheng J et al (2015) Whole-body organ-level and kidney micro-dosimetric evaluations of (64)Cu-loaded HER2/ErbB2-targeted liposomal doxorubicin ((64)Cu-MM-302) in rodents and primates. EJNMMI Res 5:24 18. Zhu P, Chertova E, Bess J et al (2003) Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions. Proc Natl Acad Sci U S A 100:15812–7 43. Nieba L, Nieba-Axmann SE, Persson A et al (1997) BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem 252:217–28 19. Zhu P, Liu J, Bess J et al (2006) Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441:847–52 20. Wang et al. Nanoscale Research Letters (2016) 11:350 Liu J, Bartesaghi A, Borgnia MJ et al (2008) Molecular architecture of native HIV-1 gp120 trimers. Nature 455:109–13 21. 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Smith TJ, Olson NH, Cheng RH et al (1993) Structure of a human rhinovirus- bivalently bound antibody complex: implications for viral neutralization and antibody flexibility. Proc Natl Acad Sci U S A 90:7015–8 26. Icenogle J, Shiwen H, Duke G et al (1983) Neutralization of poliovirus by a monoclonal antibody: kinetics and stoichiometry. Virology 127:412–25 27. Duncan R (2003) The dawning era of polymer therapeutics. Nat Rev Drug Discov 2:347–60 28. Chiu GNC, Edwards LA, Kapanen AI et al (2007) Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs. Mol Cancer Ther 6:844–55 29. Oliveira S, Schiffelers RM, van der Veeken J et al (2010) Downregulation of EGFR by a novel multivalent nanobody-liposome platform. J Control Release 145:165–75 30. Koh WWL, Steffensen S, Gonzalez-Pajuelo M et al (2010) Generation of a family-specific phage library of llama single chain antibody fragments that neutralize HIV-1. J Biol Chem 285:19116–24 31. Gorlani A, Brouwers J, McConville C, et al. (2011) Llama antibody fragments have good potential for application as HIV type 1 topical microbicides. AIDS Res Hum Retroviruses 28(2):198-205. 31. Gorlani A, Brouwers J, McConville C, et al. (2011) Llama antibody fragments have good potential for application as HIV type 1 topical microbicides. AIDS Res Hum Retroviruses 28(2):198-205. 32. Forsman A, Beirnaert E, Aasa-Chapman MMI et al (2008) Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J Virol 82:12069–81. 32. Wang et al. Nanoscale Research Letters (2016) 11:350 Conclusions 7. Wei X, Decker JM, Wang S et al (2003) Antibody neutralization and escape by HIV-1. Nature 422:307–12 This study demonstrated that antibody-conjugated lipo- somes can provide anti-HIV viral defence in two ways. First, non-covalently conjugated liposomal J3 neutralized HIV virus as efficiently as free J3. It was also shown that incorporation of the lipophilic drug dapivirine in lipo- somes resulted in a higher level of virus inactivation in vitro than observed after exposure of cells to free drug dissolved in DMSO—an effective method of solubilizing the drug, but not one which can be used in vivo. Since we have demonstrated that immunoliposomes with co- valently grafted anti-CD4 Vhhs facilitated cellular uptake of liposomes by lymphoma cells overexpressing CD4 on the surface (data not shown), it follows that a combined 8. Kwong PD, Doyle ML, Casper DJ et al (2002) HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature 420:678–82 9. Kwong PD, Wyatt R, Robinson J et al (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648–59 9. Kwong PD, Wyatt R, Robinson J et al (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648–59 10. Labrijn AF, Poignard P, Raja A et al (2003) Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77:10557–65 10. Labrijn AF, Poignard P, Raja A et al (2003) Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77:10557–65 11. Wyatt R, Desjardin E, Olshevsky U et al (1997) Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein. J Virol 71:9722–31 12. Moore JP, Sodroski J (1996) Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein. J Virol 70:1863–72 12. Moore JP, Sodroski J (1996) Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein. J Virol 70:1863–72 Page 10 of 10 Page 10 of 10 Page 10 of 10 38. (2010) Linear order release polymer. http://www.google.com/patents/ EP2244782A2?cl=en. 37. das Neves J, Sarmento B, Amiji MM, Bahia MF (2010) Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles. J Pharm Biomed Anal 52:167–72 38. (2010) Linear order release polymer. http://www.google.com/patents/ EP2244782A2?cl=en. 39. Malavia NK, Zurakowski D, Schroeder A et al (2011) Liposomes for HIV prophylaxis. Biomaterials 32:8663–8 40. Watson DS, Platt VM, Cao L et al (2011) Antibody response to polyhistidine- tagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice. Clin Vaccine Immunol 18:289–97 41. Clayton R, Ohagen A, Nicol F et al (2009) Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes. Antiviral Res 84:142–9 42. Gaddy DF, Lee H, Zheng J et al (2015) Whole-body organ-level and kidney micro-dosimetric evaluations of (64)Cu-loaded HER2/ErbB2-targeted liposomal doxorubicin ((64)Cu-MM-302) in rodents and primates. EJNMMI Res 5:24 43. Nieba L, Nieba-Axmann SE, Persson A et al (1997) BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem 252:217–28 43. Nieba L, Nieba-Axmann SE, Persson A et al (1997) BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem 252:217–28 Wang et al. Nanoscale Research Letters (2016) 11:350 Forsman A, Beirnaert E, Aasa-Chapman MMI et al (2008) Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J Virol 82:12069–81. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and benefi t from: Submit your manuscript to a journal and benefi t from: 33. McCoy LE, Quigley AF, Strokappe NM et al (2012) Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization. J Exp Med 209:1091–103 34. New RR (1990) Liposomes a practical approach. 1st ed. Oxford: Oxford University Press. o GD, Wenk MR (2012) Lipids. 1st ed. San Diego: Academic press. 36. Shew RL, Deamer DW (1985) Hemoglobin and alkaline phosphatase were each encapsulated in phosphatidylcholine liposomes using a dehydration- rehydration cycle for liposome formation. In this method, iiposomes prepared by sonication are mixed in aqueous solution with the solute desi. Science (80-) 816:1–8 36. Shew RL, Deamer DW (1985) Hemoglobin and alkaline phosphatase were each encapsulated in phosphatidylcholine liposomes using a dehydration- rehydration cycle for liposome formation. In this method, iiposomes prepared by sonication are mixed in aqueous solution with the solute desi. Science (80-) 816:1–8
https://openalex.org/W2178981046
https://www.combinatorics.org/ojs/index.php/eljc/article/download/v22i4p10/pdf
English
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Walks, Partitions, and Normal Ordering
˜The œElectronic journal of combinatorics/˜The œjournal of combinatorics
2,015
cc-by
12,054
Abstract We describe the relation between graph decompositions into walks and the nor- mal ordering of differential operators in the n-th Weyl algebra. Under several spec- ifications, we study new types of restricted set partitions, and a generalization of Stirling numbers, which we call the λ-Stirling numbers. Keywords: Decompositions of graphs; differential operators; Weyl algebra; set partitions; Stirling numbers dzhuma@hotmail.com yeldamir@gmail.com Submitted: Apr 11, 2015; Accepted: Sep 30, 2015; Published: Oct 16, 2015 Mathematics Subject Classifications: 05A18, 05A30, 11B73 the electronic journal of combinatorics 22(4) (2015), #P4.10 1 Introduction Let G = (V, E) be a digraph with an ordered set of edges E = (e1, . . . , em). A walk of G is any sequence of edges ei1 . . . eiℓsuch that the terminal vertex of eik coincides with the initial vertex of eik+1 for all k < ℓ. The walk ei1 . . . eiℓis called increasing if i1 < · · · < iℓ. + Consider decompositions of G into edge-disjoint increasing walks. This setting gen- eralizes set partitions, because when G has only one vertex and m labeled loops (1, 1), decompositions into increasing walks correspond to partitions of the set [m] := {1, . . . , m} into subsets. If G is decomposed into one walk (with distinct edges), it is an Euler tour. If G is a path and edges along the path are labeled by an arbitrary permutation σ ∈Sm, decompositions into minimal number of increasing walks index the descent set of σ. This interpretation arises from the normal ordering problem in the Weyl algebra. The n-th Weyl algebra An is an associative algebra with 2n generators x1, . . . , xn, ∂1, . . . , ∂n subject to relations [∂i, xj] = δi,j, [xi, xj] = 0, [∂i, ∂j] = 0, where [a, b] = ab −ba is the commutator and δi,j is the Kronecker delta. The element w ∈An is normally ordered if it is expressed in the form w = P k ck Q i xi Q j ∂j. For the digraph G, the special case of our normal ordering formula is −→ m Y ℓ=1 xiℓ∂jℓ= X decompositions into increasing walks with sources I Y i∈I xj Y j∈J ∂j, 1 1 2 3 e1 e2 e3 Figure 1: A digraph with an ordered set edges. 1 2 3 e1 e2 e3 Figure 1: A digraph with an ordered set edges. where (iℓ, jℓ) is an ℓ-th edge of G and J is the corresponding multiset of sinks (which is determined uniquely from the given sources I). For example, the graph in Figure 1 has four decompositions into increasing walks: e1e2e3, e1e2 ∪e3, e1 ∪e2e3, e1 ∪e2 ∪e3. This corresponds to the normal ordering This corresponds to the normal ordering x1∂2x2∂3x3∂1 = x1∂1 + x1x3∂3∂1 + x1x2∂2∂1 + x1x2x3∂2∂3∂1, where the sources and sinks of walks are exactly the indices of terms. 1 Introduction In fact, this formula is a graph-theoretic version of Olshanski’s analog of Wick’s formula [26] (cf. [27]). Collecting the terms we can rewrite where the sources and sinks of walks are exactly the indices of terms. In fact, this formula is a graph-theoretic version of Olshanski’s analog of Wick’s formula [26] (cf. [27]). Collecting the terms we can rewrite −→ m Y ℓ=1 xiℓ∂jℓ= X I SG(I) Y i∈I xj Y j∈J ∂j, where SG(I) is the number of decompositions of G into increasing walks with multiset of sources I. In the case of the first Weyl algebra A1 this formula gives the well-known expansion where SG(I) is the number of decompositions of G into increasing walks with multiset of sources I. In the case of the first Weyl algebra A1 this formula gives the well-known expansion (x∂)m = X i S(m, i)xi∂i for Stirling numbers of the second kind S(m, i). We introduced this combinatorial model in a more general setting in [15], where we studied algebraic applications, polynomial identities and commutators on Weyl algebra. In this paper we focus on combinatorial aspects of walk decompositions and various spec- ifications of the normal ordering interpretations, such as restricted set partitions and generalized Stirling numbers. We study walk decompositions and the G-Stirling func- tions which enumerate decompositions by sources of walks. As we see, the values of the G-Stirling function serve as connection constants in the normal ordering problem. The setting of decomposing graphs into walks is a source for certain types of restricted set partitions. For example, assume that digraph has 2 vertices and edges e1, e3, . . . going from vertex 1 to 2 and the remaining edges e2, e4, . . . going back from 2 to 1. Decomposi- tions on this model generate set partitions with the property that each block (when sorted) is parity alternating (i.e. odd, even, odd, etc.). We show that the total number of parity 2 the electronic journal of combinatorics 22(4) (2015), #P4.10 alternating partitions of the set [m], satisfies the formula a(m) = B⌊(m+1)/2⌋B⌈(m+1)/2⌉, where Bk is the k-th Bell number, the number of partitions of [k]. Note that the latter formula is not an obvious fact from the definition of a(m). We apply the composition of operators in Weyl algebra to compute the number of such decompositions. 1 Introduction This approach is illustrated for a new special type of set partitions, which we call the residue alternating partitions (a general version of the parity alternating partitions). The elements in every block of these partitions form a consecutive (cyclic) interval modulo n and their total number is the product of Bell numbers. We show both algebraic and bijective proofs to this fact. We then specify our interpretations to the case n = 1, for which there are many related studies, e.g. [3, 4, 12, 18, 20, 21, 24, 30] and refer to a recent book [22] on the subject of normal ordering. We introduce and study the λ-Stirling numbers. These numbers of second kind naturally appear in decompositions of graphs with one vertex and many loops. For the given sequence λ = (λ1 ⩾λ2 ⩾· · · ), the λ-Stirling numbers of the second kind Sλ(n, k) is the number of partitions of [n] into k blocks such that the first λ1 elements of [n] are in distinct blocks, the next λ2 elements are in distinct blocks, and so on. This definition is a natural generalization of the r-Stirling numbers [5] and it was studied in [25]. We show how these numbers Sλ(n, k) arise from our general graph setting as well as from differential operators in the first Weyl algebra. On the other hand, we also define the corresponding dual λ-Stirling numbers of the first kind Cλ(n, k), and show a combinatorial interpretation to them. Namely, Cλ(n, k) is the number of permutations of [n] having k cycles such that non-minimal elements of the first λ1 cycles are greater than all minimal elements of these λ1 cycles; non-minimal elements of the next λ2 cycles are greater than all minimal elements of these λ2 cycles, and so on (some of the remaining cycles are singletons). The classical Stirling numbers are defined on two parameters: the number of elements and the number of blocks (cycles). For these generalizations, one can see that the λ-sequence affects on the first parameter in λ-Stirling numbers of the second kind, and affects on the second parameter (cycles) in λ-Stirling numbers of the first kind. We obtain many properties of Sλ(n, k), Cλ(n, k) analogous to the properties of the usual Stirling numbers. 2 Decompositions into increasing walks Suppose that edges of the digraph G = (V, E) are ordered (or labeled), E = (e1, . . . , em). A k-decomposition is a decompositions of E into k edge-disjoint walks. We say that the k-decomposition E = P1 ∪. . . ∪Pk is principal if every walk Pi = eℓ1 . . . eℓs (1 ⩽i ⩽k) is increasing, i.e. we have ℓ1 < . . . < ℓs. When V = {1} and graph has m labeled loop edges (1, 1), principal decompositions correspond to partitions of the set [m] into disjoint subsets. Further, we suppose that the digraph G is presented by the vertex set V = [n]. A block (or p-block if p is specified) is a distinguished set of edges {e1, . . . , ep}. If graph is built up from several (disjoint) blocks, then we will require that the edges in each block must lie in distinct walks. 3 the electronic journal of combinatorics 22(4) (2015), #P4.10 1 2 3 4 e2 e3 e1 e4 e5 e6 Figure 2: A digraph built of three edge blocks (red, blue, black). 1 2 3 4 e2 e3 e1 e4 e5 e6 Figure 2: A digraph built of three edge blocks (red, blue, black). For example, digraph in Figure 2 which is built from three blocks B1 = {e1}, B2 = {e2, e3}, B3 = {e4, e5, e6}, has a principal 4-decomposition e1e5 ∪e2 ∪e3e4 ∪e6. Note that decomposition e1e5 ∪e2e3e4 ∪e6 cannot be used here since e2, e3 are from the same block B1 and thus cannot be in the same path. For each vertex v ∈V consider the sets In(v) = {ei1, . . . , eis}, Out(v) = {ej1, . . . , ejℓ} of its incoming and outcoming edges, respectively (loops (v, v) are included in both sets). Suppose that the edges of G are built up from some partition of E into blocks. Consider the matchings between In(v) and Out(v) defined as follows. We allow elements ei ∈In(v) and ej ∈Out(v) to be matched if i < j and ei, ej are not in the same block. A matching now is defined as some set of such matched pairs, where every edge is used at most once from In(v) and at most once from Out(v) (so, only loops can be used once for both In(v), Out(v)). position 2.1. There is a bijection between the sets M(1) × · · · × M(n) and PD(G Proposition 2.1. There is a bijection between the sets M(1) × · · · × M(n) and PD(G). Proposition 2.1. There is a bijection between the sets M(1) × · · · × M(n) and PD(G). Proof. Let us take an arbitrary matching for every vertex v (1 ⩽v ⩽n) and construct a principal graph decomposition. If ei ∈In(v) and ej ∈Out(v) are matched, then we define them to be a fragment of a path eiej. Otherwise, if ei ∈In(v) or ej ∈Out(v) are unmatched edges, then define them as final and initial edges of their corresponding paths, respectively. One can easily verify that this map defines a principal decomposition and its inverse defines a matching for every vertex. Corollary 2.2. |PD(G)| = Qn i=1 |M(i)|. Corollary 2.2. |PD(G)| = Qn i=1 |M(i)|. 2 Decompositions into increasing walks Let M(v) be the set of all possible matchings (not necessarily maximal, and including an empty matching) and M(v) be the set of maximal matchings. For example, at vertex 2 of the graph in Figure 2 we have In(2) = {e1, e2}, Out(2) = {e3, e5}, M(2) = {∅, {(e1, e3)}, {(e1, e5)}, {(e2, e5)}, {(e1, e3), (e2, e5)}}, M(2) = {(e1, e3), (e2, e5)}. Let PD(G) be the set of principal decompositions of G. the electronic journal of combinatorics 22(4) (2015), #P4.10 2.1 On general walk and path decompositions h G = (V, E), define flux(v) := in(v) −out(v), v ∈V, V + := {v ∈V | flux(v) > 0}, V −:= {v ∈V | flux(v) < 0} and the total flux as f(G) := X v∈V + flux(v) = X v∈V − −flux(v). The basic properties of walk decompositions such as existence criteria, simply rely on the usual Euler tours. We just add a new vertex so that for all v ∈V we get flux(v) = 0. This also allows to compute (or bound) the total number of walk decompositions using the BEST theorem and the Matrix-tree theorem. The basic properties of walk decompositions such as existence criteria, simply rely on the usual Euler tours. We just add a new vertex so that for all v ∈V we get flux(v) = 0. This also allows to compute (or bound) the total number of walk decompositions using the BEST theorem and the Matrix-tree theorem. Decompositions into walks have the following matroid structure studied in [23]. Sets of edges that are in distinct walks in a certain (minimal) decomposition are independent sets of a matroid of rank f(G) (adding with the number of components of G). This matroid is isomorphic to a cotransversal matroid. In fact, the sets of source (initial) edges of all f(G)-decompositions also form a collection of matroid bases. For (simple) path decompositions there are many studies (e.g. [1, 2, 8, 13, 16, 17, 19]), most of which are around Gallai’s conjecture. Note that f(G) is attainable minimal number of paths in a decomposition for acyclic digraphs; it was also shown in [1] that the minimal number of paths needed to decompose a transitive tournament digraph on n vertices is ⌊n2/4⌋. Gallai’s conjecture states that every connected simple undirected graph with n vertices can be decomposed into at most ⌈n/2⌉paths; it is known that every such graph can be decomposed into at most ⌊n/2⌋paths or cycles [19]. Corollary 2.2. |PD(G)| = Qn i=1 |M(i)|. Denote in(v) = |In(v)|, out(v) = |Out(v)|. Denote in(v) = |In(v)|, out(v) = |Out(v)|. 4 the electronic journal of combinatorics 22(4) (2015), #P4.10 Corollary 2.3. If G is acyclic digraph, then there exists a labeling of its edges such that every path is increasing. Therefore, every decomposition of G is a principal decomposition for such labeling. For every vertex v we can match some i edges from In(v) with some i edges of In(v) in in(v) i out(v) i  i! ways. Therefore, M(v) = X i⩾0 in(v) i out(v) i  i! and the total number of decompositions of the acyclic digraph G is and the total number of decompositions of the acyclic digraph G is n Y v=1 X i⩾0 in(v) i out(v) i  i! ! . 2.1 On general walk and path decompositions For a digraph G = (V, E), define flux(v) := in(v) −out(v), v ∈V, V + := {v ∈V | flux(v) > 0}, V −:= {v ∈V | flux(v) < 0} 2.1 On general walk and path decompositions 2.1 On general walk and path decompositions 3 G-Stirling functions We use the following notation for multisets: A −X is a difference, e.g. {13, 22, 3, 43} − {12, 2, 4} = {1, 2, 3, 42}; A ⊎X is a merge, e.g. {12, 2, 42} ⊎{1, 22, 3} = {13, 23, 3, 42}. We also write G −e if edge e is eliminated from G or G −B if block B is removed. We use the following notation for multisets: A −X is a difference, e.g. {13, 22, 3, 43} − {12, 2, 4} = {1, 2, 3, 42}; A ⊎X is a merge, e.g. {12, 2, 42} ⊎{1, 22, 3} = {13, 23, 3, 42}. We also write G −e if edge e is eliminated from G or G −B if block B is removed. 5 the electronic journal of combinatorics 22(4) (2015), #P4.10 For a given digraph G, let Vout := {1out(1), . . . , nout(n)}, Vin := {1in(1), . . . , nin(n)}, Mout(G) := {I | I ⊆Vout}, Vout := {1out(1), . . . , nout(n)}, Vin := {1in(1), . . . , nin(n)}, Mout(G) := {I | I ⊆Vout}, i.e., Mout is the set of all sub(multi)sets of Vout. Note that if for a k-decomposition, we have the sources I, then the corresponding sinks J = Vin ⊎I −Vout are determined uniquely. (Further, for any sources I we will just write sinks as J meaning that J = Vin ⊎I −Vout.) ) fine the G-Stirling function SG : Mout(G) →Z⩾0 as follows ) Define the G-Stirling function SG : Mout(G) →Z⩾0 as follows SG(I) := the number of principal decompositions of G with sources I. If n = 1, then SG(I) corresponds to Stirling number of the second kind S(m, k) where |I| = k and digraph G has m labeled loops (1, 1). Proposition 3.1. If I = {1ℓ1, . . . , nℓn}, then Proposition 3.1. If I = {1ℓ1, . . . , nℓn}, then Proposition 3.1. If I = {1ℓ1, . . . , nℓn}, then SG(I) = n Y i=1 Si(out(i) −ℓi), (1) (1) (1) where Si(k) the number of matchings in M(i) of size k. Proof. Since Si(k) the number of matchings in M(i) of size k, the vertex i is unmatched out(i) −k times. Thus, from the bijection of Proposition 2.1, the vertex i is used exactly out(i) −ℓi times as a source. 3 G-Stirling functions Therefore, we get the formula by considering this argument for every vertex i = 1, . . . , n and using Proposition 2.1. Theorem 3.2 ([15]). The G-Stirling function SG satisfies the following properties: (i) SG(Vout) = 1; (ii) if SG(I) > 0 for some I ⊂Vout, then for any I′, such that Vout ⊇I′ ⊃I, we have SG(I′) > 0; (ii) if SG(I) > 0 for some I ⊂Vout, then for any I′, such that Vout ⊇I′ ⊃I, we have SG(I′) > 0; (iii) Suppose that digraph G is built up from blocks B1, . . . , Bm so that the indices of edges increase with respect to the order of blocks. Let e = (i, j) ∈Bm, G′ = G −e, I′ = I −{i}. Let ki be the number of repetitions of i in (J −{j}) ⊎{i} and re be the number of edges in Bm −e that end by i. Then the following recurrence relation holds for SG(I). (iii) Suppose that digraph G is built up from blocks B1, . . . , Bm so that the indices of edges increase with respect to the order of blocks. Let e = (i, j) ∈Bm, G′ = G −e, I′ = I −{i}. Let ki be the number of repetitions of i in (J −{j}) ⊎{i} and re be the number of edges in Bm −e that end by i. Then the following recurrence relation holds for SG(I). SG(I) = SG′(I′) + (ki −re)SG′(I). (2) (2) Corollary 3.3. If G has edges e1, . . . , em (without blocks), then for em = (i, j), G′ = G −em, I′ = I −{i} and ki the number of repetitions of i in (J −{j}) ⊎{i}, we have Corollary 3.3. If G has edges e1, . . . , em (without blocks), then for em = (i, j), G′ = G −em, I′ = I −{i} and ki the number of repetitions of i in (J −{j}) ⊎{i}, we have SG(I) = SG′(I′) + kiSG′(I). (3) (3) 6 the electronic journal of combinatorics 22(4) (2015), #P4.10 6 Corollary 3.4. If G has n = 1 vertex and m loops (1, 1), then SG(I) = S(m, k) if |I| = k, where S(m, k) is Stirling number of the second kind. Relation (3) becomes the well-known recurrence Corollary 3.4. 3 G-Stirling functions If G has n = 1 vertex and m loops (1, 1), then SG(I) = S(m, k) if |I| = k, where S(m, k) is Stirling number of the second kind. Relation (3) becomes the well-known recurrence S(m, k) = S(m −1, k −1) + kS(m −1, k). Remark 3.5. The G-Stirling function SG(I) is a graph generalization of Stirling number of the second kind. Note that SG is different from Stirling (and Bell) numbers for graphs studied in [14], which count partitions of graph vertex set into independent sets. Although, for n = 1 (and several blocks) there is a correspondence between these definitions as noted in section 6. In fact, Mout(G) can be considered as a poset ordered by inclusion. Let us consider a subdomain of Mout(G) at which SG takes positive values; define the poset PG := {I | SG(I) > 0}, (4) (4) whose elements (multisets) are ordered by inclusion. Proposition 3.6. Let m(v) = |M(v)| be the size of a maximal matching. The Proposition 3.6. Let m(v) = |M(v)| be the size of a maximal matching. Then (i) PG has a unique maximal element Vout, unique minimal element V0, where V0 = Vout −{1m(1), . . . , nm(n)}. V0 = Vout −{1m(1), . . . , nm(n)}. (ii) PG is isomorphic to m(1) × · · · × m(n). (ii) PG is isomorphic to m(1) × · · · × m(n). Note that PG is isomorphic to a divisibility poset, and µ(I, J) = (−1)|J−I| if J −I is a set and 0 if J −I is a multiset. Hence we get Note that PG is isomorphic to a divisibility poset, and µ(I, J) = (−1)|J−I| if J −I is a set and 0 if J −I is a multiset. Hence we get SG(J) = X I⊆[n] (−1)|J−I|BG(J −I) and the kind of recurrence and the kind of recurrence BG = 1 − X ∅⊂I⊆[n] (−1)|Vout−I|BG(Vout −I). (ii) PG is isomorphic to m(1) × · · · × m(n). Here by m we denote the chain poset of m elements and m × s is the poset of ms elements defined as (cartesian) product of posets m, s (if s is empty, then put m×s = m). (See e.g. [29]) Proof. First, from (i), (ii) of Theorem 3.2, Vout is a unique maximal element and if V0 is some minimal element, then for all V0 ⊆I ⊆Vout. Let us prove that V0 = {1out(1)−m(1), . . . , nout(n)−m(n)} V0 = {1out(1)−m(1), . . . , nout(n)−m(n)} is a unique minimal element. From Theorem 2.1, there is a principal decomposition with sources V0, so V0 ∈PG. If there is another minimal element V ′, then iout(i)−m(i) ̸∈V ′ for some i. This means that the vertex i has a matching of size greater than m(i), which is impossible. So, both items (i), (ii) clearly imply from these arguments. is a unique minimal element. From Theorem 2.1, there is a principal decomposition with sources V0, so V0 ∈PG. If there is another minimal element V ′, then iout(i)−m(i) ̸∈V ′ for some i. This means that the vertex i has a matching of size greater than m(i), which is impossible. So, both items (i), (ii) clearly imply from these arguments. Corollary 3.7. If G is a cycle graph with edge labels given by 1 →e1 2 →e2 · · · →en−1 n →en 1, then m(1) = 0, and m(i) = 2 for i = 2, . . . , n. Hence, PG is isomorphic to 2 × · · · × 2 = 2[n−1] then m(1) = 0, and m(i) = 2 for i = 2, . . . , n. Hence, PG is isomorphic to 2 × · · · × 2 = 2[n−1]. 7 the electronic journal of combinatorics 22(4) (2015), #P4.10 7 The total number of principal decompositions |PD(G)| serves as a generalized Bell number. Let BG = |PD(G)|, then we have BG = X V0⊆I⊆Vout SG(I). (5) (5) If we now define an extension BG(J) as BG(J) := X V0⊆I⊆J SG(I), (6) (6) then applying the M¨obius inversion formula on the poset PG we get SG(J) = X I⊆J µ(I, J)BG(I) and in particular SG(Vout) = 1 = X I⊆Vout µ(I, J)BG(I). 4 Normal ordering in the Weyl algebra Let K be a field of characteristic 0. The n-th Weyl algebra An is an associative algebra over K defined by 2n generators x1, . . . , xn, ∂1, . . . , ∂n and relations [xi, xj] = [∂i, ∂j] = 0, [∂i, xj] = δi,j for 1 ⩽i, j ⩽n, where [a, b] = ab −ba is the commutator and δi,j is the Kronecker symbol. The typical example of An is the polynomial algebra with ∂i considered as partial derivations d/dxi. The elements of types xα∂β := xα1 1 · · · xαn n ∂β1 1 · · · ∂βn n with α = (α1, . . . , αn), β = (β1, . . . , βn) ∈Zn ⩾0 are called monomials. We will also write monomials in the equivalent form xi1 . . . xis∂j1 . . . ∂jp. All monomials xα∂β form a linear vector space basis of An. When the element w of An is expressed as a linear combination w = X α,β c(α, β)xα∂β, c(α, β) ∈K, the electronic journal of combinatorics 22(4) (2015), #P4.10 the electronic journal of combinatorics 22(4) (2015), #P4.10 8 we say that w is normally ordered. Define the following subspaces of An: A(p,q) n := ⟨xα∂β : |α| = p, |β| = q⟩, A(0) n := M i⩾1 A(i,i) n , where |α| = Pn i=1 αi. Note that A(0) n is the subalgebra of An. We show that combinatorial meaning of coefficients in the normal ordering can be in- terpreted in terms of graph decompositions. Furthermore, we will consider the monomials of subspace A(0) n (otherwise, we may add fictive elements as shown in subsection 6.1). We associate every monomial w = xi1 . . . xip∂j1 . . . ∂jp ∈A(0) n with the p-block of a graph in the following way: block(w) := {(i1, j1), . . . , (ip, jp)}. block(w) := {(i1, j1), . . . , (ip, jp)}. Theorem 4.1 ([15]). Let w1, . . . , wm ∈A(0) n be monomials. Then we have Theorem 4.1 ([15]). Let w1, . . . , wm ∈A(0) n be monomials. Then we have w1 · · · wm = X I⊆Vout SG(I) Y i∈I xi Y j∈J ∂j, (7) (7) where G = ([n], E) with E = (block(w1), . . . , block(wm)) (i.e. where m m1,...,mk  = m! m1!···mk! is a multinomial coefficient. Proof. From monomials x1∂2, x2∂3, . . . , xm−1∂m, xm∂m+1, the digraph G is a single path 1 →2 →· · · →m →m + 1. Taking all permutations σ ∈Sm means that we permute the edges of G up to σ. Consider decompositions into increasing walks in that case. Suppose that we break the paths at vertices 2 ⩽i1 < · · · < ik ⩽m. Then the number of permutations σ ∈Sm for which the fragments 1 →· · · →i1/i1 →· · · →i2/ · · · /ik → · · · →m+1 are all increasing, is clearly equal to m i1−1,i2−i1,...,m+1−ik  and the corresponding monomial is x1xi1 · · · xik∂i1 · · · ∂ik∂m+1 and so we obtain the formula. Remark 4.6. Note that the last formula is computationally effective; we compute 2m−1 terms instead of calculating s+ m with m! summands. 4 Normal ordering in the Weyl algebra the indices of edges increase with respect to the order of blocks) and J = Vin ⊎I −Vout. Corollary 4.2. For a digraph G = ([n], E) with E = (e1, . . . , em), we have Corollary 4.2. For a digraph G = ([n], E) with E = (e1, . . . , em), we have −→ m Y ℓ=1 xiℓ∂jℓ= X I SG(I) Y i∈I xi Y j∈J ∂j, (8) (8) where eℓ= (iℓ, jℓ) and the sum runs over all (multi)sets of sources I. where eℓ= (iℓ, jℓ) and the sum runs over all (multi)sets of sources I. Corollary 4.3. If n = 1, then (8) gives the well-known formula (x∂)m = m X i=0 S(m, i)xi∂i. where S(m, i) is Stirling number of the second kind. where S(m, i) is Stirling number of the second kind. Analogous to formula (8), the operators xi∂j + xj∂i give a decomposition formula for undirected graphs. Namely, we obtain the following result. Theorem 4.4. For an undirected graph G = ([n], E) with E = (e1, . . . , em), eℓ= (iℓ, jℓ), we have −→ m Y ℓ=1 (xiℓ∂jℓ+ xjℓ∂iℓ) = X decompositions into increasing walks with sources I Y i∈I xi Y j∈J ∂j. (9) (9) the electronic journal of combinatorics 22(4) (2015), #P4.10 9 The normal ordering decomposition formulas like (8), (9) are in particular useful when we sum through all decompositions of G or symmetrize over all permutations of the edge set. For w1, . . . , wm ∈An, let s+ m(w1, . . . , wm) := X σ∈Sm wσ(1) · · · wσ(m). s+ m(w1, . . . , wm) := X σ∈Sm wσ(1) · · · wσ(m). Then for undirected graph G we have Then for undirected graph G we have s+ m(xi1∂j1+xj1∂i1, . . . , xim∂jm+xjm∂im) = (# Euler tours i →j) xi∂j+higher order s+ m(xi1∂j1+xj1∂i1, . . . , xim∂jm+xjm∂im) = (# Euler tours i →j) xi∂j+higher order terms. Consider a case of computing the sum s+ m. Proposition 4.5. s+ m(x1∂2, x2∂3, . . . , xm−1∂m, xm∂m+1) s+ m(x1∂2, x2∂3, . . . , xm−1∂m, xm∂m+1) = x1 X 2⩽i1<···<ik⩽m  m i1 −1, i2 −i1, . . . , m + 1 −ik  k Y ℓ=1 xiℓ k Y ℓ=1 ∂iℓ ! ∂m+1, = x1 X 2⩽i1<···<ik⩽m  m i1 −1, i2 −i1, . . . , m + 1 −ik  k Y ℓ=1 xiℓ k Y ℓ=1 ∂iℓ ! ∂m+1, where m m1,...,mk  = m! m1!···mk! is a multinomial coefficient. 5 Cyclic multigraphs and residue alternating partitions Consider the digraph Cm,n with n vertices and m edges (m > n) that consecutively form a cycle (12 · · · n), i.e. the edges {e1, . . . , em} in order are e1 = (1, 2), e2 = (2, 3), . . . , en = (n, 1), en+1 = (1, 2), . . . The principal decompositions on such graph correspond to the following type of set parti- tions. Say that the set partition ∪Xi = [m] is residue alternating if for every i (1 ⩽i ⩽k) and Xi = {a1, . . . , ar} with a1 < · · · < ar, we have ai+1 −ai ≡1 (mod n) for i = 1, . . . , r (ar+1 = a1). Let A(m, n) be the number of residue alternating partitions (or the number of decom- positions of digraph Cm,n). 10 10 the electronic journal of combinatorics 22(4) (2015), #P4.10 Theorem 5.1. The following formulas hold for A(m, n) Theorem 5.1. The following formulas hold for A(m, n) A(nk, n) = Bk(Bk+1)n−1 and for 0 < r < n A(nk + r, n) = (Bk+1)n−r+1(Bk+2)r−1, where Bk is the Bell number, the number of partitions of set [k]. where Bk is the Bell number, the number of partitions of set [k]. Proof. First we examine the approach using composition of differential operators. Note that A(m, n) corresponds to the number BG = P I SG(I) (see def. (5)) of all principal decompositions of G = Cm,n. Therefore, we can use the normal ordering expansion of differential operators to calculate A(m, n) = BG. ( ) If m = nk, then the composition of operators is (x1∂2 · · · xn∂1) · · · (x1∂2 · · · xn∂1) = (x1∂2 · · · xn∂1)k = (x1∂1)k(∂2x2)k · · · (∂nxn)k. In fact, A(m, n) is the sum of coefficients in the normal ordering expansion of the last expression. The sum of coefficients in expansion (x1∂1)k is In fact, A(m, n) is the sum of coefficients in the normal ordering expansion of the last expression. 5 Cyclic multigraphs and residue alternating partitions Construct set partition of [k + 1] as follows: (1) if (ean+i−1, ebn+i) ∈M, where a ⩽b, then put a + 1, b + 2 in the same block (1) if (ean+i−1, ebn+i) ∈M, where a ⩽b, then put a + 1, b + 2 in the same block; (2) the remaining elements of [k + 1] (that were not considered yet), put in separate blocks. For example, if k = 5, In(i) = {e1, e3, e5, e7, e9}, Out(i) = {e2, e4, e6, e8, e10}, M = {(e1, e6), (e3, e4), (e5, e8)}, then (e1, e6) means that we should put 1, 4 in the same block; for (e3, e4) the elements 2, 3 are in the same block; and for (e5, e8) the elements 3, 5 are in the same block. Therefore, we have the partition {1, 4}{2, 3, 5}{6}. then (e1, e6) means that we should put 1, 4 in the same block; for (e3, e4) the elements 2, 3 are in the same block; and for (e5, e8) the elements 3, 5 are in the same block. Therefore, we have the partition {1, 4}{2, 3, 5}{6}. The inverse procedure can be described as follows: take any block of the partition, {a1 < · · · < ar} and for every j = 1, . . . , r −1 match the edges (e(aj−1)n+i−1, e(aj+1−2)n+i). One can see that this properly defines the bijection. By applying a similar argument one can show that |M(1)| = Bk and therefore, by Theorem 2.1, A(nk, n) = Bk(Bk+1)n−1. The formula for A(nk + r, n) implies analogously. 5 Cyclic multigraphs and residue alternating partitions The sum of coefficients in expansion (x1∂1)k is k X i=0 S(k, i) = Bk k X i=0 S(k, i) = Bk k X i=0 S(k, i) = Bk and the sum of coefficients in the normal ordering of (∂ℓxℓ)k is k X i=0 k i  Bi, k X i=0 k i  Bi, since since (∂ℓxℓ)k = (1 + xℓ∂ℓ)k = k X i=0 k i  (xℓ∂ℓ)i. (∂ℓxℓ)k = (1 + xℓ∂ℓ)k = k X i=0 k i  (xℓ∂ℓ)i. It remains to use the well-known recurrence for Bell numbers Bk+1 = k X i=0 k i  Bi. Bk+1 = k X i=0 k i  Bi. So, the sum of coefficients A(nk, n) in the normal ordering of (x1∂1)k(∂2x2)k · · · (∂nxn)k is Bk(Bk+1)n−1. So, the sum of coefficients A(nk, n) in the normal ordering of (x1∂1)k(∂2x2)k · · · (∂nxn)k is Bk(Bk+1)n−1. Similarly, if m = nk + r we have the composition So, the sum of coefficients A(nk, n) in the normal ordering of (x1∂1)k(∂2x2)k · · · (∂nxn)k is Bk(Bk+1)n−1. k( k+1) Similarly, if m = nk + r we have the composition +1) ilarly, if m = nk + r we have the composition ( + ) Similarly, if m = nk + r we have the composition (x1∂2 · · · xn∂1) · · · (x1∂2 · · · xn∂1)x1∂2 · · · xr∂r+1 = x1(∂1x1)k  (∂2x2)k+1 · · · (∂rxr)k+1 (∂r+1xr+1)k∂r+1  (∂r+2xr+2)k · · · (∂nxn)k and by the same argument it follows that A(nk + r, n) = (Bk+1)n−r+1(Bk+2)r−1. the electronic journal of combinatorics 22(4) (2015), #P4.10 11 the electronic journal of combinatorics 22(4) (2015), #P4.10 11 Bijective proof. Using Corollary 2.2 of Theorem 2.1 we look at matchings of edges at every vertex. Suppose m = nk and consider the vertex i, where 2 ⩽i ⩽n. We have In(i) = {ei−1, en+i−1, e2n+i−1, . . . , e(k−1)n+i−1}, Out(i) = {ei, en+i, e2n+i, . . . , e(k−1)n+i}. In(i) = {ei−1, en+i−1, e2n+i−1, . . . , e(k−1)n+i−1}, Out(i) = {ei, en+i, e2n+i, . . . , e(k−1)n+i}. We will prove that |M(i)| = Bk+1 by establishing a bijection between matchings in M(i) and partitions of set [k + 1]. Let M ∈M(i) be any matching between In(i), Out(i). the electronic journal of combinatorics 22(4) (2015), #P4.10 Proposition 5.3. Recurrence relations for a(m, k, i) are given by Proposition 5.3. Recurrence relations for a(m, k, i) ar ition 5.3. Recurrence relations for a(m, k, i) are given by a(1, 1, 0) = 1, a(m, k, i) = 0 for m < k or k < i, a(2m + 1, k, i) = a(2m, k −1, i) + (i + 1)a(2m, k, i + 1), a(2m, k, i) = a(2m −1, k −1, i −1) + (k −i + 1)a(2m −1, k, i −1). Proof. Consider parity alternating partitions of set [2m + 1] into k blocks, i of whose maximal elements are even. The element 2m + 1 can form a separate block contributing a(2m, k −1, i) ways. Otherwise, 2m + 1 can be placed into blocks of parity alternating partitions of [2m] having k blocks and (i + 1) maximal even elements (since 2m + 1 will change the parity of one maximal element); this gives (i + 1)a(2m, k, i + 1) ways. Similarly, if we look at partitions of [2m], then 2m can form a separate block in a(2m−1, k−1, i−1) ways and can be placed into other blocks in (k−i+1)a(2m−1, k, i−1) ways. 5.1 Parity alternating partitions For n = 2 we have the graph model with 2 vertices and n edges {e1, . . . , en} such that all odd-indexed e2i−1 are of type (1, 2) and all even-indexed e2i are of type (2, 1). All principal decompositions on this graph can be considered as partitions X1∪· · ·∪Xk of [m] that have the following property: For every i(1 ⩽i ⩽k) and Xi = {a1, . . . , ar} with a1 < · · · < ar, the sequence a1, . . . , ar is parity alternating (i.e. even, odd, even, odd, etc. or similarly beginning with odd). Let us call such partitions of sets parity alternating partitions. For example, the parity alternating partitions of {1, 2, 3, 4} into two blocks are {1}{2, 3, 4}; {4}{1, 2, 3}; {1, 2}{3, 4}; {1, 4}{2, 3}. Denote by a(m) = A(m, 2) the total number of parity alternating partitions of [m]. Then the following formulas hold Denote by a(m) = A(m, 2) the total number of parity alternating partitions of [m]. Then the following formulas hold a(2k) = BkBk+1, a(2k + 1) = B2 k+1. the electronic journal of combinatorics 22(4) (2015), #P4.10 12 the electronic journal of combinatorics 22(4) (2015), #P4.10 Remark 5.2. The latter formulas mean that the number of parity alternating partitions of [m] is equal to the number of partitions of [m + 1] where elements in each block have the same parity. (The second sequence appears in OEIS, A124419 [28]). Bijectively, this fact can be described as follows: x, y are successive elements in the block of parity alternating partitions iffx, y + 1 are successive elements of the second type of partitions. Note that such reduction algorithm was applied to regular (noncrossing) set partitions in [6]. The values of a(m) can also be computed by the number of blocks. Let a(m, k) be the number of parity alternating partitions into k blocks and a(m, k, i) the number of parity alternating partitions into k blocks i of which have even maximal elements. We have a(m, k) = Pk i=0 a(m, k, i), a(m) = Pn k=1 a(m, k). 5.1 Parity alternating partitions m\k 1 2 3 4 5 6 7 8 a(m) 1 1 1 2 1 1 2 3 1 2 1 4 4 1 4 4 1 10 5 1 6 11 6 1 25 6 1 10 28 26 9 1 75 7 1 14 61 86 50 12 1 225 8 1 22 136 276 236 92 16 1 780 Table 1: Small values of a(m) and a(m, k). Table 1: Small values of a(m) and a(m, k). 6.1 Normal ordering for n = 1. Combinatorial interpretations of coefficients in the normal ordering expansion xr1∂s1 · · · xrt∂st = X i SG(i)xi∂j (10) (10) 13 the electronic journal of combinatorics 22(4) (2015), #P4.10 the electronic journal of combinatorics 22(4) (2015), #P4.10 can be extracted from Theorem 4.1 as follows. The elements xri∂si are not from A(0) 1 = ⟨xi∂i : i = 0, 1, . . .⟩in general (i.e. if ri ̸= si). To deal with this situation, we add fictive |ri −si| new variables xi+1 or ∂i+1 so that the monomial will belong to A(0) 1 , and the graph scheme can be applied. For example, x2∂5 is transformed to x3 2x2∂5. Note that the new variables commute with all other and so we can freely move them in the normal ordering expansion. Using these new monomials, we construct the graph G according to the rules above. Thus, combinatorial meaning of SG(i) can be described as the number of principal decompositions of G having i sources at vertex 1. This interpretation is similar to the graph models studied in [3] (the model there is acyclic which is different to ours since for n = 1 we have loops). Remark 6.1. In fact, the normal ordering in the n-th Weyl algebra can be computed using the n = 1 case. For instance, we can restructure compositions as follows x1x2∂1∂3x2x3∂1∂3x1∂2 = (x1∂2 1x1)(x2 2∂2)(∂3x3∂3). This view also helps to refine all possible multisets of sources and sinks I, J, since what coefficients are nonzero in every composition like (10) can be found. This view also helps to refine all possible multisets of sources and sinks I, J, since what coefficients are nonzero in every composition like (10) can be found. 6.2 The λ-Stirling numbers • Cλ(n, k) is the number of permutations of [n] with k cycles such that non-minimal elements of the first λ1 cycles are greater than all minimal elements of these λ1 cycles; non-minimal elements of the next λ2 cycles are greater than all minimal elements of these λ2 cycles, and so on; and the remaining rk cycles are singletons (i.e. consist of one element). • Cλ(n, k) is the number of permutations of [n] with k cycles such that non-minimal elements of the first λ1 cycles are greater than all minimal elements of these λ1 cycles; non-minimal elements of the next λ2 cycles are greater than all minimal elements of these λ2 cycles, and so on; and the remaining rk cycles are singletons (i.e. consist of one element). By definition, the sequence λ affects on the first argument n in Sλ(n, k) and on the second argument k in Cλ(n, k). Further we will see that Sλ(n, k), Cλ(n, k) are dual to each other. Remark 6.2. For permutation σ of [n] having k cycles we associate representation in the form σ = (σ(1)) · · · (σ(k)), where σ(1), . . . , σ(k) are cycles written in increasing order of their minimal elements. For example, the permutation (5, 6, 1, 7, 3, 2, 4) is written as (1, 5, 3)(2, 6)(4, 7). So we divide the set [n] according to the integer partition n = λ1 + . . . + λq + r: So we divide the set [n] according to the integer partition n = λ1 + . . . + λq + r: [n] = {1, . . . , λ1 | {z } 1st part , . . . , λ1 + · · · + λq−1 + 1, . . . , λ1 + · · · + λq | {z } q-th part , n −λq + 1, . . . , n | {z } last r elements } [n] = {1, . . . , λ1 | {z } 1st part , . . . , λ1 + · · · + λq−1 + 1, . . . , λ1 + · · · + λq | {z } q-th part , n −λq + 1, . . . 6.2 The λ-Stirling numbers Consider the graph with n = 1 vertex and suppose it is built up from blocks of loops (1, 1) of λ1, λ2, . . . edge sizes. Principal decompositions on this model require that the edges within one block cannot lie on the same walk. This setting clearly corresponds to partitions of the set [m] (m is a total number of edges), where the first λ1 elements are in distinct subsets, the next λ2 elements are also in distinct subsets, and so on. The coefficients SG(I) present a generalization of Stirling numbers of the second kind on such restricted partitions. In this section we study these generalized Stirling numbers. We also introduce a generalization of Stirling numbers of the first kind, which can be considered as dual to the second. We call these numbers the λ-Stirling numbers. Fix the sequence λ0 = 0, λ = (λ1, λ2, . . .), λ1 ⩾λ2 ⩾· · · of nonnegative integers, and let of nonnegative integers, and let q = qn := max{i | λ0 + · · · + λi ⩽n}, r = rn := n −λ0 −· · · −λq. q = qn := max{i | λ0 + · · · + λi ⩽n}, r = rn := n −λ0 −· · · −λq. So λ is a kind of ‘infinite’ integer partition, q is the analog of quotient and r is the analog of remainder. So λ is a kind of ‘infinite’ integer partition, q is the analog of quotient and r is the analog of remainder. Definition. The λ-Stirling numbers of second and first kinds Sλ(n, k), Cλ(n, k) are defined as follows: the electronic journal of combinatorics 22(4) (2015), #P4.10 14 • Sλ(n, k) is the number of partitions of [n] into k blocks such that the first λ1 elements of [n] are in distinct blocks, the next λ2 elements are in distinct blocks, and so on; the remaining rn elements are also in distinct blocks. • Sλ(n, k) is the number of partitions of [n] into k blocks such that the first λ1 elements of [n] are in distinct blocks, the next λ2 elements are in distinct blocks, and so on; the remaining rn elements are also in distinct blocks. 6.2 The λ-Stirling numbers , n | {z } last r elements } and consider only those set partitions which restrict the elements of the same part to be in the same subset. and consider only those set partitions which restrict the elements of the same part to be in the same subset. For the case of permutations, we take the partition k = λ1 +. . .+λqk +rk and consider permutations of the following type: σ(1), . . . , σ(λ1) | {z } ∀non-min > ∀min | · · · | σ(λ1+···+λqk−1+1), . . . , σ(λ1+···+λqk) | {z } ∀non-min > ∀min | σ(k−rk+1), . . . , σ(k) | {z } last rk singletons {z ∀non-min > ∀min {z ∀non-min > ∀min Consider examples. If λ = (3, 2, 1, 1, . . 6.2 The λ-Stirling numbers .), then Sλ(6, 4) = 30 and allowed configurations of partitions of {1, 2, 3, 4, 5, 6} into 4 blocks can be described as follows: • If 1, 2, 3, 4 are blocks minima; then 5 can be put with 1, 2, 3 (except restriction with 4) and 6 with 1, 2, 3, 4 which totally gives 3 × 4 = 12 ways; If 1, 2, 3, 4 are blocks minima; then 5 can be put with 1, 2, 3 (except restriction wit 4) and 6 with 1 2 3 4 which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 4 are blocks minima; then 5 can be put with 1, 2, 3 (except restriction with 4) and 6 with 1, 2, 3, 4 which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are blocks minima; then 4 can be put with 1, 2, 3 and 6 with 1, 2, 3, 4 which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are blocks minima; then 4 can be put with 1, 2, 3 and 6 with 1, 2, 3, 4 which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 6 are blocks minima; then 4 can be put with 1, 2, 3 and 5 with 1, 2, 3 except the block with 4, which totally gives 3 × 2 = 6 ways; • If 1, 2, 3, 6 are blocks minima; then 4 can be put with 1, 2, 3 and 5 with 1, 2, 3 except the block with 4, which totally gives 3 × 2 = 6 ways; • If 1, 2, 3, 6 are blocks minima; then 4 can be put with 1, 2, 3 and 5 with 1, 2, 3 except the block with 4, which totally gives 3 × 2 = 6 ways; So, there are totally 12 + 12 + 6 = 30 ways to arrange desired partitions. Cλ(6, 4) = 36 and configurations of permutations of (1, . . . , 6) with 4 cycles can be described as follows: So, there are totally 12 + 12 + 6 = 30 ways to arrange desired partitions. Cλ(6, 4) = 36 and configurations of permutations of (1, . . . 6.2 The λ-Stirling numbers , 6) with 4 cycles can be described as follows: ( h ld l b l d l b l ) So, there are totally 12 + 12 + 6 = 30 ways to arrange desired partitions. Cλ(6, 4) = 36 and configurations of permutations of (1, . . . , 6) with 4 cycles can be described as follows: 6, 4) = 36 and configurations of permutations of (1, . . . , 6) with 4 cycles can be ed as follows: (1, 2, 3 should always be cycle minima; and cycle number 4 is singleton) 15 the electronic journal of combinatorics 22(4) (2015), #P4.10 • If 1, 2, 3, 4 are cycle minima; then 5 can be put with 1, 2, 3 and 6 can be put after 1, 2, 3, 5, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 4 are cycle minima; then 5 can be put with 1, 2, 3 and 6 can be put after 1, 2, 3, 5, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are cycle minima; then 4 can be put with 1, 2, 3 and 6 can be put after 1, 2, 3, 4, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are cycle minima; then 4 can be put with 1, 2, 3 and 6 can be put after 1, 2, 3, 4, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 6 are cycle minima; then 5 can be put with 1, 2, 3 and 4 can be put after 1, 2, 3, 5, which gives 3 × 4 = 12 ways. • If 1, 2, 3, 6 are cycle minima; then 5 can be put with 1, 2, 3 and 4 can be put after 1, 2, 3, 5, which gives 3 × 4 = 12 ways. So, there are totally 12 + 12 + 12 = 36 possible permutations with 4 cycles. So, there are totally 12 + 12 + 12 = 36 possible permutations with 4 cycles. Clearly if λ = (1, 1, . . .), then Cλ(n, k), Sλ(n, k) are just the usual Stirling numbers. The case λ = (r, 1, 1, . . .) corresponds to the r-Stirling numbers of first and second kinds introduced in [5]. 6.2 The λ-Stirling numbers Generalized Stirling numbers of the second kind Sr,s(n, k) that arise from the expansion xr1∂s1 · · · xrn∂sn = x P ri−di X k Sr,s(n, k)xk∂k. k have been studied in the bosons normal ordering problem [4, 24] (see also [12]). Our definition gives a natural (and simple) combinatorial interpretation to the case Sr,r(n, k) and also solves an inverse problem, where Stirling numbers of the first kind arise. Note that the general formulas for Sr,s(n, k) given in [24] can be used to compute our numbers SG, BG (defined in Section 2). Interpretation to Sr,r(n, k) with colorings of complete graphs introduced in [7] is very close to the meaning of Sλ(n, k) (λ = (r, r, . . .)), since any r elements that cannot be in the same subset can be viewed as proper colorings of a component complete graph Kr; on the other hand, this definition corresponds to Stirling numbers for graphs in the sense of counting partitions of a vertex set into independent sets (see [14]). The numbers Sλ(n, k) were introduced in [25] as (r1, . . . , rp)-Stirling numbers of the second kind. In order to be consistent with the corresponding Stirling numbers of the first kind Cλ(n, k), we define these numbers over a general sequence λ relating it with integer partitions, since it somehow acts (as integer partition) on first (or resp. second) argument of these Stirling numbers. In fact, by polynomial relations (18), (19) shown below, the λ-Stirling numbers correspond to a case of the multiparameter non- central Stirling numbers introduced in [11]. This also leads to a case of a general study of connection constants between persistent sequences of polynomials [10]. [ ] The numbers Sλ(n, k) also related to compositions (ordered partitions) of multisets in the following way. For n = λ1 + · · · + λq + r consider a multiset n := {1λ1, . . . , qλq, (q + 1)r}. n := {1λ1, . . . , qλq, (q + 1)r}. Then the number of ways to distribute the elements of n into k ordered nonempty sets is equal to Then the number of ways to distribute the elements of n into k ordered nonempty sets is equal to Sλ(n, k)k! λ1! · · · λq!r!. Sλ(n, k)k! λ1! · · · λq!r!. 6.2 The λ-Stirling numbers .) n\k 2 3 4 5 6 2 1 3 2 1 4 2 2 1 5 4 14 8 1 6 4 32 38 12 1 n\k 2 3 4 5 6 2 1 3 2 1 4 6 4 1 5 24 18 8 1 6 120 96 58 12 1 Sλ(n, k) for 2 ⩽n, k ⩽6 Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (3, 2, 1, . . .) n\k 3 4 5 6 3 1 4 3 1 5 6 6 1 6 18 30 11 1 n\k 3 4 5 6 3 1 4 3 1 5 12 6 1 6 60 36 11 1 Sλ(n, k) for 3 ⩽n, k ⩽6 Cλ(n, k) for 3 ⩽n, k ⩽6. Table 2: Some tables for Sλ(n, k), Cλ(n, k).1 Table 2: Some tables for Sλ(n, k), Cλ(n, k).1 6.3 Main properties 1Sλ(n, k) = Cλ(n, k) = 0 if k < λ1 or k > n. If λ = (1, 1, . . .) or λ = (r, 1, 1, . . .) then the λ-Stirling numbers are referred to the ordinary Stirling numbers (A008275, A008277, A048994 in OEIS) or to the r-Stirling numbers (A143494, A143495, A143496, A193685, A143491, A143492, A143493 in OEIS [28]), respectively. Some values of Sλ(n, k) (of second kind) are referred to A078739, A008297, A035342, A078740, A078741, A090214 in OEIS [28]. 6.2 The λ-Stirling numbers the electronic journal of combinatorics 22(4) (2015), #P4.10 16 16 the electronic journal of combinatorics 22(4) (2015), #P4.10 This easily implies from the combinatorial interpretation of Sλ(n, k). If blocks are ordered, then we multiply the number of ways by k!. The elements λ1+· · ·+λj−1+1, . . . , λ1+· · ·+λj (which all in distinct blocks) can be changed to the repetition jλj; that was calculated λj! times. λ = (2, 2, 1, 1, . . .) n\k 2 3 4 5 6 2 1 3 2 1 4 2 4 1 5 4 14 8 1 6 8 46 46 13 1 n\k 2 3 4 5 6 2 1 3 2 1 4 6 4 1 5 24 18 8 1 6 120 96 58 13 1 Sλ(n, k) for 2 ⩽n, k ⩽6 Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (2, 2, 2, . . .) n\k 2 3 4 5 6 2 1 3 2 1 4 2 2 1 5 4 14 8 1 6 4 32 38 12 1 n\k 2 3 4 5 6 2 1 3 2 1 4 6 4 1 5 24 18 8 1 6 120 96 58 12 1 Sλ(n, k) for 2 ⩽n, k ⩽6 Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (3, 2, 1, . . .) n\k 3 4 5 6 3 1 4 3 1 5 6 6 1 6 18 30 11 1 n\k 3 4 5 6 3 1 4 3 1 5 12 6 1 6 60 36 11 1 Sλ(n, k) for 3 ⩽n, k ⩽6 Cλ(n, k) for 3 ⩽n, k ⩽6. Table 2: Some tables for Sλ(n, k), Cλ(n, k).1 λ = (2, 2, 1, 1, . . .) λ = (2, 2, 1, 1, . . .) n\k 2 3 4 5 6 2 1 3 2 1 4 2 4 1 5 4 14 8 1 6 8 46 46 13 1 n\k 2 3 4 5 6 2 1 3 2 1 4 6 4 1 5 24 18 8 1 6 120 96 58 13 1 Sλ(n, k) for 2 ⩽n, k ⩽6 Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (2, 2, 2, . . 6.3 Main properties Let us define (x)0 := 1; (x)m := x(x −1) · · · (x −m + 1); (x)λ n := (x)λ0 · · · (x)λqn(x)rn. Denote Dℓ:= xℓ∂ℓ, then Denote Dℓ:= xℓ∂ℓ, then Dℓ1Dℓ2 = Dℓ2Dℓ1 Dℓ1Dℓ2 = Dℓ2Dℓ1 or more precisely or more precisely Dℓ1Dℓ2 = X i i! ℓ1 i ℓ2 i  Dℓ1+ℓ2−i. (11) (11) 1Sλ(n, k) = Cλ(n, k) = 0 if k < λ1 or k > n. If λ = (1, 1, . . .) or λ = (r, 1, 1, . . .) then the λ-Stirling numbers are referred to the ordinary Stirling numbers (A008275, A008277, A048994 in OEIS) or to the r-Stirling numbers (A143494, A143495, A143496, A193685, A143491, A143492, A143493 in OEIS [28]), respectively. Some values of Sλ(n, k) (of second kind) are referred to A078739, A008297, A035342, A078740, A078741, A090214 in OEIS [28]. the electronic journal of combinatorics 22(4) (2015), #P4.10 17 The last formula gives Dℓ= Dℓ−1D1 −(ℓ−1)Dℓ−1. (12) Dℓ= Dℓ−1D1 −(ℓ−1)Dℓ−1. (12) (12) Dℓ= Dℓ−1D1 −(ℓ−1)Dℓ−1. Let Dλ n be the operator defined as Let Dλ n be the operator defined as Dλ n := Dλ0 · · · DλqDr. Dλ n := Dλ0 · · · DλqDr. Similar to equation (12) we can obtain that Similar to equation (12) we can obtain that Similar to equation (12) we can obtain that Dλ n = Dλ n−1D1 −rn−1Dλ n−1. (13) (13) Theorem 6.3. The numbers Sλ(n, k), Cλ(n, k) have the following properties. (i) Recurrence relations Theorem 6.3. The numbers Sλ(n, k), Cλ(n, k) have the following properties. (i) Recurrence relations Sλ(n, k) = Sλ(n −1, k −1) + (k −rn−1)Sλ(n −1, k) (14) with Sλ(n, k) = 0 if k < λ1 or k > n; Sλ(n, n) = 1. Cλ(n, k) = Cλ(n −1, k −1) + (n −1 −rk)Cλ(n −1, k) (15) (14) (15) with Cλ(n, k) = 0 if k < λ1 or k > n; Cλ(n, n) = 1. (ii) Expansions with differential operators with Cλ(n, k) = 0 if k < λ1 or k > n; Cλ(n, n) = 1. (ii) Expansions with differential operators with Cλ(n, k) = 0 if k < λ1 or k > n; Cλ(n, n) = 1. (ii) Expansions with differential operators Dλ n = X k Sλ(n, k)Dk, (16) Dn = X k (−1)n−kCλ(n, k)Dλ k. 6.3 Main properties (17) Dλ n = X k Sλ(n, k)Dk, (16) (16) k Dn = X k (−1)n−kCλ(n, k)Dλ k. (17) Dn = X k (−1)n−kCλ(n, k)Dλ k. (17) (iii) Polynomial expansions (iii) Polynomial expansions (iii) Polynomial expansions (x)λ n = n X k=0 Sλ(n, k)(x)k, (18) (x)λ n = n X k=0 Sλ(n, k)(x)k, (18) (x)n = n X k=0 (−1)n−kCλ(n, k)(x)λ k (19) (x)λ n = n X k=0 Sλ(n, k)(x)k, (x)λ n = n X k=0 Sλ(n, k)(x)k, (x)n = n X k=0 (−1)n−kCλ(n, k)(x)λ k (18) (x)n = n X k=0 (−1)n−kCλ(n, k)(x)λ k (19) (iv) Orthogonality relations (iv) Orthogonality relations X k (−1)k−mSλ(n, k)Cλ(k, m) = δn,m, X k (−1)k−mCλ(n, k)Sλ(k, m) = δn,m. (20) (21) (v) Symmetric functions related formulas 2 (v) Symmetric functions related formulas 2 (v) Symmetric functions related formulas 2 Sλ(n, k) = X 1⩽i1⩽···⩽in−k⩽k n−k Y j=1 (ij −rij+j−1), (22) 2These are related to complementary symmetric functions studied in [9]. 2These are related to complementary symmetric functions studied in [9]. 2These are related to complementary symmetric functions studied in [9]. the electronic journal of combinatorics 22(4) (2015), #P4.10 the electronic journal of combinatorics 22(4) (2015), #P4.10 18 Cλ(n, k) = X 1⩽i1<···<in−k⩽n n−k Y j=1 (ij −rij+1−j). (23) (23) (vi) The general formula for Sλ(n, k) is given by (vi) The general formula for Sλ(n, k) is given by Sλ(n, k) = 1 k! k X ℓ=0 k ℓ  (−1)k−ℓ(ℓ)λ n. (24) (24) (vii) The following recurrence relations hold (viii) The formulas (viii) The formulas (viii) The formulas Sλ(n, k) = X j1,...,jqn+1  rn jqn+1  (k −jqn+1)rn−jqn+1 qn Y ℓ=1 λℓ jℓ  (k −jℓ−· · · −jqn+1)λℓ−jℓ. (27) (27) Cλ(n, k) = X j1,...,jqk+1 jqk+1 rk  (n −rk −1)jqk+1−rk qk Y ℓ=1 jℓ λℓ  (n −jℓ−· · · −jqk+1 −rℓ−1)jℓ−rℓ. ( ) j1,...,jqk+1   ℓ=1   (28) (28) the electronic journal of combinatorics 22(4) (2015), #P4.10 (vii) The following recurrence relations hold Sλ(n, k) = rn X j=0 rn j  (k −j)rn−jSλ(n −rn, k −j), (25) (25) Cλ(n, k) = n X j=rk  j rk  (n −rk −1)j−rkCλ(n −j, k −rk). (26) (26) Proof. (i) Recurrence relations. We can show that the number of described partitions has the same recurrence as (14). Note that the number of ways is 0 when k < λ1 or k > n. If we consider the element n, then two cases are possible. If n forms a separate block, then we have the number of ways to partition [n −1] into k −1 parts over partition λ. If n if placed in the block with some other elements except the restricted; this can be done in (k −rn−1) ways of any of partitions of [n −1] into k blocks over partitions λ. This argument implies the needed recurrence for Sλ(n, k). We show that the described number of ways satisfies the same recurrence as (15). Note that the number of ways is 0 when k < λ1 or k > n. Consider the element n and two cases. If n form a singleton separate cycle, then the number of corresponding ways is the number of permutations of [n −1] having k −1 cycles with the properties for λ partition of k −1. If n is in cycle with the other elements, then we can put n in cycles after any element except last rk singletons. This gives (n −1 −rk) ways for any permutation of [n −1] with k cycles and the described partition property. This argument clearly implies the needed recurrence for Cλ(n, k). 19 the electronic journal of combinatorics 22(4) (2015), #P4.10 (ii) Expansions with differential operators. The first relation n Dλ n = n X k=0 Sλ(n, k)Dk implies from our general Theorem 4.1 on graph partitions. From equation (12) we may easily obtain that implies from our general Theorem 4.1 on graph partitions. From equation (12) we may easily obtain that implies from our general Theorem 4.1 on graph partitions. g g From equation (12) we may easily obtain that Dλ n = Dλ n−1D1 −rn−1Dλ n−1. tronic journal of combinatorics 22(4) (2015), #P4.10 20 Proof. (i) Recurrence relations. For the companion expansion if for we know for n −1 For the companion expansion if for we know for n −1 Dn−1 = n−1 X k=0 (−1)n−1−kCλ(n −1, k)Dλ k, then for n we get then for n we get Dn = Dn−1D1 −(n −1)Dn−1 = n−1 X k=0 (−1)n−1−kCλ(n −1, k)Dλ kD1 − n−1 X k=0 (−1)n−1−k(n −1)Cλ(n −1, k)Dλ k = n−1 X k=0 (−1)n−1−kCλ(n −1, k)(Dλ k+1 + rkDλ k) − n−1 X k=0 (−1)n−1−k(n −1)Cλ(n −1, k)Dλ k = n X k=0 (−1)n−k(Cλ(n −1, k −1) + (n −1 −rk)Cλ(n −1, k))Dλ k = n X k=0 (−1)n−kCλ(n, k)Dλ k. (iii) Polynomial expansions. (vii) Recurrence relations. (vii) Recurrence relations. (vii) Recurrence relations. Recurrence (25). Suppose that j elements of the last rn in [n] are singleton blocks. We can choose these elements in rn j  ways. The remaining (rn −j) elements should be Recurrence (25). Suppose that j elements of the last rn in [n] are singleton blocks. We can choose these elements in rn j  ways. The remaining (rn −j) elements should be put in distinct (k −j) blocks of any of Sλ(n −rn, k −j) partitions, which can be done (k −j)rn−j times. Recurrence (26). We may choose the needed rk singleton cycles from the last j elements of [n]. This can be done in j rk  ways. The remaining (j −rk) elements should be put in the first (n −j) cycles of any of Cλ(n −j, k −rk) permutations, which can be done (n −rk −1)j−rk times. j k i) The last formulas (27), (28) imply from iterative use of relations (25), (26). j k (viii) The last formulas (27), (28) imply from iterative use of relations (25), (2 j (viii) The last formulas (27), (28) imply from iterative use of relations (25), (26). [1] B. R. Alspach and N. J. Pullman. Path decompositions of digraphs. Bull. Austral. Math. Soc., 10:421–427, 1974. (iii) Polynomial expansions. (iii) Polynomial expansions. Applying the derivation operation to the function xt with a real parameter t, expan- ( ) ( ) (iii) Polynomial expansions. Applying the derivation operation to the function xt with a real parameter t, expan- sions (16), (17) yield (t)λ0 · · · (t)λqn(t)rnxt = n X k=0 Sλ(n, k)(t)kxt, (t)nxt = n X k=0 (−1)n−kCλ(n, k)(t)λ0 · · · (t)λqk(t)rkxt. The last two identities are polynomial relations in t and hold for all t which imply (18), (19). The last two identities are polynomial relations in t and hold for all t which imply (18), (19) ) (iv) Orthogonality relations directly imply from the inverse expansions (16), (17) ( ) (iv) Orthogonality relations directly imply from the inverse expansions (16), (17). 20 the electronic journal of combinatorics 22(4) (2015), #P4.10 (v) Symmetric function related formulas can be easily obtained by induction and use of the recurrence relations (14), (15). (v) Symmetric function related formulas can be easily obtained by induction and use of the recurrence relations (14), (15). (vi) The general formula for Sλ(n, k). We will show that this formula holds using combinatorial interpretation of Sλ(n, k) and the inclusion-exclusion principle. Suppose that blocks are ordered. Let us enumerate them as 1, . . . , k. Denote by Ai (1 ⩽i ⩽k) the set of corresponding restricted (up to λ) arrangements of [n] into k ordered blocks such that the i-th block is empty. Let A be the number of all restricted arrangements of [n] into k blocks (some of them might be empty). Then it is clear that k!Sλ(n, k) = A −|A1 ∪· · · ∪Ak|. Note that Note that A = (k)λ1 . . . (k)λqn(k)rn A = (k)λ1 . . . (k)λqn(k)rn and |Ai1 ∩· · · ∩Aik−ℓ| = (ℓ)λ1 . . . (ℓ)λqn(ℓ)rn for any set of indices 1 ⩽i1 < · · · < ik−ℓ⩽k. Therefore, using the inclusion-exclusion principle we get for any set of indices 1 ⩽i1 < · · · < ik−ℓ⩽k. Therefore, using the inclusion-exclusion principle we get k!Sλ(n, k) = A −|A1 ∪· · · ∪Ak| = (k)λ1 . . . (k)λqn(k)rn − k−1 X ℓ=0 X 1⩽i1<···<ik−ℓ⩽k (−1)k−1−ℓ|Ai1 ∩· · · ∩Aik−ℓ| = (k)λ1 . . . (k)λqn(k)rn − k−1 X ℓ=0 (−1)k−1−ℓ k ℓ  (ℓ)λ1 . . . (ℓ)λqn(ℓ)rn = k X ℓ=0 (−1)k−ℓ k ℓ  (ℓ)λ1 . . . (ℓ)λqn(ℓ)rn. (vii) Recurrence relations. (vii) Recurrence relations. References [1] B. R. Alspach and N. J. Pullman. Path decompositions of digraphs. Bull. Austral. Math. Soc., 10:421–427, 1974. 21 the electronic journal of combinatorics 22(4) (2015), #P4.10 [2] P. Balister. Packing digraphs with directed closed trails. Combin. Probab. Comput., 121–15, 2003. [3] P. Blasiak and P. Flajolet. Combinatorial models of creation-annihilation. S´eminaire Lotharingien de Combinatoire, 65:Article B65c, 2011. [4] P. Blasiak, K. A. Penson, and A. I. Solomon. The boson normal ordering problem and generalized Bell numbers. Ann. Comb., 7:127–139, 2003. [5] A. Z. Broder. The r-Stirling numbers. Discrete Math., 49:241–259, 1984. [6] W. Chen, E. Deng, and R. Du. Reduction of m-regular noncrossing partitions. European J. Combin., 26:237–243, 2005. [7] P. Codara, O. M. D’Antona, and P. Hell. A simple combinatorial interpretation of certain generalized Bell and Stirling numbers. Discrete Math., 318:53–57, 2014. [8] F. R. K. Chung and R. L. Graham. Recent results in graph decompositions. Combi- natorics (HNV Temperley, ed.), London Math. Soc., Lecture Notes Series, 52:103– 124, 1981. [9] E. Damiani, O. M. D’Antona, and D. E. Loeb. The complementary symmetric functions: Connection constants using negative sets. Adv. Math., 135:207–219, 1998. [10] O. M. D’Antona and E. Munarini. A combinatorial interpretation of the connection constants for persistent sequences of polynomials. European J. Combin., 26:1105– 1118, 2005. [11] B. S. El-Desouky. The multiparameter non-central Stirling numbers. Fibonacci Quart, 32:218–225, 1994. [12] B. S. El-Desouky, N. P. Cakic, and T. Mansour. Modified approach to generalized Stirling numbers via differential operators. Appl. Math. Lett., 23:115–120, 2010. [13] A. Donald. An upper bound for the path number of a graph. J. Graph Theory, 4:189–201, 1980. [14] B. Duncan and R. Peele. Bell and Stirling numbers for graphs. J. Integer Seq. 12:Article 09.7.1, 2012 [15] A. Dzhumadil’daev and D. Yeliussizov. Path decompositions of digraphs and their applications to Weyl algebra. Adv. in Appl. Math., 67:36–54, 2015. [16] G. Fan. Path decompositions and Gallai’s conjecture. J. Combin. Theory Ser. B, 93:117–125, 2003. [17] P. Harding and S. McGuinness. Gallai’s Conjecture For Graphs of Girth at Least Four. J. Graph Theory, 75:256–274, 2014. [18] J. Katriel and M. Kibler. Normal ordering for deformed boson operators and operator-valued deformed Stirling numbers. Journal of Physics A: Mathematical and General, 25:2683, 1992. [19] L. Lov´asz. On covering of graphs. in Theory of Graphs, Proc. Coll. Tihany, 1966. 22 the electronic journal of combinatorics 22(4) (2015), #P4.10 [20] T. Mansour, M. Schork, and M. Shattuck. References On a new family of generalized Stirling and Bell numbers. Electron. J. Combin., 18:#P77, 2011. [21] T. Mansour, M. Schork and M. Shattuck. The generalized Stirling and Bell numbers revisited. J. Integer Seq., 15:Article 12.8.3, 2012. [22] T. Mansour and M. Schork. Commutation Relations, Normal Ordering, and Stirling Numbers. CRC Press, 2015. [23] C. J. H. McDiarmid. Path-partition structures of graphs and digraphs. Proc. Lond. Math. Soc., 3.4:750–767, 1974. [24] M. A. M´endez, P. Blasiak, and K. A. Penson. Combinatorial approach to generalized Bell and Stirling numbers and boson normal ordering problem. J. Math. Phys., 46:083511-1-8, 2005. [25] M. Mihoubi and M.S. Maamra. The (r1, . . . , rp)-Stirling numbers of the second kind. Integers, 12:#A35, 2012. [26] G. Olshanski. Representations of infinite-dimensional classical groups, limits of enveloping algebras, and Yangians. Topics in representation theory, Advances in Soviet Mathematics (A. Kirillov, ed.), vol. 2, AMS, Providence, RI, 1–66, 1991. [27] A. Okounkov. Young basis, Wick formula, and higher Capelli identities. Int. Math. Res. Not., 1996.17: 817-839, 1996. [28] The Online Encyclopedia of Integer Sequences (OEIS), http://oeis.org [29] R. Stanley. Enumerative Combinatorics, Vol. 1. Cambridge University Press, Cam- bridge, 2011. [30] A. Varvak. Rook numbers and the normal ordering problem. J. Combin. Theory Ser. A, 112:292–307, 2005. the electronic journal of combinatorics 22(4) (2015), #P4.10 23 the electronic journal of combinatorics 22(4) (2015), #P4.10
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Shape of Testosterone
˜The œjournal of physical chemistry letters
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Shape of Testosterone Article Recommendations * Supporting Information * sı * ABSTRACT: We have successfully characterized the structure of testosterone, one of the essential steroids, through high-resolution rotational spectroscopy. A single conformer has been detected, and a total of 404 transitions have been fitted, allowing a precise determination of the rotational constants. It allowed us to unravel that the isolated structure of testosterone adopts an extended disposition. The results obtained in this work highlight how using laser ablation techniques in combination with Fourier transform microwave techniques allow the study of large biomolecules or common pharmaceuticals. It is an important step toward studying relevant biomolecules and developing new analytical techniques with unprecedented sensitivity and resolution. T estosterone (17β-hydroxyandrost-4-en-3-one, mp = 155 °C), shown in Scheme 1, is one of the most relevant T magnetic resonance (NMR) spectroscopic studies have been conducted.5,6 These high-resolution studies conducted under the solid-state 13C NMR technique show the existence of two species. However, the authors highlight how care should be taken when transferring conformational information from crystalline to the solution states,6 since the conformational landscape of a molecule can be perturbed due to the surrounding solvent effects. An effective way of removing such effects is the use of gas-phase spectroscopic techniques, most notably supersonic expansions. The absence of any solvent provides the conformational panorama unbiased by perturbing agents.7−10 Scheme 1. Chemical Structure of Testosteronea aThe labels a−d are used to reference the different rings in refs 5 and 6. Scheme 1. Chemical Structure of Testosteronea Scheme 1. Chemical Structure of Testosteronea aThe labels a−d are used to reference the different rings in refs 5 and 6. A significant limitation occurring in gas-phase studies is the size of a molecule: as growing molecular size reduces the vapor pressure, i.e., the larger the molecular size and the lower the vapor pressure, more complex organic molecules cannot be driven into the gas phase. It is essential for biomolecules that cannot be studied using conventional heating methods due to their thermolability and low vapor pressure. One exception is estradiol, a vital steroid hormone. Its rotational spectrum has been recently reported, and three conformers have been characterized.11 In a first attempt, we tried to measure the rotational spectrum of testosterone through heating, but no spectral signature was obtained. Shape of Testosterone Iker León,* Elena R. Alonso, Santiago Mata, and José L. Alonso Cite This: J. Phys. Chem. Lett. 2021, 12, 6983−6987 Read Onlin ACCESS Metrics & More Article Recommendation The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Table 1. Experimental Spectroscopic Parameters for Testosterone’s Detected Conformer along with Plausible Configurations I and IIa Calculated at the B3LYP-GD3/6- 311++G(d,p) Level of Theory configuration I configuration II experimental 1 2 3 4 5 6 Ab 785.3463(11)h 789 787 788 667 665 666 B 168.66869(21) 168 168 169 182 182 182 C 153.72059(18) 153 153 153 171 171 172 |μa| observed 3.4 3.8 4.9 3.0 3.6 4.4 |μb| observed 0.8 0.7 0.6 0.5 1.2 0.9 |μc| observed 1.1 2.8 0.8 2.1 3.6 1.4 σc 19.0 Nd 404 ΔEe 0 24 73 492 523 556 ΔEZPEf 0 45 64 546 595 598 ΔGg 0 67 62 596 664 642 aSee text for details. bA, B, and C represent the rotational constants (in MHz); μa, μb, and μc are the electric dipole moment components (in D). cRMS deviation of the fit (in kHz). dNumber of measured transitions. eEnergies (in cm−1) relative to the global minimum calculated at the B3LYP-GD3BJ/6-311++G(d,p) level of theory. fEnergies (in cm−1) relative to the global minimum, taking into account the zero-point energy (ZPE), calculated at the B3LYP- GD3BJ/6-311++G(d,p) level of theory. gGibbs energies (in cm−1) relative to the global minimum calculated at 298 K at the B3LYP- GD3BJ/6-311++G(d,p) level of theory. hStandard error in paren- theses in units of the last digit. pubs.acs.org/JPCL Letter Table 1. Experimental Spectroscopic Parameters for Testosterone’s Detected Conformer along with Plausible Configurations I and IIa Calculated at the B3LYP-GD3/6- 311++G(d,p) Level of Theory Table 1. Experimental Spectroscopic Parameters for Testosterone’s Detected Conformer along with Plausible Configurations I and IIa Calculated at the B3LYP-GD3/6- 311++G(d,p) Level of Theory methodology, we were successful in obtaining the first rotational spectrum of testosterone. We used our laser ablation chirped-pulse Fourier transform microwave (LA-CP-FTMW) spectrometer14,15 to obtain the microwave spectrum of testosterone in the 1.5−6.5 GHz frequency range as shown in Figure 1a. Obtaining the laser- configuration I configuration II experimental 1 2 3 4 5 6 Ab 785.3463(11)h 789 787 788 667 665 666 B 168.66869(21) 168 168 169 182 182 182 C 153.72059(18) 153 153 153 171 171 172 |μa| observed 3.4 3.8 4.9 3.0 3.6 4.4 |μb| observed 0.8 0.7 0.6 0.5 1.2 0.9 |μc| observed 1.1 2.8 0.8 2.1 3.6 1.4 σc 19.0 Nd 404 ΔEe 0 24 73 492 523 556 ΔEZPEf 0 45 64 546 595 598 ΔGg 0 67 62 596 664 642 b Figure 1. The Journal of Physical Chemistry Letters (a) Broadband spectrum of testosterone in the 1.5−6.5 GHz frequency region using the LA-CP-FTMW spectrometer, highlighting the a-type R-branch progressions (J' ←J) originated by a single conformer of testosterone. (b) A comparison between a selected range of the experimental spectrum with the simulated one, with selected rotational transitions (J' ́K'a,K'c ←JKa,Kc). As can be seen, there is an excellent agreement between theory and experiment. aSee text for details. bA, B, and C represent the rotational constants (in MHz); μa, μb, and μc are the electric dipole moment components (in D). cRMS deviation of the fit (in kHz). dNumber of measured transitions. eEnergies (in cm−1) relative to the global minimum calculated at the B3LYP-GD3BJ/6-311++G(d,p) level of theory. fEnergies (in cm−1) relative to the global minimum, taking into account the zero-point energy (ZPE), calculated at the B3LYP- GD3BJ/6-311++G(d,p) level of theory. gGibbs energies (in cm−1) relative to the global minimum calculated at 298 K at the B3LYP- GD3BJ/6-311++G(d,p) level of theory. hStandard error in paren- theses in units of the last digit. To help identify the observed rotamer of testosterone, we performed a conformational search using molecular mechanics methods. A total of 8 molecular structures were screened within an energy window of 2500 cm−1. These structures were reoptimized using DFT methods (B3LYP-GD3/6-311++G- (d,p)), and the six low-energy structures, those below 1000 cm−1, belong to the two configurations shown in Figure 2. In configuration I, rings b and c (see Scheme 1) adopt a chair conformation, while ring a takes a half-chair disposition. Ring a is almost in the same plane as rings b, c, and d, and the molecule adopts an extended form. Configuration II is in a semifolded arrangement shown in Figure 2b, with the a ring almost perpendicular to the b−c−d rings’ plane. The three lowest-energy conformers of either configuration differ in the hydroxyl group’s orientation for each configuration, as indicated in Figure S1. As shown in Table 1, the three conformers belonging to configuration I are considerably more stable than those belonging to configuration II, resulting in extra stability of 600 cm−1. Figure 1. (a) Broadband spectrum of testosterone in the 1.5−6.5 GHz frequency region using the LA-CP-FTMW spectrometer, highlighting the a-type R-branch progressions (J' ←J) originated by a single conformer of testosterone. (b) A comparison between a selected range of the experimental spectrum with the simulated one, with selected rotational transitions (J' ́K'a,K'c ←JKa,Kc). Shape of Testosterone This problem can be overcome by using laser ablation techniques, which have proven to be successful for many biomolecules.12,13 Using this steroids.1,2 It is a sex hormone that plays an essential role in the body. It regulates sex drive (libido), fat distribution, body hair, bone mass, muscle mass, and red blood cells in humans. It also plays a crucial role in sperm production and developing male reproductive tissues such as the testes and prostate. It is biosynthesized from cholesterol through a series of steps, and a small amount of circulating testosterone is transformed into estradiol, a form of estrogen. Though it is also present in females to a lesser extent, they are more sensitive to it. As men age, the testosterone levels decrease, producing less estradiol. Thus, changes attributed to testosterone deficiency might be partly or entirely due to the accompanying decline in estradiol. Additionally, testosterone is also used as a medication for breast cancer treatment in women and low testosterone levels in men. Received: June 2, 2021 Accepted: July 16, 2021 Published: July 20, 2021 https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 Received: June 2, 2021 Accepted: July 16, 2021 Published: July 20, 2021 Received: June 2, 2021 Accepted: July 16, 2021 Published: July 20, 2021 It is well-known that there is a direct relationship between a simple or macromolecule’s structure and its particular function or properties.3,4 Obtaining the precise structure of testosterone is therefore needed to understand its activity. So far, nuclear © 2021 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 6983 Letter Letter https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 The Journal of Physical Chemistry Letters The barriers to hydroxyl conformer interconversion are below 400 cm−1, suggesting that neon should be capable of relaxing structure 2 into structure 3 and the latter into structure 1. Nevertheless, at room temperature, testosterone should participate in a dynamic equilibrium of the three conformers. We unambiguously determined that the extended form of testosterone is the most stable configuration. Subsequently, this knowledge allows for some conclusions about its biological activity due to the structure−property correspondence. Explanation of epimers’ activity differences is likely to be related to the substituents’ gross spatial orientation rather than to subtle conformational changes in the steroid skeleton.6 From our results in Figure 2, it is clear that the semifolded form of testosterone, in which ring a is out of plane from the rest of the structure, is not suitable to establish a hydrogen bond between subsequent testosterone molecules. Further- more, it would not allow direct interactions between stacked molecules, thus precluding van der Waals interactions between different ribbons. The extended form, on the other hand, allows both types of interactions. It is in excellent agreement y Having deduced a plausible assignment of testosterone’s observed rotamer, we conclude the spectral analysis by explaining why we observe only a single conformer, turning our attention to the properties of a supersonic expansion. By examining the behavior of a variety of molecules, Ruoffet al. noted that, when a low potential energy barrier separates the conformational species, the noble-gas collisions in the initial stages of the expansion provide the energy required for their interconversion.19,20 We used theoretical calculations to perform the relaxed potential energy surface (PES) scan shown in Figure 3 by rotating the C−C−O−H dihedral angle. The barriers to hydroxyl conformer interconversion are below 400 cm−1, suggesting that neon should be capable of relaxing structure 2 into structure 3 and the latter into structure 1. Nevertheless, at room temperature, testosterone should participate in a dynamic equilibrium of the three conformers. b l d d h h d d f f Figure 3. Relaxed PES rotating the C−C−O−H dihedral angle of the three lowest-energy conformers of testosterone within configuration I. The low barrier separating them explains why a single conformer is seen. A close-up view of the hydroxyl group position is shown, while a larger view of the structures can be found in the SI. The Journal of Physical Chemistry Letters The Journal of Physical Chemistry Letters The Journal of Physical Chemistry Letters Letter pubs.acs.org/JPCL Figure 2. A comparison between the two plausible configurations of testosterone. A top and side view of (a) structure 1 within the extended configuration and (b) structure 4 within the semifolded configuration. For each configuration, three conformers are possible differing in the hydroxyl group’s orientation, as indicated by the green arrow. Figure 2. A comparison between the two plausible configurations of testosterone. A top and side view of (a) structure 1 within the extended configuration and (b) structure 4 within the semifolded configuration. For each configuration, three conformers are possible differing in the hydroxyl group’s orientation, as indicated by the green arrow. Figure 3. Relaxed PES rotating the C−C−O−H dihedral angle of the three lowest-energy conformers of testosterone within configuration I. The low barrier separating them explains why a single conformer is seen. A close-up view of the hydroxyl group position is shown, while a larger view of the structures can be found in the SI. are directly connected with the selection rules and intensity of the observed transitions. We estimate relative intensities of 6.5:1.0:1.5 for the a-, b-, anc c-type transitions, respectively. The intensity of the a-type transitions is significantly larger than those of b- and c-type in good agreement with the dipole moment selection rules predicted for structures 1 and 3 but in sharp contrast to intense c-type transitions expected for structure 2, which therefore can be ruled out. The distinction between structures 1 and 3 is not definitive. However, calculations using both DFT and MP2 methods predict structure 1 to be the global minimum and, therefore, is most likely the detected structure of testosterone. y Having deduced a plausible assignment of testosterone’s observed rotamer, we conclude the spectral analysis by explaining why we observe only a single conformer, turning our attention to the properties of a supersonic expansion. By examining the behavior of a variety of molecules, Ruoffet al. noted that, when a low potential energy barrier separates the conformational species, the noble-gas collisions in the initial stages of the expansion provide the energy required for their interconversion.19,20 We used theoretical calculations to perform the relaxed potential energy surface (PES) scan shown in Figure 3 by rotating the C−C−O−H dihedral angle. https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 The Journal of Physical Chemistry Letters As can be seen, there is an excellent agreement between theory and experiment. ablated rotational spectrum of such a large biomolecule is not easy and requires careful control of the experimental parameters as well as its fragmentation to be minimized.12 As can be seen, the spectrum shows very intense rotational lines. Initially, lines corresponding to known photofragment species and water clusters were identified and removed.14,16,17 The remaining rotational spectrum still showed many rotational transitions, subsequently attributed to testosterone. At first glance, the characteristic pattern of an a-type R-branch progression arising from a dominant rotameric species (see Figure 1a) was quickly identified. The first set of rotational constants determined from a rigid rotor analysis18 helped us quickly locate b- and c-type transitions with new predictions. A total of 404 rotational transitions were assigned and measured (Table S1 of the Supporting Information, SI), allowing us to determine very accurate values of the rotational constants. Figure 1b shows excellent matching between experimental and simulated spectra using the fitted values, which are listed in the first column of Table 1. After the rotational lines of this rotamer were removed, no significant signals remained unassigned in the spectrum. Therefore, no spectral searches for other conformational candidates were conducted. We used two different indicators for conformer identi- fication: rotational constants and dipole moment components. The rotational constants provide information on the rotamer’s mass distribution and are fundamental in obtaining conforma- tional structures. The comparison of Table 1 between the experimental rotational constants with the theoretically predicted values unambiguously assigns the observed rotamer to one of the three conformers of configuration I, i.e., the extended form. Unfortunately, the values of the rotational constants are not able to discern between the three structures 1, 2, and 3 that differ only in the orientation of the terminal hydroxyl group. This small difference does not cause a significant change in the mass distribution and, consequently, in the rotational constants’ values. The missing information can be obtained from the dipole moment components, which 6984 The Journal of Physical Chemistry Letters with the crystal testosterone’s structural arrangement:6 The testosterone’s unit cell consists of two extended parallel ribbons of the molecules extended into a sheet. These ribbons are hydrogen-bonded sequentially between the hydroxyl group of a molecule and the next molecule’s ketone group. Strong van der Waals interactions bind both ribbons together, which are in a head-to-tail disposition. It is important to note that these results correlate very well with the arrangement observed in steroids in the condensed phase.21,22 We unambiguously determined that the extended form of testosterone is the most stable configuration. Subsequently, this knowledge allows for some conclusions about its biological activity due to the structure−property correspondence. Explanation of epimers’ activity differences is likely to be related to the substituents’ gross spatial orientation rather than to subtle conformational changes in the steroid skeleton.6 From our results in Figure 2, it is clear that the semifolded form of testosterone, in which ring a is out of plane from the rest of the structure, is not suitable to establish a hydrogen bond between subsequent testosterone molecules. Further- more, it would not allow direct interactions between stacked molecules, thus precluding van der Waals interactions between different ribbons. The extended form, on the other hand, allows both types of interactions. It is in excellent agreement An interesting comparison is that of testosterone with estradiol. Estradiol is similar to testosterone but with ring a differing considerably: the methyl group in ring a is lost and it has a hydroxyl group instead of the ketone group, leaving ring a as a phenolic ring. This change makes estradiol lose the two possible ring configurations in testosterone, which is now planar. Effectively, the structure of estradiol has a single configuration but six possible conformers that differ in the hydroxyl groups’ orientations. The rotational spectrum of estradiol11 shows two predominant structures that are similar 6985 Letter pubs.acs.org/JPCL The Journal of Physical Chemistry Letters Letter Santiago Mata −Grupo de Espectrocopía Molecular (GEM), Edificio Quifima, Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, Parque Científico UVa, Universidad de Valladolid, 47011 Valladolid, Spain; to our characterized structure but differing in the two possible orientations of the hydroxyl group in ring a. This is another point supporting our assignment. Additionally, a third conformer is found, which is much weaker than expected due to conformational interconversion. It further confirms our assignment and discussion about conformational interconver- sion. * sı Supporting Information * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.1c01743. (5) Hayamizu, K.; Kamo, O. Complete Assignments of the 1H and 13C NMR Spectra of Testosterone and 17α-methyltestosterone and the 1H Parameters Obtained from 600 MHz Spectra. Magn. Reson. Chem. 1990, 28, 250−256. Detailed experimental and theoretical section; detailed view of the configurations of testosterone (Figure S1); measured frequencies for the 404 transitions of the testosterone’s detected rotamer (Table S1) (PDF) (6) Roberts, P. J.; Pettersen, R. C.; Sheldrick, G. M.; Isaacs, N. W.; Kennard, O. Crystal and Molecular Structure of 17β-Hydroxyandrost- 4-En-3-One (Testosterone). J. Chem. Soc., Perkin Trans. 2 1973, 2, 1978−1984. (7) Robertson, E. G.; Simons, J. P. Getting into Shape: Conforma- tional and Supramolecular Landscapes in Small Biomolecules and Their Hydrated Clusters. Phys. Chem. Chem. Phys. 2001, 3, 1−18. Corresponding Author Iker León −Grupo de Espectrocopía Molecular (GEM), Edificio Quifima, Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, Parque Científico UVa, Universidad de Valladolid, 47011 Valladolid, Spain; orcid.org/0000-0002-1992-935X; Email: Iker.leon@ uva.es Iker León −Grupo de Espectrocopía Molecular (GEM), Edificio Quifima, Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, Parque Científico UVa, Universidad de Valladolid, 47011 Valladolid, Spain; id /0000 0002 1992 935X E il Ik l @ (9) Oomens, J.; Steill, J. D.; Redlich, B. Gas-Phase IR of Deprotonated Amino Acids. J. Am. Chem. Soc. 2009, 131, 4310−4319. ( ) (10) Caminati, W. Nucleic Acid Bases in the Gas Phase. Angew. Chem., Int. Ed. 2009, 48, 9030−9033. orcid.org/0000-0002-1992-935X; Email: Iker.leon@ uva.es (11) Zinn, S.; Schnell, M. Flexibility at the Fringes: Conformations of the Steroid Hormone β-Estradiol. ChemPhysChem 2018, 19, 2915− 2920. ■AUTHOR INFORMATION (8) De Vries, M. S.; Hobza, P. Gas-Phase Spectroscopy of Biomolecular Building Blocks DFT: Density-Functional Theory. Annu. Rev. Phys. Chem. 2007, 58, 585−612. Notes The authors declare no competing financial interest. The authors declare no competing financial interest. The Journal of Physical Chemistry Letters Comparing both steroids is interesting, because the characterized predominant species are very similar but have entirely different biological functionalities. Because the only structural difference is ring a, it must be responsible for their ligand−protein binding in different receptors. The variation from a carbonyl to a hydroxyl group probably forces a binding with a receptor capable of a proton donor or proton acceptor, respectively. Additionally, the receptor must be ready to adapt to the dispersive forces or sterical effects caused by the methyl group in testosterone. This comparison is another illustrative example of the importance of the structure−property relation- ship. Santiago Mata −Grupo de Espectrocopía Molecular (GEM), Edificio Quifima, Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, Parque Científico UVa, Universidad de Valladolid, 47011 Valladolid, Spain; orcid.org/0000-0002-1892-5015 José L. Alonso −Grupo de Espectrocopía Molecular (GEM), Edificio Quifima, Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, Parque Científico UVa, Universidad de Valladolid, 47011 Valladolid, Spain; orcid org/0000 0002 3146 8250 José L. Alonso −Grupo de Espectrocopía Molecular (GEM), Edificio Quifima, Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, Parque Científico UVa, Universidad de Valladolid, 47011 Valladolid, Spain; id /0000 0002 3146 8250 orcid.org/0000-0002-3146-8250 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jpclett.1c01743 ■REFERENCES (1) Mooradian, A. D.; Morley, J. E.; Korenman, S. G. Biological Actions of Androgens. Endocr. Rev. 1987, 8, 1−28. (1) Mooradian, A. D.; Morley, J. E.; Korenman, S. G. Biological Actions of Androgens. Endocr. Rev. 1987, 8, 1−28. (2) Rommerts, F. F. G. Testosterone: An Overview of Biosynthesis, Transport, Metabolism and Nongenomic Actions. Testosterone 1998, 1. (3) Lehninger, A.; Nelson, D.; Cox, M. Lehninger Principles of Biochemistry, 5th ed.; W. H. Freeman: New York, 2008. (4) Park, Y.; Helms, V. On the Derivation of Propensity Scales for Predicting Exposed Transmembrane Residues of Helical Membrane Proteins. Bioinformatics 2007, 23, 701−708. ■ACKNOWLEDGMENTS The financial fundings from the Ministerio de Ciencia e Innovación (CTQ2016-76393-P and PID2019-111396GB- I00), Junta de Castilla y León (Grants VA077U16 and VA244P2), and European Research Council under the European Union’s Seventh Framework Programme (FP/ 2007-2013)/ERC-2013-SyG, Grant Agreement n. 610256 NANOCOSMOS, are gratefully acknowledged. E.R.A. ac- knowledges the Ministerio de Ciencia e Innovación for a Juan de la Cierva postdoctoral grant (FJC2018-037320-I). In summary, we have been able to transfer testosterone, a large biomolecule, from its solid into the gas phase using laser ablation techniques. It has allowed us to characterize the structure of a relevant biomolecule such as testosterone by rotational spectroscopy for the first time. This technique is one of the most powerful spectroscopic techniques for structural determination due to the direct relation of shape and spectral position of 404 rotational lines. Interestingly, out of the two possible configurations, the extended form is the predominant one. Supersonic-jet analytical techniques combined with laser ablation can be used to determine a sample’s composition without dissolving the sample in a suitable medium that would usually require chemical manipulations. Additionally, there is no challenging or tedious preparation of the sample requiring lengthy analysis times. As we show, the sensitivity reached using our experimental procedure is sufficient to detect large biomolecules or common pharmaceuticals with unrivaled structural determination. https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 Authors (12) Alonso, E. R.; León, I.; Alonso, J. L. The Role of the Intramolecular Interactions in the Structural Behavior of Biomole- cules: Insights from Rotational Spectroscopy. Intra- and Intermolecular Interactions Between Non-covalently Bonded Species 2021, 93. Elena R. Alonso −Instituto Biofisika (UPV/EHU, CSIC), University of the Basque Country, 48940 Leioa, Spain; Departamento de Química Física, Facultad de Ciencia y Tecnología, Universidad del País Vasco, 48940 Leioa, Spain; orcid.org/0000-0001-5816-4102 (13) Mata, S.; Pena, I.; Cabezas, C.; López, J. C.; Alonso, J. L. A Broadband Fourier-Transform Microwave Spectrometer with Laser 6986 https://doi.org/10.1021/acs.jpclett.1c01743 J. Phys. Chem. Lett. 2021, 12, 6983−6987 Letter The Journal of Physical Chemistry Letters pubs.acs.org/JPCL The Journal of Physical Chemistry Letters Ablation Source: The Rotational Spectrum of Nicotinic Acid. J. Mol. Spectrosc. 2012, 280, 91−96. p (14) Cabezas, C.; Varela, M.; Alonso, J. L. The Structure of the Elusive Simplest Dipeptide Gly-Gly. Angew. Chem. 2017, 129, 6520− 6525. (15) León, I.; Alonso, E. R.; Cabezas, C.; Mata, S.; Alonso, J. L. Unveiling the N→π* Interactions in Dipeptides. Commun. Chem. 2019, 2, 3. (16) Peña, I.; Cabezas, C.; Alonso, J. L. The Nucleoside Uridine Isolated in the Gas Phase. Angew. Chem., Int. Ed. 2015, 54, 2991− 2994. (17) Bermúdez, C.; Mata, S.; Cabezas, C.; Alonso, J. L. Tautomerism in Neutral Histidine. Angew. Chem., Int. Ed. 2014, 53, 11015−11018. g (18) Pickett, H. M. The Fitting and Prediction of Vibration-Rotation Spectra with Spin Interactions. J. Mol. Spectrosc. 1991, 148, 371−377. (19) Ruoff, R. S.; Klots, T. D.; Emilsson, T.; Gutowsky, H. S. Relaxation of Conformers and Isomers in Seeded Supersonic Jets of Inert Gases. J. Chem. Phys. 1990, 93, 3142−3150. (20) Godfrey, P. D.; Brown, R. D.; Rodgers, F. M. The Missing Conformers of Glycine and Alanine: Relaxation in Seeded Supersonic Jets. J. Mol. Struct. 1996, 376, 65−81. (21) Herbert, R. B. Terpenes and Steroids. The Biosynthesis of Secondary Metabolites 1981, 50. y (22) Weeks, C. M.; Cooper, A.; Norton, D. A.; Hauptman, H.; Fisher, J. The Crystal and Molecular Structures of 5α-Androstan-3β- Ol-17-One and 5β-Androstane-3α,L7β-Diol. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 1971, 27, 1562−1572. 6987
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ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge
IACR transactions on cryptographic hardware and embedded systems
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Document status and date: Published: 31/08/2022 Document status and date: Published: 31/08/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. p • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. 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If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: Document license: CC BY Document license: CC BY DOI: 10.46586/tches.v2022.i4.527-552 Document status and date: Published: 31/08/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There ca important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visi DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication DOI: 10.46586/tches.v2022.i4.527-552 ECDSA White-Box Implementations Citation for published version (APA): Barbu, G., Beullens, W., Dottax, E., Giraud, C., Houzelot, A., Li, C., Mahzoun, M., Ranea, A., & Xie, J. (2022). ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge. IACR Transactions on Cryptographic Hardware and Embedded Systems, 2022(4), 527-552. https://doi.org/10.46586/tches.v2022.i4.527-552 Citation for published version (APA): Barbu, G., Beullens, W., Dottax, E., Giraud, C., Houzelot, A., Li, C., Mahzoun, M., Ranea, A., & Xie, J. (2022). ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge. IACR Transactions on Cryptographic Hardware and Embedded Systems, 2022(4), 527-552. https://doi.org/10.46586/tches.v2022.i4.527-552 Download date: 24. Oct. 2024 www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 IACR Transactions on Cryptographic Hardware and Embedded Systems ISSN 2569-2925, Vol. 2022, No. 4, pp. 527–552. DOI:10.46586/tches.v2022.i4.527-552 Guillaume Barbu1 , Ward Beullens2, Emmanuelle Dottax1 , Christophe Giraud1 , Agathe Houzelot1,3 , Chaoyun Li4 , Mohammad Mahzoun5, Adrián Ranea4 and Jianrui Xie4 1 IDEMIA, Cryptography & Security Labs, Pessac, France firstname.lastname@idemia.com 2 IBM Research, Zurich, Switzerland wbe@zurich.ibm.com 3 LaBRI, CNRS, Université de Bordeaux, Bordeaux, France 4 imec-COSIC, KU Leuven, Leuven, Belgium firstname.lastname@esat.kuleuven.be 5 Eindhoven University of Technology, Eindhoven, Netherlands m.mahzoun@tue.nl Abstract. Despite the growing demand for software implementations of ECDSA secure against attackers with full control of the execution environment, scientific literature on ECDSA white-box design is scarce. The CHES 2021 WhibOx contest was thus held to assess the state-of-the-art and encourage relevant practical research, inviting developers to submit ECDSA white-box implementations and attackers to break the corresponding submissions. In this work, attackers (team TheRealIdefix) and designers (team zerokey) join to describe several attack techniques and designs used during this contest. We explain the methods used by the team TheRealIdefix, which broke the most challenges, and we show the efficiency of each of these methods against all the submitted implementations. Moreover, we describe the designs of the two winning challenges submitted by the team zerokey; these designs represent the ECDSA signature algorithm by a sequence of systems of low-degree equations, which are obfuscated with affine encodings and extra random variables and equations. The WhibOx contest has shown that securing ECDSA in the white-box model is an open and challenging problem, as no implementation survived more than two days. In this context, our designs provide a starting methodology for further research, and our attacks highlight the weak points future work should address. Keywords: ECDSA · White-Box Cryptography · WhibOx Contest Keywords: ECDSA · White-Box Cryptography · WhibOx Contest Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15 Accepted: 2022-06-15 1 Introduction Cryptographic techniques are primarily designed to be secure in a context where the confidentiality of secret keys is ensured with black-box access to the algorithm – only inputs and outputs are available to the attacker. Confidence in security is built from detailed studies, carefully defined security notions, and security proofs. Such a strong level of confidence is now a standard expectation. However, real-life scenarios for implementations might jeopardize initial assumptions, where attackers have access to additional information via side channels (e.g., timing or power consumption) or can modify the algorithm execution Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15 Accepted: 2022-06-15 Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15 Accepted: 2022-06-15 Published: 2022-08-31 Published: 2022-08-31 528 Attacks and Designs from CHES 2021 Challenge and exploit faulty results. This is called the grey-box model. Developers have to put countermeasures in place to reach the originally expected security level. and exploit faulty results. This is called the grey-box model. Developers have to put countermeasures in place to reach the originally expected security level. In the context of mobile applications – contactless payments, cryptocurrency wallets, streaming services – or connected objects, devices often lack secure storage to protect secret keys, and their generally open execution environment exposes a large attack surface. This hostile environment is captured by the white-box model, which assumes an attacker having control of every aspect of the implementation: execution flow, memory content and addresses. The first white-box implementations were proposed in the early 2000s by Chow et al. [CEJvO02,CEJv03], and the field has continuously developed since then, with design proposals [BG03,BCD06,XL09,Kar11,DFLM18,RW19,SEL21,BCC21], attacks [BGEC04,GMQ07,WMGP07,MGH09,DWP10,DRP13,LRD+14,AMR19,GRW20] and efforts to define security notions [SWP09,DLPR14,AABM20]. The industry shows a growing interest in white-box cryptography owing to the widespread usage of security-related applications on connected devices. The WhibOx contest, attached to the CHES conference, has been held biennially since 2017 to encourage practical experiments both from the designer and attacker perspectives. It lasts several months, inviting coders to post white-box implementations and attackers to break them. Participants can remain anonymous and silent about any detail on their work. The first two editions in 2017 and 2019 focused on white-box implementations of AES and exhibited the community’s strong interest in this subject. Some candidates survived all attacks in the second edition in 2019, showing a certain maturity for this algorithm. 1Guillaume Barbu, Emmanuelle Dottax, Christophe Giraud and Agathe Houzelot are part of the team TheRealIdefix; Ward Beullens, Chaoyun Li, Mohammad Mahzoun, Adrián Ranea and Jianrui Xie compose the team zerokey. 2 Rules of the WhibOx 2021 Contest Designers were required to post challenges computing ECDSA signatures on the NIST P-256 curve under a hard-coded, freely chosen key, and accepting as input any 256-bit message digest e = H(m). Notice that the cryptographic hash function H is excluded from the intended white-box implementation of ECDSA and the message m is also not provided. At the same time, attackers were encouraged to extract the private keys. In addition, acceptance of submitted implementations was conditioned on some requirements: • the public key corresponding to the embedded private key, as well as a proof of knowledge of the private key, had to be provided, • submissions had to be source code in portable C, • linking to external libraries was forbidden, except for the GNU Multi Precision library [Gt20], • the signature algorithm had to be deterministic, • the execution time was limited to 3 seconds, the program size to 20 MB, and the RAM usage to 20 MB as well. • the execution time was limited to 3 seconds, the program size to 20 MB, and the RAM usage to 20 MB as well. There was an elaborate system with scoreboards to reward designers and attackers. A challenge gains strawberries as time goes by till broken. Challenges with a higher performance score (measured in terms of execution time, code size, and RAM usage) gain strawberries faster. Eventually, the challenge with the highest number of strawberries wins the competition. Accordingly, when submitting a matching private key to the system, attackers receive bananas, the number of which is determined by the number of strawberries of the challenge at the time of the break. More detailed information can be found on the contest website [CHE]. 1 Introduction Section 5 discloses the designs of Challenges 226 and 227 proposed by the team zerokey, and Section 6 concludes this paper. Outline. The paper is organized as follows. Section 2 outlines the rules of the WhibOx 2021 contest. Section 3 recalls the ECDSA algorithm and the state-of-the-art regarding white-box implementations. Section 4 presents the different methods that have been used by the team TheRealIdefix to break various implementations and some statistics regarding the success rate of these methods. Section 5 discloses the designs of Challenges 226 and 227 proposed by the team zerokey, and Section 6 concludes this paper. 1 Introduction In 2021, organisers changed the target and decided to consider the ECDSA signature algorithm, whose white-box implementation is of substantial interest to the industry but virtually lacks scientific literature. From May 17th to August 22nd 2021, 97 candidate implementations were submitted for scrutiny by 37 (teams of) attackers. All challenges were broken within 35 hours, suggesting the difficulty of achieving a secure white-box implementation of ECDSA. Thus, studying the attacks would help to discern weak points inside the implementations. Besides, the analysis of the design of the most resistant challenges, which successfully defeated most attackers, would also give directions for future designs. Contributions. In this paper, teams TheRealIdefix — who broke the most challenges — and zerokey — who proposed the two winning challenges — join to present how they proceeded during the contest1. On the attack side, we describe a strategy to achieve efficient attacks. As reverse engineering is a time-consuming task, automated attacks are desirable. We consider different attack paths against ECDSA white-boxes: the ones inherited from traditional cryptanalysis, the extensions of attacks in the grey-box model, and the logical attacks of the software. We discuss the feasibility of automating each attack path and provide detailed information regarding which attacks succeeded (or failed) on each candidate. Our results show that, with few exceptions, it was sufficient to fully recover the secret value by these automated attacks. On the design side, we describe the methodology we used to build the two winning challenges, Challenges 226 and 227. It includes modifying the implicit framework [RVP22] originally proposed for block ciphers, applying techniques from multivariate public-key cryptosystems, and obfuscating the resulting C code with a C obfuscator. Our design thus turns the ECDSA signature algorithm into a sequence of systems of low-degree equations which are obfuscated with large affine encodings and additional variables and equations. Finally, we show how to break Challenge 226 with automated attacks and how to break Challenge 227 once reverse-engineered. 529 Barbu et al. Outline. The paper is organized as follows. Section 2 outlines the rules of the WhibOx 2021 contest. Section 3 recalls the ECDSA algorithm and the state-of-the-art regarding white-box implementations. Section 4 presents the different methods that have been used by the team TheRealIdefix to break various implementations and some statistics regarding the success rate of these methods. 3.1 ECDSA In 1992, Vanstone introduced a variant of DSA based on elliptic curves. The result- ing public-key signature algorithm is called Elliptic Curve Digital Signature Algorithm (ECDSA) [Van92]. Its parameters are an elliptic curve E over a field Fq, a point G of prime order n, and a cryptographic hash function H. The private key d is randomly drawn from J1, n −1K, and the public key consists of the point Q = [d]G where [d]G corresponds to the scalar multiplication of the point G by the scalar d. The ECDSA signature is described in Algorithm 1, where Rx and Ry denote the coordinates of the point R. Note that the key d is not the only sensitive value in that scheme. Indeed, the recovery of the nonce k allows the computation of d from the signature (r, s) and the message m: d = (ks −H(m))r−1 mod n . (1) d = (ks −H(m))r−1 mod n . (1) Attacks and Designs from CHES 2021 Challenge 530 Algorithm 1: ECDSA signature Input : the message m Output : the signature (r, s) 1 e ←H(m) 2 k $←−J1, n −1K 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Return (r, s) 1 e ←H(m) 2 k $←−J1, n −1K 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Return (r, s) The nonce must not only remain secret but also differ for each execution of the algorithm. Indeed, an efficient way to recover its value is to find another signature (r′, s′) of a different message m′ ̸= m using the same nonce, that is with k′ = k. In that case, we also have r′ = r, so the adversary may compute k = (H(m′) −H(m))(s′ −s)−1 mod n . (2) (2) In the black-box model, the security of ECDSA is based on the difficulty of the Elliptic Curve Discrete Logarithm Problem (ECDLP), i.e., on the difficulty of computing the scalar k (resp. d) from the points G and R = [k]G (resp. Q = [d]G). 3.1 ECDSA To ensure that this problem is difficult to solve, there are several standards to define elliptic curves, e.g. [Loc10,Sta10,JOR11,FIP13]. However, there is a gap between the security of ECDSA in theory and that of ECDSA implementations. Many grey-box attacks have been described in the literature (see for example [FV12]). Some of them directly target the key d while others aim at recovering some information on the nonce k. As explained previously, the knowledge of the nonce allows an adversary to compute the secret key. Recovering a few bits of the nonces associated to different signatures may be enough for an attacker. Indeed, this allows the construction of a system of equations that can be solved using lattice-based algorithms [BH19,JSSS20] or Bleichenbacher’s FFT-based approach [ANT+20]. These bits could, for example, be recovered via side-channel analysis if the implementation is not protected or simply guessed if the nonce is not drawn uniformly at random. These attacks show that it is already complicated to achieve a secure implementation of ECDSA in the grey-box model, and of course, things get worse in the white-box context. 4 Breaking the Challenges White-box implementations usually rely on encodings and other theoretically sound approaches to protect the secret values and their manipulations. It is also very often the case that code obfuscation techniques are used to make understanding the design a time-consuming and challenging task. Extensive use of such obfuscation techniques in the submitted source files causes independently reverse-engineering each challenge to be overwhelming in time. We thus focused on designing attack methods that could be efficient and easily automated. This section looks at the different attacks that can be automated in a white-box context and gives the rationale for using and discarding them. We then present the results of applying the selected methods to the whole set of submissions. 3.2 White-box Implementation of ECDSA The white-box model assumes that the attacker has total access to the executable: he can read and modify it at will. He also has access to all the memory used during execution, so a white-box designer does not only have to protect his implementation against grey-box attacks but also against an adversary who can dump the memory and search for sensitive values such as k or d. The first technique to prevent secret data from appearing in plain was introduced by Chow et al. in [CEJv03]. Their idea is to embed the key into the algorithm, and each operation is performed with the help of look-up tables protected by carefully crafted encodings. Informally, the algorithm is split into low-level operations, and each operation op is replaced by f −1 ◦op ◦f ′, where f and f ′ are bijections called respectively input and output encodings. The drawback of this technique is that the required memory drastically increases with the algorithm’s complexity. Using it to secure operations as 531 Barbu et al. complex as scalar multiplications or inversions while remaining efficient is thus a real challenge. complex as scalar multiplications or inversions while remaining efficient is thus a real challenge. Another challenge in white-box cryptography is the impossibility of relying on any external source of randomness. An attacker could simply disable such a source and fix its output to a constant value. For example, in the context of AES, this renders some countermeasures against side-channel or fault attacks based on randomization techniques completely inefficient. When one considers ECDSA signatures, disabling the source of randomness yields multiple uses of the same nonce and, thus, easy recovery of the private key, as seen in Sect. 3.1. The solution is to compute k as a function of the only source of randomness available, the input message: k = f(m). In order to maintain the security of the signature scheme, this mapping must be computationally indistinguishable from what a randomly and uniformly chosen function would return. We will see in the next section that many challenges of the WhibOx competition did not fulfill this requirement. 2https://man7.org/linux/man-pages/man8/ld.so.8.html 4.1.1 Hooking Shared Libraries The contest rules were a clear incentive for developers to use the GMP library for big number arithmetic operations. A first attempt to break the submitted challenges was then to search if sensitive values were manipulated in clear by the GMP library. In order to perform this automatically, our approach has been to hook the calls to GMP functions thanks to the so-called LD_PRELOAD trick. Pre-loading is a feature of the dynamic linker on UNIX systems that allows loading a specific shared library before all other libraries linked to a given executable binary2. In our specific case, we built a shared library defining the same function as the GMP library (e.g. mpz_mul, mpz_mod or mpz_invert). Each of these functions simply updates a log of the given parameters before calling the real GMP function, explicitly using the dynamic linker (thanks to the <dlfcn.h> module) to ensure the correct execution of the white-box implementation. It is then only necessary to add our shared library to the LD_PRELOAD environment variable of the dynamic linker on our system before calling the ECDSA binary to have our custom functions called in place of the genuine GMP ones. The corresponding log is analysed in a second step to eventually reveal the secret key if d, k or related values such as r · d or e + r · d are found in the log. Such an approach allowed us to break 32% of the challenges. As a side note, this technique also jeopardizes implementations relying on system- dependent random generators such as srand or mpz_XrandomX functions, or on other sources such as time. 2https://man7.org/linux/man-pages/man8/ld.so.8.html 532 Attacks and Designs from CHES 2021 Challenge Attacks and Designs from CHES 2021 Challenge 4.1.2 Biased Nonces To sum up, the relations we used for our lattice attacks are the following (with ei ranging from 0 to 999): • assuming l = 6 known most- or least-significant bits of the ephemeral key: kmsb2L + klsb = s−1(e + rd) mod n , (3) (3) with L = 256 −l for the MSB case and L = l for the LSB case (we considered both cases where the known value is 0 or 63 = 26 −1), with L = 256 −l for the MSB case and L = l for the LSB case (we considered both cases where the known value is 0 or 63 = 26 −1), • assuming the ephemeral keys are ki = tκi:  t = κ−1 0 (e0 + r0d) mod n, κi = t−1(s−1 i ei −s−1 i rir−1 0 e0) + κ0(s−1 i rir−1 0 s0) mod n, (4) (4) with κi < 2248 and t an unknown constant scalar. with κi < 2248 and t an unknown constant scalar. with κi < 2248 and t an unknown constant scalar. Such an approach allowed us to break 72% of the challenges. 4.1.2 Biased Nonces As explained in Sect. 3.2, white-box designers usually generate the nonce k from the input. In the case of the WhibOx contest, the nonce is thus computed as a function of the hash, i.e. k = f(e). However, if the function f is not carefully selected, it could happen that the ki’s generated from different ei’s are not uniformly random. In the worst case, we have collisions such that different hash values e0 and e1 produce the same nonce (k0 = k1). If such a collision occurs, one can recover the private key d as explained in Sect. 3.1. Furthermore, collisions can be efficiently detected by looking at the r part of the signature. To efficiently browse a subset of hash values in search of such collisions, we limited ourselves to hash values with a Hamming weight equal to 1 or 2. We thus considered 32 896 hash values and were able to break 60% of the challenges with this technique. In those cases where we did not find any collision, we looked for biases in the nonce generation. We used well-known lattice attacks derived from [NS03] and [FGR13] to exploit such a potential weakness. Such attacks can recover an ECDSA private key only with the knowledge of a few bits of the ephemeral keys of several signatures. A concrete example showing why such techniques can succeed in our context consists in considering f = Id. Then ki = ei and with providing ei ranging from 0 to 99 we obtained 100 signatures for which the 249 most-significant bits of the nonces are 0. This bias is more than enough for a lattice attack to recover the private key d. Lattice-based attacks can also be applied when the ephemeral key is the product of a small random κ by another (large) constant scalar t. Such a design allows to efficiently perform the scalar multiplication as R = [κ]T = [k]G, with T = [t]G a precomputed value. The point is that the small size of κ reduces the cost of the scalar multiplication. 4.1.4 Fault Injections Another attack method is to disturb the algorithm execution and exploit the resulting faulty output. In the white-box context, faults can be easily induced since the attacker can modify the binary or use debugging tools to stop the execution and, for example, skip an instruction or modify the value of a particular register. Again, this attack can be automated and does not require an earlier reverse engineering step. All the fault attacks that can be performed in the grey-box context are obviously also a potential threat in the white-box context. In the case of ECDSA, different faults can be induced on different variables to give an exploitable result. The most obvious attack is to force the use of a weak elliptic curve during the scalar multiplication by disturbing the curve parameters [BMM00] in order to solve the discrete logarithm problem easily. The attacker can also force the use of biased nonces, for instance, by sticking a 32-bit word of k at zero during several executions. The corresponding signatures can then be used to obtain information on the key using lattice-based algorithms. Finally, modifying one byte of d during the computation of rd may allow one to recover information on the key, as shown in [GK04]. In addition, the white-box model offers new possibilities [PSS+18,ABF+18,DGH21]. They arise from the fact that deterministic versions of the scheme have to be implemented due to the impossibility of relying on a source of randomness in this context. When the algorithm is used twice on the same message, the same nonce k is derived. The attacker may thus obtain a correct signature for a given digest e, and an erroneous one by modifying a second execution of the same signature. To break the challenges of the WhibOx contest, we mainly disturbed the computation of the first part of the signature r, obtaining faulty results ˜r and ˜s = k−1(e + ˜rd) mod n. Some secret information can be deduced from the correct and faulty signatures: (r −˜r)(s −˜s)−1 ≡(r −˜r)(k−1d(r −˜r))−1 ≡kd−1 mod n . (5) (5) Let α = kd−1 mod n. The adversary can then compute the private key: Let α = kd−1 mod n. The adversary can then compute the private key: d = e(αs −r)−1 mod n . (6) (6) It is also possible to disturb other variables, but still, the faulty value must be known to exploit the result. 4.1.3 DCA In 2016, Bos et al. showed that although firstly described for the grey-box context, the well-known side-channel attacks could be very well adapted to the white-box model. The resulting attack [BHMT16] is called Differential Computational Analysis (DCA). The principle is very similar to classical side-channel attacks: secret values are extracted from leakage traces obtained during several executions of a cryptographic algorithm with the help of statistical tools. The only difference relies upon the nature of the traces. Whereas in the grey-box context, one can record the power consumption of the device in which the algorithm is implemented, a white-box attacker can simply use software execution traces. By instrumenting the binary, he can record completely noiseless traces of all accessed addresses and data over time, leading to much more efficient attacks. 533 Barbu et al. In theory, this attack is particularly devastating since it can be fully automated and does not require any earlier reverse engineering step. In practice, it is quite difficult to apply because of the size of the traces, in particular for cryptosystems such as ECDSA that have relatively long execution time. Indeed, if the whole white-box execution were to be recorded, each trace would easily reach several gigabytes. For instance, tracing n 64-bit registers on a 3GHz machine during 3 seconds would lead to a single trace of 9 ∗8 ∗n Gb. Therefore, iterating over dozens of traces for a CPA would be overwhelming in time and memory. A time-consuming step of reverse engineering allowing to select a smaller window of the implementation before the attack is thus required, which is why we did not use this technique to break the challenges of the WhibOx contest. 4.1.4 Fault Injections Interestingly, when one modifies the first part of the signature, if no countermeasure is implemented, the faulty value is just given to the attacker as part of the output. Furthermore, the attack surface is huge: the fault may happen anywhere during the scalar multiplication. This is why we considered only this perturbation in the context of this competition. This approach is the most successful one, allowing us to break 75% of the challenges. 534 Attacks and Designs from CHES 2021 Challenge 4.2 Attacks Results When applying the various attack methods described above, we obtain the results presented in Table 1. We observe that lattice and fault attacks are very efficient. Collision attacks also give good results. Table 1: Success rate of each attack on the 97 challenges. Attack type Percentage of broken challenges Hooking 32% Bad nonce - Collision 60% - Lattice 72% Fault Injection 75% We give in Appendix A the specific vulnerabilities of each of the 97 submitted challenges as well as the corresponding private key. However, we noticed that many challenges had a low level of security, some of which were even plain implementations. We thus excluded 30 challenges3 where the nonce and/or the private key were manipulated in plain. Table 2 illustrates the efficiency of the attacks presented in Sect. 4.1 on the remaining 67 challenges. We observe that hooking gives no significant result anymore, collision and lattice attacks become less efficient, and fault injection seems the most powerful attack. Table 2: Success rate of each attack on the 67 strongest challenges. Table 2: Success rate of each attack on the 67 strongest challenges. Attack type Percentage of broken challenges Hooking 1% Bad nonce - Collision 49% - Lattice 61% Fault Injection 69% Among the 67 strongest challenges, Challenges 226 and 227 are the winning ones. In the next section, we present the design of these two white-box implementations. 3The challenges 3, 4, 8, 10, 11, 32, 45, 54, 55, 57, 85, 97, 114, 135, 136, 139, 153, 157, 174, 185, 187, 231, 235, 267, 274, 299, 307, 320, 321, and 323 are considered as weak. 5.1 Implicit White-box Implementations The implicit framework is a method to obtain a white-box implementation of a block cipher. Its main idea is to represent the round functions of the cipher by implicit functions of low degree and to protect these implicit functions with large affine encodings. Before introducing implicit white-box implementations, we need to introduce the notions of encoding, encoded implementation, and quasilinear implicit functions. While these notions are originally defined in [RVP22] for vectorial functions over the binary field, we extend these notions for an arbitrary finite field. Let Fq be the finite field with q elements. A vectorial function F from the vector space (Fq)l to (Fq)l′ is called a (l, l′) function over Fq, and its l′ component functions are denoted by (F1, F2, . . . , Fl′). The degree of an (l, l′) function F denotes the maximum polynomial degree of the l′ multivariate polynomials uniquely representing the component functions of F. Definition 1. Let F be an (l, l′) function over Fq, A be an (l, l) permutation over Fq and B be an (l′, l′) permutation over Fq. The function F = B ◦F ◦A is called an encoded function of F, and A and B are called the input and output encodings respectively. Definition 2. Let F = F (t) ◦F (t−1) ◦· · ·◦F (1) be a vectorial function over Fq. An encoded implementation of F, denoted by F, is an encoded function of F composed of encoded functions of F (i), that is, F = F (t) ◦· · · ◦F (1) = (B(t) ◦F (t) ◦A(t)) ◦· · · ◦(B(1) ◦F (1) ◦A(1)) , where the input and output encodings (A(i), B(i)) are permutations over Fq such that A(r+1) = B(r)−1. The first and last encodings (A(1), B(t)) are called the external encodings. where the input and output encodings (A(i), B(i)) are permutations over Fq such that A(r+1) = B(r)−1. The first and last encodings (A(1), B(t)) are called the external encodings. Definition 3. Let F be an (l, l′) function over Fq. A (l + l′, l′′) function T is called an implicit function of F if it satisfies T(u1, u2, . . . , ul, v1, v2, . . . , vl′) = 0 ⇐⇒F(u1, u2, . . . , ul) = v1, v2, . . . , vl′ . 5 Design of the Winning Challenges In this section, we describe the designs of the two winning challenges of the WhibOx contest: Challenges 226 and 227. The designs of both challenges were inspired from the white-box implicit framework [RVP22], which allows encoding the whole state with large affine permutations efficiently. We implemented both challenges with the same methodology; they only differ in some additional countermeasures used. As mentioned in Sect. 2, in the WhibOx contest, a challenge gains strawberries quadrat- ically with time before being broken. The rule is that challenges that are either smaller, faster, or less memory-consuming gain strawberries faster. As a result, we strategically 535 Barbu et al. posted two challenges with different trade-offs between security level and implementa- tion cost. Challenge 227, our lightweight variant, was the winning implementation of the contest, obtaining the highest number of strawberries (20.39). On the other hand, Challenge 226, our hardened but heavier variant, achieved second place in the contest with the second-highest number of strawberries (11.19). However, it stood unbroken for the longest time (35 hours). Note that these challenges were specifically built for the WhibOx contest, where attackers did not know the design. Against an attacker who knows the design details, these challenges are easy to break once reverse-engineered. This section first introduces the implicit framework, then describes the shared design approach of both challenges, and finally explains the additional countermeasures used in each challenge. For access to the underlying software used to build these challenges, please contact the authors from the team zerokey. 5.1 Implicit White-box Implementations An implicit implementation of F with underlying encoded implementation F is a set of quasilinear implicit functions {T (1), T (2), . . . , T (t)} where T (i) is an implicit function of F (i). 5.1 Implicit White-box Implementations In this case, T is said to be quasilinear if for any (u1, u2, . . . , ul) ∈(Fq)l, the function (v1, v2, . . . , vl′) 7→T(u1, u2, . . . , ul, v1, v2, . . . , vl′) is affine over Fq. The following lemma from [RVP22] describes how the composition of affine permutations translates to implicit functions. 536 Attacks and Designs from CHES 2021 Challenge Lemma 1. Let F be an (l, l′) function over Fq and T be a quasilinear implicit (l + l′, l′′) function of F. Let A be an affine (l, l) permutation over Fq, B be an affine (l′, l′) permutation over Fq, and M be a linear (l′′, l′′) permutation over Fq. Then, T ′ = M ◦T ◦ (A, B−1) is a quasilinear implicit function of F ′ = B ◦F ◦A. Lemma 1. Let F be an (l, l′) function over Fq and T be a quasilinear implicit (l + l′, l′′) function of F. Let A be an affine (l, l) permutation over Fq, B be an affine (l′, l′) permutation over Fq, and M be a linear (l′′, l′′) permutation over Fq. Then, T ′ = M ◦T ◦ (A, B−1) is a quasilinear implicit function of F ′ = B ◦F ◦A. The quasilinear property allows the implicit evaluation of F in a point (u1, u2, . . . , ul) by solving the affine system T(u1, u2, . . . , ul, v1, v2, . . . , vl′) = 0 for the variables v1, v2, . . . , vl′. We are ready to present the definition of an implicit implementation. Definition 4. Let F = F (t) ◦F (t−1) ◦· · · ◦F (1) be a vectorial function over Fq, and let F = F (t)◦F (t−1)◦· · ·◦F (1) be an encoded implementation of F. An implicit implementation of F with underlying encoded implementation F is a set of quasilinear implicit functions {T (1), T (2), . . . , T (t)} where T (i) is an implicit function of F (i). Definition 4. Let F = F (t) ◦F (t−1) ◦· · · ◦F (1) be a vectorial function over Fq, and let F = F (t)◦F (t−1)◦· · ·◦F (1) be an encoded implementation of F. 5.2 White-boxing ECDSA Signature Algorithm Using the Implicit Frame work In the WhibOx contest, designers submitted white-box implementations of the ECDSA signature algorithm on the NIST P256 curve. As opposed to the standard ECDSA algorithm (cf. Algorithm 1), the algorithm for the WhibOx contest (hereafter denoted by E) takes as input the 256-bit message digest. The private key is not an input of the algorithm; it is freely chosen by the designer, but it is fixed (hard-coded) in the implementation. Algorithm 2 depicts a high-level overview of this deterministic variant of ECDSA, where the deterministic nonce derivation mechanism is chosen freely by the designer. Algorithm 2: Deterministic ECDSA signature algorithm for WhibOx contest Input : 256-bit message digest e Output : the signature (r, s) 1 state ←e 2 k, state ←NonceDerivation(state) 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Return (r, s) Algorithm 2: Deterministic ECDSA signature algorithm for WhibOx contest Input : 256-bit message digest e Output : the signature (r, s) 1 state ←e 2 k, state ←NonceDerivation(state) 3 R = (Rx, Ry) ←[k]G 4 r ←Rx mod n 5 s ←k−1(e + rd) mod n 6 if r = 0 or s = 0 then 7 Go to step 2 8 end 9 Ret ( ) The main steps of E can be represented by the functions E(1) and E(2). The Fp-function E(1) is given by E(1)(e) = (Rx, k, e) , (7) (7) which takes as input e ∈Fp and computes the scalar multiplication R = [k]G over Fp. On the other hand, the Fn-function E(2) can be written as E(2)(R′ x, k′, e′) = (r, s) , (8) (8) which takes as input (R′ x, k′, e′) = (Rx mod n, k mod n, e mod n) and computes (r, s) = (R′ x, k−1(e + rd)) over Fn. Inspired from the implicit framework, we built the white-box implementations of Challenges 226 and 227 by encoding E(1) and E(2) with affine permutations and obtaining low-degree implicit round functions of E(1) and E(2), the encoded functions of E(1) and E(2). We will first describe the implicit implementation of E(1) and then that of E(2). 537 Barbu et al. 5.2.1 White-boxing the Scalar Multiplication To build an implicit implementation of E(1), we need first to decompose E(1) as the composition of Fp-functions that we call round functions. Then we explain how to encode these round functions and how to obtain low-degree quasilinear implicit functions of the encoded round functions. Decomposing E(1) into round functions. The function E(1)(e) = (Rx, k, e), mainly consists of the scalar multiplication r = [k]G of the nonce k and the point G. For the scalar multiplication, we perform the following subroutine. First, we precompute and store a list of t random point pairs on the curve, i.e., (Gi,0 = [ki,0]G, Gi,1 = [ki,1]G) for 1 ≤i ≤t . Then, for each pair we select one of the two points together with its logarithm, denoted as (Gi,bi, ki,bi), where bi ∈{0, 1} and 1 ≤i ≤t . We add the selected points and the selected logarithms, obtaining the scalar multiplication G1,b1 + · · · + Gt,bt = [k1,b1 + · · · + kt,bt]G = [k]G , (9) (9) where k = k1,b1 + · · · + kt,bt. This selection is done in a deterministic way depending on the bits (e1, e2, . . . , e256) of the hash e, the only source of entropy in the algorithm. Moreover, the selection is done with Fp-arithmetic operations rather than with conditional instructions, so that each iteration only performs Fp operations. The subroutine is given in Algorithm 3. It is worth pointing out that the values ki,j are chosen such that the sum of max(ki,0, ki,1) for all i is always smaller than n. That is, we have k < n. Hence, r and s are never 0. In this way, we avoid the trivial case, i.e., avoid going to Step 7 in Algorithm 2. Algorithm 3: Round-based scalar multiplication used in E(1) Input : the bits (e1, e2, . . . 5.2.1 White-boxing the Scalar Multiplication , e256) of the hash e (little-endian order) Output : x-coordinate of [k]G and the message-dependent scalar k /* Round 1: input e1, k1,0, k1,1, G1,0, G1,1 embedded values */ 1 R ←[1 −e1]G1,0 + [e1]G1,1 2 k ←(1 −e1)k1,0 + e1k1,1 /* Round i: input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values */ 3 for 2 ≤i ≤t do 4 R ←R + [1 −ei]Gi,0 + [ei]Gi,1 5 k ←k + (1 −ei)ki,0 + eiki,1 6 end 7 return Rx, k // (Rx, Ry) = R = [k]G /* Round 1: input e1, k1,0, k1,1, G1,0, G1,1 embedded values 1 R ←[1 −e1]G1,0 + [e1]G1,1 k ←(1 )k + k [ ] , [ ] , 2 k ←(1 −e1)k1,0 + e1k1,1 /* Round i: input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values 3 for 2 ≤i ≤t do /* Round i: input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values 3 for 2 ≤i ≤t do 4 R ←R + [1 −ei]Gi,0 + [ei]Gi,1 5 k ←k + (1 −ei)ki,0 + eiki,1 6 end // (Rx, Ry) = R = [k]G 7 return Rx, k By considering the precomputed points Gi,j and their logarithms ki,j as fixed values and by representing the elliptic curve additions by operations over Fp, we can represent E(1) given by Algorithm 3 as an iterated function over Fp, that is, E(1) = F (t) ◦· · · ◦F (2) ◦F (1) , (10) (10) where each (4, 4) round function F (i) is given by the following component functions F (i) 1,2(u1, u2, u3, u4) = (u1, u2) + [1 −ei]Gi,0 + [ei]Gi,1 F (i) 3 (u1, u2, u3, u4) = u3 + (1 −ei)ki,0 + eiki,1 , F (i) 4 (u1, u2, u3, u4) = u4 + ei2i . (11) (11) F (i) 4 (u1, u2, u3, u4) = u4 + ei2i . 538 Attacks and Designs from CHES 2021 Challenge Note that the input value of F (1) is (0, 0, 0, 0), and each round function F (i) takes the hash bit ei as an additional input value. The pair of component functions F (i) 1,2 return a point on the elliptic curve with F (i) 1 and F (i) 2 the x- and y- coordinates respectively. 5.2.1 White-boxing the Scalar Multiplication The component function F (i) 3 updates and returns the current nonce, while F (i) 4 updates the current hash. The hash e is recomputed so that E(1) can output the hash e and provide it in an encoded form to E(2) . Encoding the round functions. To protect the round functions, we encode each round with random Fp-affine permutations A(i), obtaining the encoded round functions F (i) = A(i) ◦F (i) ◦(A(i−1))−1, 1 ≤i ≤t . (12) (12) In other words, the input and output encodings of F (i) are (A(i−1))−1, A(i) , and the composition of the round functions cancels all intermediate encodings except (A(0))−1 and A(t), that is, E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 , (13) is the number of rounds. The input encoding (A(0))−1 of F (1) is set as the identity g to preserve the input-output behaviour of E. E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 , (13) is the number of rounds The input encoding (A(0))−1 of F (1) is set as the identity E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 , (13) (13) where t is the number of rounds. The input encoding (A(0))−1 of F (1) is set as the identity mapping to preserve the input-output behaviour of E. Obtaining the implicit round functions. Now we proceed to obtain an implicit round function T (i) of each encoded round function F (i). To this end, we first show how to derive an implicit function of the elliptic curve addition. Let ADD(Px, Py, Qx, Qy) = (Rx, Ry) be the vectorial Fp-function denoting the elliptic curve addition P + Q = R where P and Q are not the point at infinity and where P and Q have different x-coordinates4. In this case, R can be written as [KL14] Rx = (Qy −Py)2((Qx −Px)2)−1 −Px −Qx Ry = (Qy −Py)(Px −Rx)(Qx −Px)−1 −Py . (14) (14) From Eq. (14) it is easy to see that P + Q = R holds if and only if the relations From Eq. 4The only elliptic curve additions performed in E(1) are the additions between the random points Gi,j, and the probability that these additions involve points at infinity or points with the same x-coordinate is negligible. 5.2.2 White-boxing the Computation of s Now we turn our attention to E(2), the second step of the signing algorithm, where we compute r = Rx mod n and s = k−1(e + dRx) mod n, and output the signature (r, s). As opposed to E(1), we do not decompose E(2) but build a single (vectorial) quasilinear implicit function of E(2) = E(2) ◦(A(t))−1, the encoded version of E(2). ( ) The vectorial Fn-function T (t+1) defined as ( ) The vectorial Fn-function T (t+1) defined as ( T (t+1) 1 (Rx, Ry, k, e; s, r) = ks −e −dRx T (t+1) 2 (Rx, Ry, k, e; s, r) = r −Rx (19) (19) is a quasilinear implicit function of E(2). In other words, the polynomial system T (t+1) = {T (t+1) 1 , T (t+1) 2 } implicitly defines E(2) because (s, r) = E(2)(Rx, Ry, k, e) if and only if T (t+1)(Rx, Ry, k, e; s, r) = 0. Moreover, the system is affine in r and s, so after plugging in values for Rx, Ry, k and e, the system can be solved for r, s efficiently. The encoded version E(2) gets as input u = A(t)(Rx, Ry, k, e), where A(t) is the affine function that protects the last round of E(1). By Lemma 1, we build the implicit round function of E(2) as T (t+1)(u; s, r) = M · T (t+1)((A(t))−1(u); s, r) , (20) (20) where (A(t))−1 is the inverse of A(t) mod n, and where M is a random invertible 2-by-2 matrix mod n. The function T (t+1) is quasilinear, and we can implicitly evaluate E(2) on input u = A(t)(Rx, Ry, k, e) by plugging u in the first slot of T (t+1) and solving the remaining system (which is affine) for r and s over Fn. However, the fact that E(1) works in Fp while E(2) works in Fn causes a problem. The input to E(2) is u = A(t)(Rx, Ry, k, e) reduced by mod p, so (A(t))−1(u) is in general not equal to (Rx, Ry, k, e) mod n if there are overflows in the computation of u. is a quasilinear implicit function of F (i) for 1 ≤i ≤t. The white-box implementations of Challenges 226 and 227 contain this implicit imple- mentation of E(1), with underlying encoded implementation E(1), given by the t implicit round functions {T (1), . . . , T (t)} in Eq. (18). Moreover, E(1) is evaluated in our white-box implementations by implicitly evaluating the encoded round functions F (i). In other words, given the output u of the round i−1, the output v of the ith round is computed by finding the solution of the affine system T (i)(u; v) = 0 for v. Barbu et al. is a quasilinea The white- mentation of E round function implementatio given the outp the solution of 5.2.2 White Now we turn compute r = As opposed to implicit functi The vector is a quasilinea {T (t+1) 1 , T (t+1) 2 T (t+1)(Rx, Ry in values for R The encode function that function of E( where (A(t))− matrix mod n on input u = remaining syst However, t input to E(2) equal to (Rx, R vector of overfl then (A(t))−1( affine map A(t To deal wit vector o and s If the guess is r, s will be rec to get a candi output the firs need to protec If A(t) was would be very 539 Barbu et al. is a quasilinear implicit function of F (i) for 1 ≤i ≤t. 5.2.1 White-boxing the Scalar Multiplication (14) it is easy to see that P + Q = R holds if and only if the relations From Eq. (14) it is easy to see that P + Q = R holds if and only if the relations (Px + Qx + Rx)(Qx −Px)2 = (Qy −Py)2 (15) (Ry + Py)(Qx −Px) = (Qy −Py)(Px −Rx) (16) (15) (16) (15) (16) (16) hold. Note that these relations have degree 3 (degree 1 over the variables Rx and Ry), while Eq. (14) has a high degree due to the inversion over Fp. g p Px, Py, Qx, Qy, Rx, Ry) = (IMP0, IMP1) defined by ( ) p Thus, the function IMP(Px, Py, Qx, Qy, Rx, Ry) = (IMP0, IMP1) defined by IMP0 = (Qy −Py)2 −(Px + Qx + Rx)(Qx −Px)2 IMP1 = (Qy −Py)(Px −Rx) −(Ry + Py)(Qx −Px) (17) (17) is a quasilinear implicit round function of ADD with degree 3, assuming none of the points is the point at infinity and assuming the x-coordinates of the points are different. From the above implicit function of the elliptic curve addition, it is easy to derive a quasilinear implicit function T (i) of each round function F (i). Then, we sample a linear permutation M (i) for each round i, and by Lemma 1 the function T (i) = M (i) ◦T (i) ◦ (A(i−1))−1, (A(i))−1 (18) (18) 4The only elliptic curve additions performed in E(1) are the additions between the random points Gi,j, and the probability that these additions involve points at infinity or points with the same x-coordinate is negligible. 5.2.2 White-boxing the Computation of s Let o be the vector of overflows mod p, such that u = A(t)(Rx, Ry, k, e) −po , (21) (21) then (A(t))−1(u) = (Rx, Ry, k, e) −pL−1 t (o) mod n, where Lt is the linear part of the affine map A(t) (i.e., A(t)(x) = Lt(x) + c for some constant term c). To deal with this problem, we correct for the overflow mod p by guessing the overflow vector o and setting u′ = u + po before plugging u′ into T (t+1)(u; s, r) to solve for (r, s). If the guess is correct, then u′ is equal to A(t)(Rx, Ry, k, e) over the integers, so the correct r, s will be recovered. Therefore, we repeatedly run the last step with random guesses of o to get a candidate signature (r, s). Then we run the verification algorithm on (r, s) and output the first (r, s) for which the verification algorithm succeeds. Note that we do not need to protect the verification algorithm because it does not use secret information. If A(t) was a random affine map with entries of size up to p, then guessing o correctly would be very unlikely. Therefore, we choose the affine map A(t) with small entries. For Attacks and Designs from CHES 2021 Challenge 540 xample, we could use A(t)(Rx, Ry, k, e) =     1 0 1 2 1 1 2 0 0 1 2 1 1 2 0 1         Rx Ry k e    + c . (22) (22) With this choice, the weight of each row is four, so there are at most four overflows mod p in each entry of u, which means o can be guessed more easily. Not all guesses are equally likely, (e.g., o = [4, 4, 4, 4] only occurs if Rx, Ry, k, e are all quite big, which is unlikely). Rather than inefficiently guessing o ∈[0, 4]4 at random, we precompute a list of guesses L ordered from more likely to be correct to less likely, and we iterate through the list of guesses in that order. The white-box implementations of Challenges 226 and 227 contain the implicit function T (t+1), which allows the implicit evaluation of E(2), together with the correction for the overflow mod p described above and summarized in Algorithm 4. 5.2.2 White-boxing the Computation of s Note that the severe restriction on the size of the entries of A(t) makes the conversion from Fp to Fn one of the most vulnerable points in the white-box implementation. In particular, an attacker knowing the specifications of the design can easily recover A(t) by exhaustive search if no additional countermeasures are used. Algorithm 4: White-box implementation of ECDSA signature algorithm for winning challenges Input : 256-bit hashed message digest e Output : the signature (r, s) 1 e ←e mod p 2 (v1, v2, v3) ←E(1)(e) // implicit evaluation 3 for o in L do 4 (u1, u2, u3) ←(v1, v2, v3) + p · o 5 (r, s) ←E(2)(u1, u2, u3) // implicit evaluation 6 if VerifySignature(r, s, e) = valid then 7 return (r, s) 8 end 9 end Algorithm 4: White-box implementation of ECDSA signature algorithm for winning challenges // implicit evaluation 5https://tigress.wtf/transformations.html 6https://github.com/CryptoExperts/whibox_contest_submission_server 5.3 Additional Countermeasures The representation of the implicit round functions as systems of multivariate polynomials allows applying countermeasures from multivariate public-key cryptosystems. In fact, Challenges 227 and 226 only differ in the additional countermeasures used. In particular, we considered two techniques. First, we obfuscated the components (seen as polynomials) of the implicit round functions T (i) by multiplying them with random polynomials in the input variables. Note that the multiplication of input variables preserves the quasilinear property. Moreover, the image of a random polynomial is non-zero with high probability, and multiplying an equation with a non-zero value does not change its solution set. In the unlikely case that one of the added polynomials vanishes, the output of the corresponding implicit function will be invalid, and no valid signature will be obtained. To prevent this extreme case, we made the first implicit round function dependent on an initial value; if no valid signature is found, we simply repeated the whole process with a different initial value. 541 Barbu et al. This first technique increases the degree of the implicit round functions, significantly increasing the implementation size. Thus, for the lightweight Challenge 227 we only applied this technique to raise the degree of the components to the total degree of the functions, but for Challenge 226 we multiplied with polynomials of higher degree to increase the total degrees of the implicit round functions. The final degrees are listed in Tables 3 and 4. The second technique we used was adding additional variables and components to the implicit round functions but preserving the input-output behaviour of the underlying encoded round functions. In particular, to avoid the bias in the most significant part of the nonce k due to the constraint k < n (see Section 5.2.1), we duplicated the nonce variable and its equations so that E(1) outputs an additional nonce variable k′ similarly to k, k′ = k′ 1,b1 + · · · + k′ t,bt, X i max(k′ i,0, k′ i,1) < n , and E(2) uses the sum of the nonce variables k + k′ as the final nonce. On top of that, instead of e, the input L(e) is given to E(1) for some hard-coded low-degree encoding L, and its inverse L(−1) is composed to E(2) to recover e. 5.3 Additional Countermeasures Note that this is a minor trick since the encoding L is not merged or composed with other functions (as opposed to the other encodings A(i)), and the computation L(e) is done in clear. Since adding additional variables and equations also introduces significant overhead in the implementation size, we only applied the second technique to Challenge 226. In particular, we added two variables and two equations in the implicit round functions of E(1), and two variables and one equation in those of E(2). We also used Tigress [Col] for both challenges to obfuscate the C source code. Tigress is an obfuscator for C language that protects programs against dynamic and static reverse engineering attacks. We used the transformations5 Flatten (flattens the code to remove structured flow), AntiTaintAnalysis (disrupts tools that make use of dynamic taint analysis), AddOpaque (adds opaque predicates), EncodeLiterals (replaces integers and strings with run-time expressions) and CleanUp (renames variables and functions). 5.4.2 Security Analysis Challenge 227 can be broken in several ways. Here, we explain how the attacks of Sect. 4 allow one to recover the secret key of Challenge 227 or why they do not work. Hooking shared libraries. During the implicit evaluation of E(2) for the valid input u = A(t)(Rx, Ry, k, e), the affine system T (t+1)(u; s, r) is solved for r and s. By denoting the entries of M as M =  m0 m1 m2 m3  , it is easy to see that this affine system is given by the equations the equations ( c1s + c3r −c5 = 0 c2s + c4r −c6 = 0 (23) (23) where the coefficients ci are given by c1 = m0k, c3 = m1, c5 = m0e −m0dRx −m1Rx c2 = m2k, c4 = m3, c6 = m2e −m2dRx −m3Rx . (24) (24) We stress that k, e, and Rx do not appear in the clear. They are expressed as linear combinations of the input u = A(t)(Rx, Ry, k, e) of E(2). Nevertheless, the coefficients ci are operated in the clear during the Gaussian elimination, and the adversary can obtain their values. Some of these coefficients are sensitive. In particular, if the attacker manages to find c1 during the computation of two different signatures (r, s) and (r′, s′), he may solve the following system of two equations with two unknowns (m0 and d) in Fn: ( m0(e + rd) = c1s m0(e′ + r′d) = c1s′ . (25) (25) Therefore, recovering the value c1 = m0k mod n for two different signatures allows an attacker to compute the private key. Finding the interesting values inside the white-box may seem difficult without a reverse engineering step. However, the attack turns out to be easily automated on challenges that use the GMP library, such as Challenge 227. Indeed, one of the coefficients of s, say c1 = m0k, will be inverted modulo n during the resolution of the system, and finding this inversion is easy when one can simply trace the calls to the function mpz_invert(). The team TheRealIdefix was thus able to efficiently apply this attack on Challenge 227 during the contest without any reverse engineering step. 5.4.1 Description Following the method described in Section 5.2, we built Challenge 227 (keen_ptolemy) as a lightweight white-box implementation. The only additional countermeasures from Section 5.3 included in Challenge 227 are the degree increase of each component to the total degree of the corresponding vectorial function and the code obfuscation by Tigress. Challenge 227 was the winning implementation of the WhibOx contest; it achieved the highest number of strawberries (20.39) and stood for 33 hours as the second-longest. ( ) Table 3 describes the memory complexity of Challenge 227 (after applying the additional countermeasures) by describing {T (1), . . . , T (t)} and T (t+1), the implicit round functions of E(1) and E(2) respectively. The number of coefficients in Table 3 denotes the maximum number of non-zero coefficients of a quasilinear vectorial function with a given number of input variables, components, and degrees. If each coefficient is represented with 256 bits, {T (1), . . . , T (t)} and T (t+1) require in total roughly 4 MB. After obfuscating the code with Tigress, the size of the final C source code of Challenge 227 is 4.4 MB. In a modern personal laptop with the environment6 provided by the competition, the size of the compiled binary is 4.42 MB, and the average running time and 542 Attacks and Designs from CHES 2021 Challenge Table 3: Information of the implicit round functions T (i) of Challenge 227. T (1) {T (2), . . . , T (t−1)} T (t) T (t+1) input variables 2+4 5+4 5+3 3+2 number of components 4 4 3 2 degree 3 3 4 2 number of coefficients 27 × 4 130 × 4 255 × 3 18 × 2 Table 3: Information of the implicit round functions T (i) of Challenge 227. RAM consumed are 0.04 seconds and 6.14 MB respectively. The code obfuscation did not impact the running time but increase the binary size by 8% and the average RAM by 3%. 5.4.2 Security Analysis This attack may seem very specific, but multiplying the nonce with a constant may appear as an easy way to protect the inversion step, and could very well be used by designers. This attack shows it is not a robust countermeasure. 543 Barbu et al. Biased nonces. There exists a more generic way of breaking Challenge 227. Indeed, the way the ephemeral key is constructed (see Sect. 5.2.1) opens the way for an attack using lattice reduction techniques. Given that the ephemeral key k is obtained by summing 256 scalars ki,j according to each bit of the input, one can obtain the following signatures by selecting a couple of hashes (e0, ei), with e0 = 0 and ei = 2i :            s0 = 255 P j=0 kj,0 !−1 (e0 + r0d) mod n si = ki,1 + 255 P j=0,j̸=i kj,0 !−1 (ei + rid) mod n , (26) (26) which allows us to construct 256 equations involving only one of the ki,j: ki,1 + 255 P j=0,j̸=i kj,0 − 255 P j=0 kj,0 = s−1 i (ei + rid) −s−1 0 (e0 + r0d) mod n ki,1 −ki,0 = s−1 i ei −s−1 0 e0 + (s−1 i ri −s−1 0 r0)d mod n . (27) (27) Now, the additional constraint k < n lets us estimate that each ki,j is sampled from J0, ⌊n/256⌋K. Consequently, |ki,1 −ki,0|n = |s−1 i ei −s−1 0 e0 + (s−1 i ri −s−1 0 r0)d|n < j n 256 k , (28) (28) with |y|n := min a∈Z |y −an| to denote the distance of y ∈R to the closest integer multiple of n. with |y|n := min a∈Z |y −an| to denote the distance of y ∈R to the closest integer multiple of n We recognize in Eq. (28) an instance of the Hidden Number Problem (HNP) [BV96]. Indeed, we are given many HNP inequalities of the form: |αti −ui|n < j n 256 k , (29) (29) with ti = s−1 i ri −s−1 0 r0, ui = s−1 0 e0 −s−1 i ei and the hidden number α is the private key d. Solving HNP instances in the context of ECDSA given inequalities such as Eq. (29) has been described numerous times in the literature. We refer the reader to [JSSS20] for a more detailed description7. 7We also highlight that the authors of [JSSS20] made their code available at https://github.com/crocs- muni/minerva. 5.4.2 Security Analysis In particular, the authors detail the reduction of the HNP instance to a Closest Vector Problem instance in a specific lattice as well as the construction of this lattice. Finally, we use 75 relations such as Eq. (28) (out of the 255 we can establish) to build a lattice whose reduction allows us to recover the private key d. DCA. As explained in Sect. 4, we did not mount this side-channel attack during the contest. With the design of Challenge 227 in hand, we can see that it would have been unsuccessful, at least at the first order, thanks to the linear masking scheme used to protect all the implementation. Fault injections. None of the faults injected on Challenge 227 during the contest were exploitable. This can be explained by the presence of the signature verification that is used to check if the guess for the overflow between E1 and E2 is correct. If a fault is induced, the signature is rejected and recomputed. g Of course, a reverse engineering step could be performed to get rid of this verification, but this would be quite time-consuming. Furthermore, even without this verification, 544 Attacks and Designs from CHES 2021 Challenge the fault attack is still not trivial to perform because of the linear masking scheme. In particular, Rx is not manipulated directly in E2. It is expressed as a linear combination of the input A(t)(Rx, Ry, k, e), so modifying one of the shares would probably also fault e, k or Ry, making the resulting faulty signature unexploitable. 5.5.1 Description Challenge 226 (clever_kare) was the second white-box implementation that we built following the method described in Section 5.2 and including all the additional countermea- sures from Section 5.3. While this challenge stood for the longest (35 hours), Challenge 226 achieved the second-highest number of strawberries (11.19) due to its higher time and memory complexity than Challenge 227. Table 4 describes the memory complexity of {T (1), . . . , T (t)} and T (t+1) of Challenge 226 after applying the additional countermeasures. Given each coefficient as a 256-bit value, {T (1), . . . , T (t)} and T (t+1) require in total roughly 15 MB. The impact of the additional countermeasures on the number of equations, the degree, and the number of variables can be seen by comparing this table with Table 3. Table 4: Information of the implicit round functions T (i) of Challenge 226. Table 4: Information of the implicit round functions T (i) of Challenge 226. T (1) {T (2), . . . , T (t−1)} T (t) T (t+1) input variables 2+6 7+6 7+5 5+2 number of components 6 6 5 2 degree 3 3 4 5 number of coefficients 37 × 6 322 × 6 854 × 5 504 × 2 The size of the final C source code of Challenge 226 is 17.54 MB, the size of the compiled binary is 15.44 MB, and the average running time and RAM consumed are 0.15 seconds and 17.27 MB, respectively. The code obfuscation did not significantly impact the performance of Challenge 226; the running time, the binary size, and the average RAM increased by less than 1%. in d in order to obtain the secret key. in d in order to obtain the secret key. Therefore, this challenge can be easily broken once reverse-engineered. Nevertheless, such an attack is quite time-consuming, and resisting TheRealIdefix’s automated attacks on ECDSA in the white-box contest is already an achievement considering that only 5 challenges resisted these attacks during the contest. 5.5.2 Security Analysis During the WhibOx contest, the team theRealIdefix did not manage to break Challenge 226 with any of the automated attacks presented in Sect. 4.1. DCA and fault injection, which fail to break Challenge 227, are also not applicable to Challenge 226 since it is designed to be more secure. Moreover, the two attacks presented in Sect. 5.4.2 also fail to recover any secret information. Hooking shared libraries. As mentioned in Sect. 5.3, Challenge 226 implements an additional countermeasure which consists in multiplying the components of the implicit round functions T (i) with random polynomials in the input variables. Hence, the coefficients of s in the system T (t+1)(u; s, r) for the valid input u are no longer fixed multiples of k, and the attack cannot be mounted anymore. Biased nonces. Likewise, the additional countermeasures implemented in Challenge 226 makes the lattice attack described in Sect. 5.4.2 fail. The additional variable k′ alone would only reduce by 1 bit the bias observed in Eq. 29 and the attack would still be 545 Barbu et al. practical. However, without the knowledge of the encoding L introduced in this challenge it is impossible to exhibit such a bias leading to key recovery. practical. However, without the knowledge of the encoding L introduced in this challenge it is impossible to exhibit such a bias leading to key recovery. As explained, none of the automated attacks that the TheRealIdefix team used during the contest were successful on Challenge 226. Nevertheless, with the design in hand, one could easily break this challenge. Indeed, knowing that the matrix M of the last affine encoding A(t) contains small entries, the attacker could, for example: • Compute two signatures (r1, s1) and (r2, s2) for two messages e1 and e2 and extract the two valid E(2) inputs u1 = A(t)(v1) and u2 = A(t)(v2) from the execution. Note that v1 −v2 contains the nonce difference κ = k1 −k2. • Compute two signatures (r1, s1) and (r2, s2) for two messages e1 and e2 and extract the two valid E(2) inputs u1 = A(t)(v1) and u2 = A(t)(v2) from the execution. Note that v1 −v2 contains the nonce difference κ = k1 −k2. • Find κ by exhaustive search over M; for each guess M ′, obtain a candidate v1 −v2 = (M ′)−1(u1 −u2) and check if one of its entries κ satisfies (κG)x = r1 −r2. 5.5.2 Security Analysis • Find κ by exhaustive search over M; for each guess M ′, obtain a candidate v1 −v2 = (M ′)−1(u1 −u2) and check if one of its entries κ satisfies (κG)x = r1 −r2. • Solve the equation s−1(e1 + r1d) −s−2(e2 + r2d) = κ (30) (30) s−1(e1 + r1d) −s−2(e2 + r2d) = κ 6 Conclusion This work describes several attack techniques and designs used in the WhibOx 2021 contest. We explained the attack methods used by the team TheRealIdefix, which broke the most challenges, and we showed the success of each method against all the implementations in the contest. Fault attacks were the most efficient and effective ones; collision and lattice attacks were slightly less efficient, and hooking succeeded against weak implementations only. Among the white-box implementations that resisted these attacks, the one with the highest score was Challenge 226 (clever_kare). This challenge, together with Challenge 227 (keen_ptolemy), was submitted by the team zerokey, and they obtained the second- highest and the highest score in the contest, respectively. In this work, we described the design methodology of these two challenges, which was inspired by the implicit white-box framework. The large number of implementations broken by our automated attacks and the fact that no challenge survived more than two days show that securing ECDSA in the white-box model is a challenging problem. White-box attacks benefit from the huge progress in side-channel and fault attacks against ECDSA implementations, but not much research has been done on the design part. To this end, our designs provide insightful examples for future works, and our attacks highlight the weak points future research should address. One of the main challenges specific to white-boxing ECDSA is the conversion from Fp to Fn. While grey-box countermeasures can protect this step (e.g., with Arithmetic-to-Boolean and Boolean-to-Arithmetic mask conversions), these techniques rely on randomness, which is ineffective in white-box implementations. In particular, the conversion from Fp to Fn is one of the weakest points in our designs, and further research in white-boxing the field conversion is needed. 546 Attacks and Designs from CHES 2021 Challenge Acknowledgment The authors would like to thank the other members of the TheRealIdefix team: Yannick Bequer, Luk Bettale, Laurent Castelnovi, Thomas Chabrier, Nicolas Debande, Roch Lescuyer, Sarah Lopez and Nathan Reboud. Adrián Ranea is supported by a PhD Fellowship from the Research Foundation – Flanders (FWO) under grant No. 11E1921N. Chaoyun Li is an FWO post-doctoral fellow under grant No. 1283121N. Ward Beullens is an FWO post-doctoral fellow under grant No. 1S95620N. 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A Attacks Summary Table g Challenge Hooking Collision Fault Lattice Key 3 ✓ ✓ ✓ ✓ 45189C81EADEE03202BFA06EAA15831789F0C76575508A563E1A739CA37B87BE 4 ✓ ✓ ✓ ✓ 22BEF7AC4C31B2B98227D95B5EB49AF23343004CF2713FED48BEC3B5B7C3D24D 8 ✓ ✓ ✓ ✓ F484955872415A32B1B5B731EA1A8C729458055C17DC5FE9C57BCB39D1A40BFE 10 ✓ ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494CFD5112E303BA1413388126EA33BB60AEFC 11 ✓ ✓ ✓ ✓ E7F3287D91B528D78BF19D5E62828C845E1A4027A3E1F988B62B7407EBF5CF38 12 ✓ ✓ 773F0C0FFACB531F50FAE0987D2B8972FE1B9231BBF46859F475BAFB45257FED 13 ✓ ✓ 034332A23341538143FDB88F314FD942501FF8B6BA6A14D5013F1FC0984924BE 15 ✓ ✓ 3F77C51259E1C8CC48217A66998CCF3212A17120B0FCA09163E300576DFCD9E7 16 ✓ 23773F0BECFACB534250FAE0987D2B8969D1AFD7EF942F148746DC73A3C6B39A 32 ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 33 ✓ CD9540B70C2F92B2894594CABC4E724203A615B9144C459714758BC3CAA12242 34 ✓ ✓ ✓ 70253E6587D04D7A9A30A1461A80FCD235B28FBFC11FE8534CDFCE0A341C9257 36 ✓ 10D7EF92F06DF6EB94F2F344085DAD51D3A550E24A4569922460F579CB5DF11A 38 ✓ 70C3A9F11773C8DD795FD7942B5DB448FDFA5D12E6EC387691A19B6E523AE6AE 42 ✓ 1BEDDC1DD79F8856BF2E1FD66EB194073D60FEC658C5D0E2C8BAE02DC72ADF65 44 ✓ B519BB44EC5BF3380CB2DF555F39ED836CDBF4961E43A66C218FADB211BF468C 45 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 50 ✓ 7A7AA97370B1EE16D64C71C7C5BC8C9F9456FBEA603780883399D89DA43F8A15 54 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E22222222222F126EA33F6E49790 55 ✓ ✓ ✓ ✓ 00498594859849584954E92752494FFD5112E2222222EE22F126EA33F6E49790 57 ✓ ✓ ✓ ✓ 7D1BBD475A8EB5AF7DDB238CD8A67F86B601E0EA101C04036849B31F96CA6083 58 ✓ ✓ ✓ BD3026C700A75B5970807802E2B47C2A892DF85E3CE57366D335EEBABCAAE255 61 ✓ ✓ ✓ F4DDC95A88146CF52DEC752E737F8E3FB16AE4F6B7E726068946F3B0BA0C8E95 62 ✓ ✓ ✓ 0A99EB20F9DE4DD7607288B8B766F6217FE5D2CE6DDD51C6159941066AF192ED 66 ✓ ✓ ✓ 8836AC84AA148440A20628810CA65EB038BB625841275CC11590D8F5BC7F1BAC 70 ✓ ✓ ✓ 21A35C57E23B2D23ADDA19EA30325F1B532DA645489E29E47A13E92CA1F6670C 71 ✓ ✓ ✓ 588BEED930355AF54EEBAFAA46A7D26DA378A36EF5CD15D1F876D753A395F8AF 72 ✓ ✓ ✓ B7A9B0F7661FC9A1DEC001F2C2C9EAE08748AEB187E1247726663E3DD1AB36BF 73 ✓ ✓ ✓ 4DAA29CBD634F28137499B9557104FDD36D4D4EFDFE87EFC0D8BD03555F8497F 74 ✓ ✓ ✓ 12691AAC55A079F529FE81205DF775EF297A14CA81499BF0857643E694CF8816 76 ✓ ✓ ✓ F5178EEC7A9779E13CE01B35C8264BF32C094B172051CA32156DC61485718318 77 ✓ ✓ ✓ A0543814F86D1C4AF6A08094CD0246F606F7E76CEE47EC052B62328038146D93 78 ✓ ✓ ✓ 511128DCBF369E985B99D07CC1668A2D28F4BA535CF7AC7926D4C5F696C3D35F 79 ✓ ✓ ✓ 595AD4C8A0EB2FDA798BC01D322F4C5ED098A2E749004B2B54FD815215F46686 80 ✓ ✓ ✓ 8E938EA9BE9E51A28DFD30BD6EDB9D6765C1272B8F7048CE81021194759C3E52 81 ✓ ✓ ✓ F134975C5A989635F1D9FA7469C848A953622E9DA1BED7E12455DCD2AFA070BE 84 ✓ ✓ ✓ 36A990B9F35B79934FB25C64681DE3A83FC178DC2383C585FFCFDDD7C1F6C2B7 85 ✓ ✓ ✓ ✓ AB700D75274336FD26A1FE49D400ACEAE89F0FDBFE4BDE9A70373CA693003CA8 87 ✓ ✓ ✓ 9A4D4A94A1FE0FA1C559764C85D06496BD752498E0B5A2459624211013B9A088 89 ✓ ✓ C80682FCB2D78B2515A70A70D17C47A8512E24A127E797C073566D54586B9482 94 ✓ ✓ A04B6199A1DFE39EF35F6302454D71C872771A2F02A27AB5EC8130DA226F6F90 96 ✓ ✓ AFAAABE59B2EBB4FE15274E4EB5D1999C0554CC2D498BC92C59A3F6CD8FE2BC0 97 ✓ ✓ ✓ ✓ 0754CA8EA936675EC3F64782A14E1A75B3D357044D4B2C434C6011279D17E829 100 ✓ 7F58EDB783C1F3FA7FF424CF7F5DF6D4BCDCF18D8A98CE4559EC22EB17030578 101 ✓ 25D31D3AFF5773799ECF43DEC1882B8F05D9231697BDDA5482DE05B14FB8A63B 103 ✓ ✓ CC977E0748722D615B845C1B10EA554B69DFCA640440CA5C468BBEF84B8C0442 104 ✓ ✓ ✓ 638C9DFBF9F376CBB3E3B01DF27960EC53A689D2FF4DFF23D97EE5351ED4A3D0 105 ✓ ✓ ✓ D29E9D130016D930BF830BCAD071BC6503F877FB207922A9E495CF71A79631FE Key C H C Fa La K 3 ✓ ✓ ✓ ✓ 45189C81EADEE03202BFA06EAA15831789F0C76575508A563E1A739CA37B87BE 4 ✓ ✓ ✓ ✓ 22BEF7AC4C31B2B98227D95B5EB49AF23343004CF2713FED48BEC3B5B7C3D24D 8 ✓ ✓ ✓ ✓ F484955872415A32B1B5B731EA1A8C729458055C17DC5FE9C57BCB39D1A40BFE 10 ✓ ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494CFD5112E303BA1413388126EA33BB60AEFC 11 ✓ ✓ ✓ ✓ E7F3287D91B528D78BF19D5E62828C845E1A4027A3E1F988B62B7407EBF5CF38 12 ✓ ✓ 773F0C0FFACB531F50FAE0987D2B8972FE1B9231BBF46859F475BAFB45257FED 13 ✓ ✓ 034332A23341538143FDB88F314FD942501FF8B6BA6A14D5013F1FC0984924BE 15 ✓ ✓ 3F77C51259E1C8CC48217A66998CCF3212A17120B0FCA09163E300576DFCD9E7 16 ✓ 23773F0BECFACB534250FAE0987D2B8969D1AFD7EF942F148746DC73A3C6B39A 32 ✓ ✓ ✓ 32D67733DF0D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 33 ✓ CD9540B70C2F92B2894594CABC4E724203A615B9144C459714758BC3CAA12242 34 ✓ ✓ ✓ 70253E6587D04D7A9A30A1461A80FCD235B28FBFC11FE8534CDFCE0A341C9257 36 ✓ 10D7EF92F06DF6EB94F2F344085DAD51D3A550E24A4569922460F579CB5DF11A 38 ✓ 70C3A9F11773C8DD795FD7942B5DB448FDFA5D12E6EC387691A19B6E523AE6AE 42 ✓ 1BEDDC1DD79F8856BF2E1FD66EB194073D60FEC658C5D0E2C8BAE02DC72ADF65 44 ✓ B519BB44EC5BF3380CB2DF555F39ED836CDBF4961E43A66C218FADB211BF468C 45 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC 50 ✓ 7A7AA97370B1EE16D64C71C7C5BC8C9F9456FBEA603780883399D89DA43F8A15 54 ✓ ✓ ✓ ✓ 32D67733DF3D0257DA78E92752494FFD5112E22222222222F126EA33F6E49790 55 ✓ ✓ ✓ ✓ 00498594859849584954E92752494FFD5112E2222222EE22F126EA33F6E49790 57 ✓ ✓ ✓ ✓ 7D1BBD475A8EB5AF7DDB238CD8A67F86B601E0EA101C04036849B31F96CA6083 58 ✓ ✓ ✓ BD3026C700A75B5970807802E2B47C2A892DF85E3CE57366D335EEBABCAAE255 61 ✓ ✓ ✓ F4DDC95A88146CF52DEC752E737F8E3FB16AE4F6B7E726068946F3B0BA0C8E95 62 ✓ ✓ ✓ 0A99EB20F9DE4DD7607288B8B766F6217FE5D2CE6DDD51C6159941066AF192ED 66 ✓ ✓ ✓ 8836AC84AA148440A20628810CA65EB038BB625841275CC11590D8F5BC7F1BAC 70 ✓ ✓ ✓ 21A35C57E23B2D23ADDA19EA30325F1B532DA645489E29E47A13E92CA1F6670C 71 ✓ ✓ ✓ 588BEED930355AF54EEBAFAA46A7D26DA378A36EF5CD15D1F876D753A395F8AF 72 ✓ ✓ ✓ B7A9B0F7661FC9A1DEC001F2C2C9EAE08748AEB187E1247726663E3DD1AB36BF 73 ✓ ✓ ✓ 4DAA29CBD634F28137499B9557104FDD36D4D4EFDFE87EFC0D8BD03555F8497F 74 ✓ ✓ ✓ 12691AAC55A079F529FE81205DF775EF297A14CA81499BF0857643E694CF8816 76 ✓ ✓ ✓ F5178EEC7A9779E13CE01B35C8264BF32C094B172051CA32156DC61485718318 77 ✓ ✓ ✓ A0543814F86D1C4AF6A08094CD0246F606F7E76CEE47EC052B62328038146D93 78 ✓ ✓ ✓ 511128DCBF369E985B99D07CC1668A2D28F4BA535CF7AC7926D4C5F696C3D35F 79 ✓ ✓ ✓ 595AD4C8A0EB2FDA798BC01D322F4C5ED098A2E749004B2B54FD815215F46686 80 ✓ ✓ ✓ 8E938EA9BE9E51A28DFD30BD6EDB9D6765C1272B8F7048CE81021194759C3E52 81 ✓ ✓ ✓ F134975C5A989635F1D9FA7469C848A953622E9DA1BED7E12455DCD2AFA070BE 84 ✓ ✓ ✓ 36A990B9F35B79934FB25C64681DE3A83FC178DC2383C585FFCFDDD7C1F6C2B7 85 ✓ ✓ ✓ ✓ AB700D75274336FD26A1FE49D400ACEAE89F0FDBFE4BDE9A70373CA693003CA8 87 ✓ ✓ ✓ 9A4D4A94A1FE0FA1C559764C85D06496BD752498E0B5A2459624211013B9A088 89 ✓ ✓ C80682FCB2D78B2515A70A70D17C47A8512E24A127E797C073566D54586B9482 94 ✓ ✓ A04B6199A1DFE39EF35F6302454D71C872771A2F02A27AB5EC8130DA226F6F90 96 ✓ ✓ AFAAABE59B2EBB4FE15274E4EB5D1999C0554CC2D498BC92C59A3F6CD8FE2BC0 97 ✓ ✓ ✓ ✓ 0754CA8EA936675EC3F64782A14E1A75B3D357044D4B2C434C6011279D17E829 100 ✓ 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A Attacks Summary Table Challenge Hooking Collision Fault Lattice Key 107 ✓ ✓ ✓ 4E420B6AA9E9F07F19CF7ED97497871C1223BC2A68E83716575C235DE6D63E17 108 ✓ 60609404F0B9086D3A995AF0680D048724CF2B1AF2B33CEA8DD4AF4B62A5DDBB 114 ✓ ✓ ✓ 0000000000000000000000000000000000000000000000000000000000000005 127 ✓ 1144D82B9568581405D10CF8B219FF7E94E4559E0832B06056F1F87D43C75777 135 ✓ ✓ ✓ ✓ 0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33576B3123CECBB6406837BF51F5 136 ✓ ✓ ✓ 0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33476B3123CECBB6406837BF51F4 139 ✓ ✓ ✓ 000000000000000000000000000000004319055358E8617B0C46353D039CDAA9 153 ✓ ✓ 9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875C3 157 ✓ ✓ ✓ ✓ F04DBFD1147F9D43747538C1C9256DD2BC20562F9D92B83E9AFA751299B160A4 165 ✓ ✓ ✓ 84DAF8B6620FC6669BF1EE264D1B214A4FBECACEADDFDC0DCBC89CF4B6E3232B 166 ✓ ✓ C746740A4A6BCBD462D9041023A0FEF5CCF0328FF80D9C50132682030D77D33C 172 ✓ ✓ ✓ 285E57F7BDDAAA6201D8870A0B9B168C7A5D8200085F62504EE3EBFCC11EF150 174 ✓ ✓ ✓ ✓ 9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 185 ✓ ✓ ✓ ✓ 7729EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 187 ✓ ✓ ✓ ✓ 7779EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC 192 ✓ 09302BDFA5313312B9A665316F7E9365DCC57DA7E21FD8612CDCD553BABB51FE 193 ✓ E0FE06BE0684455EDD2F5134A3AE8B9F6852561C821672FA16606986233BF811 209 ✓ 6E3A09F8EC613B8A524F7608CB80B2D3C510E27506AD84FA14C3B6D018E659F7 212 ✓ D663E156F036F11D4E73CC0EC09A952DEAED316947DF73EB28467EC623C5740D 2268 6F1D9093F3D5AE7C5F133659295914C9AF22E54B4ADE38CA421CA9BBD3D48A50 227 ✓ ✓ ADA6C6A1049825989811C9495D83681A68C67AB5E8EBDDC126CEE77056A7BB27 228 ✓ EA7BA345EB9D99F54261D01AE6319B184769E5745621706D77018E0DB46DDAFA 231 ✓ ✓ ✓ ✓ 8ADE24EE6413C6E408784DBB4D81D04F33238AB503CBE35C77400517EE5ABC96 235 ✓ ✓ ✓ ✓ 000000000000000000000000000000000000000000000000D0FACADE0DEFACED 251 ✓ ✓ ✓ DDE098A74086ECBB4DBA1848511BEA924145D1A9ED2EC9E64E0C5934BAAC97AE 253 ✓ ✓ 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B Some Remarks on the Challenges Among the various submissions, we notice the following facts: • Challenges 15 and 16 have a very small code size, only 194 bytes! To obtain such tiny implementations, the designers use a fixed nonce k = 1 (i.e. r = Gx) and a private key d such that dr ≡2i mod n. In such a case, the signature of a hash e is equal to (Gx, e + 2i). • Challenge 114 uses a very small private key, indeed d114 = 5 • Some designer teams modify a few bits only of the private key in several challenges (cf. Challenges 174, 185 and 187 for instance). In such a case, if one implementation is broken, then the private keys of the other challenges of the same team could be recovered by brute force search. • Despite what is indicated in the rules (cf. Sect. 2), some challenges are not deter- ministic9, i.e. the two signatures of the same message could be different. All these challenges use the time() function to obtain some randomness. However, it is easy to hook such calls and return a constant value.
https://openalex.org/W2741088600
https://www.nature.com/articles/cddis2017336.pdf
English
null
Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells
Cell death and disease
2,017
cc-by
10,228
1College of Veterinary Medicine, Northwest A&F University, 22 Xinong Road, Yangling 712100, China and 2Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, 3 Taicheng Road, Yangling 712100, China *Corresponding author: H Wu or Y Zhang, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China. Tel/Fax: +86 29 87080092; E-mail: hbwu029@nwsuaf.edu.cn or zhangy1956@sina.com Received 22.3.17; revised 21.5.17; accepted 14.6.17; Edited by y Shi Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells Zhao1,2, Beibei Fu1,2, Songna Yin2, Chao Song1,2, Jingcheng Zhang1,2, Shanting Zhao1 and Yong Zhang*,1, aibo Wu*,1,2, Jiamin Zhao1,2, Beibei Fu1,2, Songna Yin2, Chao Song1,2, Jingcheng Zhang1,2, Shanting Zhao MicroRNAs (miRNAs) regulate critical cell processes, such as apoptosis, proliferation, and development. However, the role of miRNAs in embryonic stem cell (ESC) neural differentiation induced by retinoic acid (RA) and factors that govern neural directional differentiation remain poorly understood. In this study, we demonstrated that miR-219 is sufficient in promoting mouse ESCs to undergo neural differentiation. We discovered that Foxj3 and Zbtb18, two target genes of miR-219, are not able to determine the process of RA-induced differentiation, however they prevent ESCs from differentiating into neural cells. We identified four downstream genes, namely, Olig1, Zic5, Erbb2, and Olig2, which are essential to the gene interaction networks for neural differentiation. These data explain the mechanism of RA-induced neural differentiation of mESCs on the basis of miRNAs and support the crucial role of miR-219 in neurodevelopment. pp p Cell Death and Disease (2017) 8, e2953; doi:10.1038/cddis.2017.336; published online 27 July 2017 MicroRNAs (miRNAs) are a class of non-coding, single- stranded, and 22–25-nucleotide RNA molecules that nega- tively regulate gene expression in plants and animals.1 MiRNAs mainly pair with the 3′-untranslated regions (UTRs) of a target gene and consequently degrade the target mRNA or inhibit translation. MiRNAs are involved in the regulation of embryonic development, apoptosis, and proliferation.2–7 Moreover, some miRNAs, as oncogenes or tumor suppressor genes, are closely related to tumor formation.8–11 we demonstrated that miR-219 can promote embryonic stem cells (ESCs) to differentiate into neural cells, and Foxj3 and Zbtb18, as targets of miR-219, are dominant factors in neural differentiation. These results provide a theoretical basis for RA application in ESC research and make a contribution to the understanding of neural regulatory networks. Citation: Cell Death and Disease (2017) 8, e2953; doi:10.1038/cddis.2017.336 Official journal of the Cell Death Differentiation Association Citation: Cell Death and Disease (2017) 8, e2953; doi:10.1038/cddis.2017.336 Official journal of the Cell Death Differentiation Association OPEN OPEN www.nature.com/cddis Results All-trans retinoic acid (RA), a metabolic compound derived from Vitamin A, is important in cellular differentiation and neurogenesis.12–17 RA is the most used morphogen for producing neural progenitor cells and neurons from pluripotent stem cells in vitro.18–21 The action of RA on differentiation is probably mediated by RA receptors (RARs) in the nucleus. This action is done through the binding of the receptors to DNA sequences located in the promoter regions of RA-responsive genes.17,22,23 Furthermore, the molecular signaling pathways involved in neuronal differentiation are complex. The specific mechanism of RA-induced neural differentiation in pluripotent stem cells is not well characterized, and the role of miRNAs in this process remains unknown.24–26 RA-induced miRNA expression changes in ESCs. RA is widely used to induce neurogenesis.18–20 We determined whether or not miRNAs are involved in RA-induced neural directional differentiation by performing an array-based miRNA profiling on mouse J1 ESCs (gene expression omnibus (GEO) accession number: GSE54145; all tested miRNAs sorted by names are listed in Supplementary Data s03). The results showed that 43 miRNAs were upregulated and 281 miRNAs were downregulated after 48 h of RA treatment in ESCs. Of the 43 upregulated miRNAs (Supplementary Table S1), 18 exhibited more than a five- fold change, and five other miRNAs that are well known to be functionally related with development were then confirmed by qRT-PCR.32–34 Consistent with the microarray findings, the 18 changed miRNAs were upregulated by RA treatment at varying degrees(Figure 1a). Three of them, namely, miR-10a- 5p, miR-219-5p, and miR-219-2-3p, showed highly significant fold changes. As previously reported, RA-induced differentia- tion of mESCs is powerful and irreversible, and the first 36–48 h of induction is critical.12 Consequently, the expres- sion levels of these three significantly changed miRNAs were detected at different time points during RA induction Forkhead box J3 (Foxj3) belongs to a gene family of transcription factors that regulate skeletal muscle and peripheral artery developments.27,28 Meanwhile, zinc-finger and BTB domain containing 18 (Zbtb18), also known as Zfp238, is involved in skeletal muscle differentiation and myogenesis.29,30 When combined with PDGFRα and Sox6, Foxj3 and Zbtb18 can control oligodendrocyte differentiation and myelination.31 However, the roles of Foxj3 and Zbtb18 in stem cell differentiation are poorly understood. In this study, MiR-219 promotes neural differentiation H Wu et al Figure 1 MiRNA expression changes in J1 ESCs induced by RA. Results (a) J1 ESCs were treated with RA for 48 h, and qRT-PCR was performed to determine the relative expression levels of 23 miRNAs. U6 served as the internal control. (b–d) Relative expression levels of miR-10-5p (b), miR-219 (c), and miR-219-2-3p (d) at different time points after RA treatment on ESCs. All data are presented as mean ± S.D. and derived from three independent experiments MiR-219 promotes neural differentiation H Wu et al 2 2 Figure 1 MiRNA expression changes in J1 ESCs induced by RA. (a) J1 ESCs were treated with RA for 48 h, and qRT-PCR was performed to determine the relative expression levels of 23 miRNAs. U6 served as the internal control. (b–d) Relative expression levels of miR-10-5p (b), miR-219 (c), and miR-219-2-3p (d) at different time points after RA treatment on ESCs. All data are presented as mean ± S.D. and derived from three independent experiments Foxj3 and Zbtb18 are the targets of miR-219. To investi- gate the underlying mechanism, we examined the potential targets of miR-219 by searching the PicTar, miRanda, and targetScan databases. Among the candidate targets, the 3′-UTR of mouse Foxj3 and Zbtb18 contains putative regions that match the miR-219 seed sequence, which is conserved in humans and rats (Figure 3a). To confirm the predicted results, the 3′-UTRs of Foxj3 and Zbtb18 containing the putative regions were amplified and inserted into the psicheck-2 vector. They were then transfected to NIH/3T3 fibroblast cell line for dual luciferase reporter (DLR) assays. As shown in Figure 3b, the miR-219 mimics dramatically suppressed the activities of wild-type (WT) 3′-UTRs of Foxj3 and Zbtb18. By contrast, the double mutation type (MUT) group (site 1+2 mut) with mutated seed sequences was unaffected. qRT-PCR and western blot were performed to examine the mRNA and protein levels of Foxj3 and Zbtb18 in ESCs transfected with miR-219 mimics or inhibitors. The results showed that the miR-219 mimics considerably decreased the protein levels of Foxj3 and Zbtb18 rather than the mRNA levels of these genes (Figures 3c–e). Thus, miR-219 regulates the expression levels of Foxj3 and Zbtb18 at the post-transcriptional level. These results indicated that Foxj3 and Zbtb18 are the targets of miR-219. (Figures 1b–d). The results showed that miR-219 was dramatically and highly expressed during the initial 48 h, and its expression decreased gradually over time (Figure 1c). Results (b, c) ESCs were transfected with miR-219 mimics for 48 h; the relative levels of Oct4, Nestin, and Map2 were detected through qRT-PCR (b) and western blot (c). (d) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. (e, f) ESCs were pretreated with RA for 48 h and transfected with miR-219 inhibitors. The relative levels of Oct4, Nestin, and Map2 were detected using qRT-PCR (e) and western blot (f). (g) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. **Po0.01; NS, no significance Figure 2 MiR-219 mediates ESCs to differentiate into neural cells. (a) Confirmation of the effects of miR-219 mimics and inhibitors. (b, c) ESCs were transfected with miR-219 mimics for 48 h; the relative levels of Oct4, Nestin, and Map2 were detected through qRT-PCR (b) and western blot (c). (d) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. (e, f) ESCs were pretreated with RA for 48 h and transfected with miR-219 inhibitors. The relative levels of Oct4, Nestin, and Map2 were detected using qRT-PCR (e) and western blot (f). (g) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. **Po0.01; NS, no significance and Zbtb18 are involved in neural differentiation. Foxj3 and Zbtb18 were transiently transfected to ESCs, and the relative abundance of Nestin was detected. As expected, the Foxj3 or Zbtb18 disrupted the upregulation of Nestin after the miR-219 mimics treatment. Particularly, the synergistic effect of Foxj3 and Zbtb18 returned Nestin expression to basal levels (Figures 4a and b; Supplementary Figures S1F–K). Knock- down experiments were then conducted with small interfering RNA (siRNA) to verify the results. The results showed that Nestin expression was upregulated from 3.5- to 4.5-fold when Foxj3 or Zbtb18 was knocked down, and knockdown of both Foxj3 and Zbtb18 at one time intensified neural differentiation (Supplementary Figures 4C, D). These results suggested that Foxj3 and Zbtb18 prevent the differentiation of ESCs into neural cells. Results This result was verified in D3 and B6 ESCs (Supplementary Figure S1A), suggesting that miR-219 plays an important role in RA-induced ESC differentiation. MiR-219 mediates ESCs to differentiate into neural cells. Next, miR-219 mimics and inhibitors were used to investigate whether or not miR-219 is involved in RA-induced differentiation. The validity of miR-219 mimics and inhibitors were verified in Figure 2a. As expected, the mESCs differentiated into neural-type cells 48 h after transfection of miR-219 mimics. The stem cell marker Oct4 was reduced considerably, whereas Nestin (a marker for neural stem cells), and Map2 and Tuj1 (markers for neural cells) were increased (Figures 2b–d). Furthermore, the ESCs transfected with miR-219 inhibitors resisted the RA-induced neural differentiation. As shown in Figures 2e–g, the miR-219 inhibitors blocked the RA-induced upregulation of Nestin, Map2, and Tuj1. The ESCs were differentiated after RA treatment regardless of the presence of miR-219 inhibitors, as characterized by the decreased Oct4 and loss of tight colony morphology (Figure 2g). Interestingly, the miR-219 inhibitors prevented ESCs from differentiating in a neural directional manner (Figures 2e–g; Supplementary Figures S1B–E). These findings suggested that miR-219 mediates ESCs to differentiate into neural cells. Foxj3 and Zbtb18 prevent ESCs from differentiating into neural cells. MiR-219 promotes the neural differentiation ESCs (Figures 2e–g), targeting Foxj3 and Zbtb18 (Figures 3b–d). We then investigated whether or not Foxj3 Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al 3 Figure 2 MiR-219 mediates ESCs to differentiate into neural cells. (a) Confirmation of the effects of miR-219 mimics and inhibitors. (b, c) ESCs were transfected with miR-219 mimics for 48 h; the relative levels of Oct4, Nestin, and Map2 were detected through qRT-PCR (b) and western blot (c). (d) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. (e, f) ESCs were pretreated with RA for 48 h and transfected with miR-219 inhibitors. The relative levels of Oct4, Nestin, and Map2 were detected using qRT-PCR (e) and western blot (f). (g) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. **Po0.01; NS, no significance Figure 2 MiR-219 mediates ESCs to differentiate into neural cells. (a) Confirmation of the effects of miR-219 mimics and inhibitors. Results The ES cell lines that stably expressed Foxj3 and Zbtb18 were produced by pCDH-Puro-Foxj3/Zbtb18 lentivirus to investigate the functional roles of Foxj3 and Zbtb18 in neural differentiation. The resulting cell lines were used for in vitro differentiation under RA treatment. Compared with normal ESCs, Foxj3/Zbtb18-overexpressing (OE) ESCs Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al 4 ure 3 Foxj3 and Zbtb18 are the targets of miR-219. (a) 3′-UTR analysis of Foxj3 and Zbtb18 containing putative regions that match the seed sequence of miR-219. (b) A after NIH/3T3 fibroblast cells were transfected with miR-219 mimics, luciferase reporter constructs containing WT or MUT-type UTRs were transfected as indicated. Ce es were harvested for DLR assays. (c–e) MiR-219 mimics or inhibitors were transfected to ESCs. After 48 h, cells were harvested for qRT-PCR (c) and western blot (d) t the relative levels of Foxj3 and Zbtb18. The relative intensities of the protein bands (e) were quantified with Image J software and calculated using the samples normalize actin. All data are presented as mean ± S.D. and derived from three independent experiments. Scale bars, 30 μm. **Po0.01 igure 3 Foxj3 and Zbtb18 are the targets of miR-219. (a) 3′-UTR analysis of Foxj3 and Zbtb18 containing putative regions that match the seed sequence of miR-219. (b 4 h after NIH/3T3 fibroblast cells were transfected with miR 219 mimics luciferase reporter constructs containing WT or MUTtype UTRs were transfected as indicated Figure 3 Foxj3 and Zbtb18 are the targets of miR-219. (a) 3′-UTR analysis of Foxj3 and Zbtb18 containing putative regions that match the seed sequence of miR-219. (b) At 24 h after NIH/3T3 fibroblast cells were transfected with miR-219 mimics, luciferase reporter constructs containing WT or MUT-type UTRs were transfected as indicated. Cell lysates were harvested for DLR assays. (c–e) MiR-219 mimics or inhibitors were transfected to ESCs. After 48 h, cells were harvested for qRT-PCR (c) and western blot (d) to detect the relative levels of Foxj3 and Zbtb18. The relative intensities of the protein bands (e) were quantified with Image J software and calculated using the samples normalized to β-actin. All data are presented as mean ± S.D. and derived from three independent experiments. Scale bars, 30 μm. **Po0.01 differentiated but not in a neural directional manner, as characterized by morphology and the expression levels of neural markers Tuj1 and NeuN (Figure 4e). Cell Death and Disease Results Foxj3 and Zbtb18 (Supplementary Table S2, pretreated Foxj3/ Zbtb18-ESC 5.56% versus pretreated ESC 38.9%; represen- tative neural-like tissues are shown in Supplementary Figure S2). Moreover, the Foxj3/Zbtb18-ESCs pretreated with RA retained the ability to form epidermis-like tissues (Figure 4f), suggesting that Foxj3 and Zbtb18 affected neuroectodermal development rather than the development of the entire ectoderm. In addition, the Foxj3/Zbtb18-OE ESCs generated less ectoderm-specific tissues (21.1% versus 52.2%) and more mesoderm-specific (24.4% versus 13.3%) and endoderm-specific (28.9% versus 15.6%) tissues (Supplementary Table S2) compared with RA-pretreated Teratoma formation tests in nude mice were performed to confirm this finding. The Foxj3/Zbtb18-OE ESCs were pretreated with RA for 48 h and injected into nude mice to generate teratomas. The teratomas were harvested 14 days later for further examination. The hematoxylin/eosin- (H&E) stained samples showed that the RA-pretreated ESCs tend to form neuronal-like tissues (Supplementary Table S2, pre- treated 38.9% versus untreated 12.2%), and the neuronal differentiation potential of ESCs was mostly suppressed by Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al 5 Figure 4 Foxj3 and Zbtb18 prevent ESCs from differentiating into neural cells. (a, b) ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of pCMV-Foxj3 or pCMV-Zbtb18 as indicated. After 48 h, the relative level of Nestin was detected through qPCR (a) and western blot (b). Actin served as the loading control. (c, d) ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of Foxj3-siRNA or Zbtb18-siRNA. After 48 h, the relative level of Nestin was detected through qPCR (c) and western blot (d). (e) Foxj3/Zbtb18-OE ESCs were induced by RA for 10 days. The morphology was observed and photographed. Scale bars: 100 μm. (f) Control ESCs or Foxj3/Zbtb18-OE ESCs were pretreated with RA for 48 h and injected into nude mice to generate teratomas. The differentiations of the three germ layers were analyzed through H&E staining. Scale bars: 100 μm. **Po0.01; NS, no significance Figure 4 Foxj3 and Zbtb18 prevent ESCs from differentiating into neural cells. (a, b) ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of pCMV-Foxj3 or pCMV-Zbtb18 as indicated. After 48 h, the relative level of Nestin was detected through qPCR (a) and western blot (b). Actin served as the loading control. Results This finding was supported by the immunohistochemistry analysis results. In the WTembryos, Oct4 was restricted in the posterior region, and Nestin was expressed in the anterior region because of forebrain formation (Figures 5B, a and b). However, Oct4 disappeared and Nestin was detected in the entire ectoderm of the miR-219-injected embryos (Figures 5B, c and d). These results indicated that miR-219 induces neural development in the mouse embryos. On the contrast, embryos injected with Foxj3/Zbtb18 mRNAs formed egg cylinders at E6.5 (Figures 5A, e and f), but 48.8% (40/82) of the sectioned embryos showed quick degeneration and various abnormal phenotypes, 23.2% (19/82) of the embryos were resorbed completely or left a trace of residual pyknotic tissue by E7.5 (Figure 5B, e and f; Supplementary Figure S4). The extremely high rate of mutants in the miR-219- and Foxj3/Zbtb18- injected embryos implied that the normal expression of Foxj3/ Zbtb18 is crucial in early embryonic development in mice. ( pp y g pp y ) To locate the core regulatory genes involved in neural regulatory networks, gene co-expression networks were constructed with the overlapping genes in the Foxj3-ES and Zbtb18-ES (Figure 6a). Core regulatory factors were then identified on the basis of the degree of differences between the control and Foxj3/Zbtb18-ES groups. The genes with a difference degree of more than eight were selected. Five genes, namely, Olig1, Zic5, Erbb2, Numbl, and Olig2, met the specification in the Zbtb18-ES group. Meanwhile, eight genes, namely, Olig1, Shank1, Erbb2, Zic5, Smarcd3, Ina, Chd7, and Olig2, met the specifications in the Foxj3-ES group. In addition, the following genes were present in both groups: Olig1, Zic5, Erbb2, and Olig2 (Figures 6b and c). We then performed qRT-PCR to validate the relationships of these genes with miR-219, Foxj3, and Zbtb18. As expected, Olig1, Zic5, Erbb2, and Olig2 were upregulated two-fold to five-fold when the ESCs were induced by RA treatment or miR-219 mimics, whereas they were dramatically inhibited when Foxj3 or Zbtb18 was overexpressed (Figure 6d). These findings were consistent with the RNA-seq results. To understand the roles of Foxj3, Zbtb18, and the four genes involved in transcription factor networks during RA-dependent neural Olig1 and Olig2 are important in miR-219-mediated neural differentiation. The total mRNAs of Foxj3-ES and Zbtb18-ES were extracted and used for RNA-seq and transcriptome analyses to understand the specific mechan- ism by which Foxj3 and Zbtb18 control neural differentiation of ESCs. Results (c, d) ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of Foxj3-siRNA or Zbtb18-siRNA. After 48 h, the relative level of Nestin was detected through qPCR (c) and western blot (d). (e) Foxj3/Zbtb18-OE ESCs were induced by RA for 10 days. The morphology was observed and photographed. Scale bars: 100 μm. (f) Control ESCs or Foxj3/Zbtb18-OE ESCs were pretreated with RA for 48 h and injected into nude mice to generate teratomas. The differentiations of the three germ layers were analyzed through H&E staining. Scale bars: 100 μm. **Po0.01; NS, no significance of miR-219 and Foxj3/Zbtb18 on developing embryos between egg-cylinder and primitive streak stages. MiR-219 agomirs or Foxj3/Zbtb18 mRNAs were injected into the zygote cytoplasm. Microinjected embryos that reached the blastocyst stage were transferred to pseudopregnant female recipients. The Oct4 and Nestin expression patterns in the serially sectioned E6.5– E7.5 embryos were then examined by immunohistochemistry analysis (Figure 5). At E6.5, Nestin expression in the miR-219- injected embryos occurred earlier than the control group (Figures 5A,b and d). At E7.5, the differences between the control and miR-219-injected embryos became more appar- ent. Approximately 27.8% (15/54) of the miR-219-injected ESCs. We then tested the expression levels of mesoderm- specific (Brachyury, Flk1) and endoderm-specific (Sox17, Foxa2) markers in ESCs treated with RA or miR-219 antagomirs. The results showed that the increase in the expression levels of the mesoderm- and endoderm-specific markers in ESCs transfected with miR-219 antagomirs were larger than that in the RA-treated cells (Supplementary Figure S3). These data indicated that RA-induced ESCs tend to differentiate into mesodermal and endodermal cells upon miR-219 inhibition. To elucidate the role of miR-219-Foxj3/Zbtb18 in the regulation of neural induction in vivo, we examined the effects Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al 6 Figure 5 MiR-219 induces neural development in mouse embryos. MiR-219 agomirs, Foxj3/Zbtb18 mRNAs, and their corresponding negative controls were microinjected into the cytoplasm of zygotes as indicated. The expression patterns of Oct4 and Nestin in the serially sectioned E6.5 and E7.5 embryos were examined by immunohistochemistry analysis. (A) Immunohistochemistry analysis of the E6.5 embryos. Representative images are shown. Black dashed frames in E6.5 embryos indicate the embryonic region. (B) Immunohistochemistry (a–d) and H&E staining (e, f) analysis of E7.5 embryos. Results a and b show the representative control embryo and indicate how the embryonic region is organized and how the ectodermal and endodermal tissues can be distinguished. c and d show the representative abnormal miR-219-injected embryo, which exhibits a severe overdevelopment in its anterior region. (e, f) show representative abnormal Foxj3/Zbtb18 mRNA-injected embryo with disorganized embryonic region, apparent arrested development, or resorption. Full images of serially sectioned embryos are shown in Supplementary Figure S4. a, anterior; ac, amniotic cavity; al, allantois; d, distal; exc, exocoelomic cavity; p, posterior; px, proximal. Scale bars, 200 μm Figure 5 MiR-219 induces neural development in mouse embryos. MiR-219 agomirs, Foxj3/Zbtb18 mRNAs, and their corresponding negative controls were microinjected into the cytoplasm of zygotes as indicated. The expression patterns of Oct4 and Nestin in the serially sectioned E6.5 and E7.5 embryos were examined by immunohistochemistry analysis. (A) Immunohistochemistry analysis of the E6.5 embryos. Representative images are shown. Black dashed frames in E6.5 embryos indicate the embryonic region. (B) Immunohistochemistry (a–d) and H&E staining (e, f) analysis of E7.5 embryos. a and b show the representative control embryo and indicate how the embryonic region is organized and how the ectodermal and endodermal tissues can be distinguished. c and d show the representative abnormal miR-219-injected embryo, which exhibits a severe overdevelopment in its anterior region. (e, f) show representative abnormal Foxj3/Zbtb18 mRNA-injected embryo with disorganized embryonic region, apparent arrested development, or resorption. Full images of serially sectioned embryos are shown in Supplementary Figure S4. a, anterior; ac, amniotic cavity; al, allantois; d, distal; exc, exocoelomic cavity; p, posterior; px, proximal. Scale bars, 200 μm differentiation, and thus the genes located in the four overlapping biological processes (i.e., nervous system development, central nervous system development, neuron fate commitment, and forebrain development) of the enriched GO terms were selected for analysis. A total of 37 candidate genes in the Foxj3-ES group and 39 candidate genes in the Zbtb18-ES group were present. Between these groups, 17 genes overlapped, namely Arid1a, Camk2b, Chd7, Erbb2, Glis2, Gnao1, Id2, Ina, Map1s, Ndn, Numbl, Olig1, Olig2, Shank1, Smarcd3, Zic2, and Zic5. We hypothesized that these genes contribute to RA-mediated neural differentiation. Their expression levels were verified through qRT-PCR (Supplementary Figure S5C; Supplementary Table S3). embryos were resorbed or had no significant progress in their development, and 38.9% (21/54) of the embryos showed varying degrees of overdevelopment in the anterior region. Results Compared with control, 1331 genes were differen- tially expressed (Po0.05) by two-fold in the Foxj3-ESCs (557 were underexpressed and 774 were overexpressed), and 1175 genes were differentially expressed (Po0.05) by two- fold in Zbtb18-ESCs (548 were underexpressed and 627 were overexpressed). Gene ontology (GO) analysis indicated that 53 GO terms in Foxj3-ES and 54 GO terms in Zbtb18-ES were enriched (Po0.01; Supplementary Figures S5A, B). Foxj3 and Zbtb18 work synergistically to control neural Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al Figure 6 Olig1, Zic5, Erbb2, and Olig2 participate in neural regulatory networks. (a) Co-expression networks of differentially expressed genes in the control, Foxj3-ES, and Zbtb18-ES groups. The overlapping genes were selected to construct gene co-expression networks. Sixteen genes located in the four focused GO terms are arrow-shaped. Solid lines, positively corrected; dashed lines, negatively corrected. (b, c) The genes with difference degrees above eight between Foxj3-ES (b) or Zbtb18-ES (c) and control ESCs. (d) The relative levels of Olig1, Zic5, Erbb2, and Olig2 in the ESCs were detected through qRT-PCR. The ESCs were treated or transfected with RA, pCMV-Foxj3, pCMV-Zbtb18, or miR-219 for 48 h. (e–g) Relative levels of Nestin and Map2 were detected through qRT-PCR (e) and western blot (f) in ESCs transfected with Olig1-, Zic5-, Erbb2-, or Olig2- encoding plasmids for 48 h. Relative intensities of protein bands (g) were quantified by using Image J software and calculated by using the samples normalized to β-actin. All data are presented as mean ± S.D. and derived from three independent experiments. *Po0.05; **Po0.01; NS, no significance Figure 6 Olig1, Zic5, Erbb2, and Olig2 participate in neural regulatory networks. (a) Co-expression networks of differentially expressed genes in the control, Foxj3-ES, and Zbtb18-ES groups. The overlapping genes were selected to construct gene co-expression networks. Sixteen genes located in the four focused GO terms are arrow-shaped. Solid lines, positively corrected; dashed lines, negatively corrected. (b, c) The genes with difference degrees above eight between Foxj3-ES (b) or Zbtb18-ES (c) and control ESCs. (d) The relative levels of Olig1, Zic5, Erbb2, and Olig2 in the ESCs were detected through qRT-PCR. The ESCs were treated or transfected with RA, pCMV-Foxj3, pCMV-Zbtb18, or miR-219 for 48 h. (e–g) Relative levels of Nestin and Map2 were detected through qRT-PCR (e) and western blot (f) in ESCs transfected with Olig1-, Zic5-, Erbb2-, or Olig2- encoding plasmids for 48 h. Discussion these four factors are involved in neural regulatory networks. Notably, only Olig1 and Olig2 facilitate the expression levels of Map2 and Tuj1 (neural cell markers) in the ESCs (Figures 6e–g). We hypothesized that Olig1 and Olig2 are the most dominant downstream factors of miR-219-mediated neural differentiation. Olig1/2 overexpression and knockdown were performed to demonstrate the involvement of Olig1 and Olig2 in the downstream of the RA-miR-219-Foxj3/Zbtb18 pathway. The results showed that Nestin, Map2, and Tuj1 dramatically increased in ESCs with overexpressed Olig1/Olig2, and the levels of neural differentiation were approximate to RA treatment (Figures 7a and b). Moreover, Olig1/2-knockdown ESCs cultured in the presence of RA or transfected with miR-219 mimics failed to differentiate into neural cells, as characterized by the reduction of Oct4 and absence of significant increase in neural cell markers (Map2 and Tuj1) (Figures 7c–e). These results indicated that Olig1 and Olig2 are the main factors regulated by miR-219 in RA-induced neural differentiation. these four factors are involved in neural regulatory networks. Notably, only Olig1 and Olig2 facilitate the expression levels of Map2 and Tuj1 (neural cell markers) in the ESCs (Figures 6e–g). We hypothesized that Olig1 and Olig2 are the most dominant downstream factors of miR-219-mediated neural differentiation. Olig1/2 overexpression and knockdown were performed to demonstrate the involvement of Olig1 and Olig2 in the downstream of the RA-miR-219-Foxj3/Zbtb18 pathway. The results showed that Nestin, Map2, and Tuj1 dramatically increased in ESCs with overexpressed Olig1/Olig2, and the levels of neural differentiation were approximate to RA treatment (Figures 7a and b). Moreover, Olig1/2-knockdown ESCs cultured in the presence of RA or transfected with miR-219 mimics failed to differentiate into neural cells, as characterized by the reduction of Oct4 and absence of significant increase in neural cell markers (Map2 and Tuj1) (Figures 7c–e). These results indicated that Olig1 and Olig2 are the main factors regulated by miR-219 in RA-induced neural differentiation. RA can induce various types of cells in a concentration- dependent manner. For example, a high RA concentration increases the rate of neural differentiation, whereas a low RA concentration induces cardiomyocyte differentiation of mESCS.35 Okada et al.36 reported that RA concentration regulates dorsoventral identity, that is, high RA concentration induces a dorsal phenotype, and low RA concentrations induces a ventral phenotype during in vitro mESC differentia- tion. Results Relative intensities of protein bands (g) were quantified by using Image J software and calculated by using the samples normalized to β-actin. All data are presented as mean ± S.D. and derived from three independent experiments. *Po0.05; **Po0.01; NS, no significance differentiation, we examined the relative expression levels of these genes in the first 10 days of RA-induced differentiation (Supplementary Figure S6). The results showed that Foxj3 and Zbtb18 exhibited a similar expression pattern, that is, their expression levels rapidly declined in the first 2 days, and the temporary increased, and finally returned to a relatively low level during neural differentiation (Supplementary Figures S6A, B). However, the expression levels of Olig1 and Olig2 progressively increased and a sudden rise was observed around day 7 after RA-induced neural differentiation (Supplementary Figures S6C, F). This effect may be attributed to the presence of glial cells and neurons on that day. Furthermore, we evaluated whether these four genes are involved in the neural differentiation. As shown in Figures 6e– g, ESCs were differentiated into neural-type cells 48 h after expression of plasmids that encode Olig1, Zic5, Erbb2, or Olig2 were transfected, as characterized by the dramatic Oct4 reduction and Nestin increase. These results suggested that Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al 8 8 Figure 7 Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. (a, b) Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (a) and western blot (b) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. (c, d) ESCs were transfected with siRNAs that were targeted to Olig1/Olig2 for 12 h and treated with RA or miR-219 mimics for another 36 h. Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (c) and western blot. (e) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. **Po0.01; NS, no significance Figure 7 Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. (a, b) Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (a) a western blot (b) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. (c, d) ESCs were transfected with siRNAs that were targeted to Olig1/Olig2 for 12 h and treat with RA or miR-219 mimics for another 36 h. Results Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (c) and western blot. (e) Immunofluorescence shows t abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. **Po0.01; NS, no significance Figure 7 Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. (a, b) Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (a) and western blot (b) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. (c, d) ESCs were transfected with siRNAs that were targeted to Olig1/Olig2 for 12 h and treated with RA or miR-219 mimics for another 36 h. Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (c) and western blot. (e) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. **Po0.01; NS, no significance Discussion The most familiar mechanism is the binding of RA to RAR, which is bound to a DNA region called the RA response elements and affects the binding of other transcription factors.39–41 Moreover, RA acts through the Hox genes, which ultimately control the anterior and posterior patterns in early developmental stages.13 RA also affects the changes in the epigenetic marks on histones and creates a heritable change in chromatin responsiveness.42–44 Kashyap et al.45 reported that coincident with the RA-induced transcrip- tional activation of the Hoxa and Hoxb cluster genes, the epigenomic configuration of these clusters is rapidly remo- deled by H3K4me3 and acH3. Notably, RA treatment stabilizes p53 in human ESCs, and p53 activates the expression levels of miR-34a and miR-145, which then repress the stem cell factors Oct4, Klf4, Lin28a, and Sox2 and accelerate ESC differentiation.46 These findings sug- gested that miRNAs are important in the regulation of RA- associated differentiation of ESCs. Recent studies have suggested that miR-219 regulates NPC proliferation and differentiation.49,50 However, the specific role of miR-219 in RA-associated ESC differentiation is not elucidated. In this study, we found that miR-219 is sufficient in promoting neural differentiation by targeting Foxj3 and Zbtb18. On the basis of the results of the RNA-seq performed on Foxj3-ESCs and Zbtb18-ESCs, we constructed three gene co-expression networks and identified 17 core genes that are functionally related to neurodevelopment. Interestingly, 16 of these genes were upregulated during neural differentiation, showing that most factors that dominate neural differentiation can be induced. We then demonstrated that Foxj3 and Zbtb18 are the molecular switches in the neurodevelopment system. Furthermore, we identified four critical genes by constructing gene co-expression networks. Two of them, namely, Olig1 and Olig2 might be the most essential elements in neural differentiation of ESCs. Olig1/2-knockdown ESCs cultured in the presence of RA or transfected with miR-219 mimics failed to differentiate into neural cells, as characterized by the absence of increase of neural cell markers (Map2 and Tuj1). Notably, Nestin (neural stem cell marker) appeared in the Olig1/2-knockdown ESCs treated with RA or miR-219 mimics. This finding may be attributed to other factors or mechanisms involved in the miR-219-mediated neural differentiation apart from Olig1/2. However, this hypothesis requires validation through further experiments. Moreover, the mechanism of how RA upregulates miR-219 is not well understood. Discussion We speculate that histone demethylases accelerate DNA demethylation at the promoter region of miR-219, thereby increasing the expression level of miR-219 and facilitating neural differentia- tion. In addition to epigenetic modifications, other transcription factors can possibly mediate the effects of RA on miR-219 upregulation.51 Thus the involvement of histone demethylases or other transcription factors with the RA and miR-219 pathway is interesting for further investigation. To this end, we characterized the expression of miRNAs in mESCs with RA treatment for 48 h. Our previous studies showed that ESCs start to differentiate irreversibly after RA pretreatment for 48 h and continued to do so even after RA withdrawal.12 Therefore, miRNAs with high expression levels during the early stage of RA induction may be critical in RA- induced neural directional differentiation of mESCs. We performed most of the experiments in J1 ESCs to investigate the miR-219-dependent neural regulatory networks, which was later confirmed in D3 and B6 ESCs and in C57BL/6 mice, to ensure that this finding is not a particular characteristic of a single cell line. MiR-219 is necessary in promoting oligodendrocyte differ- entiation, and partially rescues oligodendrocyte differentiation defects caused by total miRNA loss.31 Kocerha et al.47 reported that miR-219 is important in the expression of behavioral aberrations associated with NMDA receptor hypofunction. Santa-Maria et al.48 found that miR-219 down- regulation promotes neurodegeneration by targeting Tau. Overall, we discovered that functionally important gene expression changes mediated by miRNA contribute to RA-induced differentiation. We demonstrated that Foxj3 and Zbtb18, two target genes of miR-219, are the main controls in neural differentiation of ESCs (Figure 8). Our findings illustrate Figure 8 Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional differentiation of ESCs was determined. TF, transcription factor Figure 8 Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional differentiation of ESCs was determined. TF, transcription factor Figure 8 Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Discussion A similar phenomenon was observed in human pluripo- tent stem cells, in which low-dose RA increased hematopoietic progenitor cell generation, whereas low-dose RA abrogated blood cell generation in vitro.37 Here, we found that 1 μM RA is sufficient to induce mESCs that cultured without any stromal cell line, neural culture medium, or neural growth factors undergo neural differentiation. During early embryonic development, RA can facilitate embryonic positioning along the embryonic axis by acting as Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al MiR-219 promotes neural differentiation H Wu et al 9 an intercellular signaling molecule that guides the develop- ment of the posterior portion of the embryo.18,38 Efforts have been undertaken to uncover the mechanism of the effects of RA in embryonic development. The most familiar mechanism is the binding of RA to RAR, which is bound to a DNA region called the RA response elements and affects the binding of other transcription factors.39–41 Moreover, RA acts through the Hox genes, which ultimately control the anterior and posterior patterns in early developmental stages.13 RA also affects the changes in the epigenetic marks on histones and creates a heritable change in chromatin responsiveness.42–44 Kashyap et al.45 reported that coincident with the RA-induced transcrip- tional activation of the Hoxa and Hoxb cluster genes, the epigenomic configuration of these clusters is rapidly remo- deled by H3K4me3 and acH3. Notably, RA treatment stabilizes p53 in human ESCs, and p53 activates the expression levels of miR-34a and miR-145, which then repress the stem cell factors Oct4, Klf4, Lin28a, and Sox2 and accelerate ESC differentiation.46 These findings sug- gested that miRNAs are important in the regulation of RA- associated differentiation of ESCs. an intercellular signaling molecule that guides the develop- ment of the posterior portion of the embryo.18,38 Efforts have been undertaken to uncover the mechanism of the effects of RA in embryonic development. Materials and Methods Ethi t t t Thi t Double-stranded mmu- miR-219-5p (miRBase accession number MIMAT0000664, 5'-UGAUUGUCCA AACGCAAUUCU-3') mimics, single-stranded mmu-miR-219-5p inhibitors and their corresponding negative controls were purchased from GenePharma (GenePharma, Shanghai, China). Negative control siRNA and siRNAs that target Foxj3, Zbtb18, Olig1, and Olig2 were chemically synthesized by Ribobio (Guangzhou, Guangdong, China). The knockdown efficiencies of siRNAs were verified by qRT-PCR and western blot (Supplementary Figure S8). The miRNAs and siRNAs were transfected with Lipofectamine RNAiMAX reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. RA (all-trans-Retinoic acid, product number: R2625) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of 1 mM RA was prepared in absolute ethanol, stored at −20 °C, and protected from light. The working concentration of RA was 1 μM. Culture medium and RA were changed daily and ESCs were passaged every 2–3 days at a 1 : 3–1 : 5 ratio according to cell density. A direct induction system without any stromal cell line, neural culture medium, or neural growth factors as neural inducer was performed to render the normal cultured ESCs a perfect control. RNA-seq and data analysis. Total RNA was extracted from Foxj3-ESCs or Zbtb18-ESCs by Trizol reagent (Invitrogen) separately. Then, the RNA samples were sent to Novel Bioinformatics company (Shanghai, China) for RNA-seq. The RNA quality was checked by Bioanalyzer 2200 (Agilent Technologies, Santa Clara, CA, USA) and kept at −80 °C. The RNA with RIN48.0 is right for cDNA library construction. RNA libraries were prepared for sequencing using IonProton. The cDNA libraries were processed for the proton sequencing process according to the commercially available protocols. Data were submitted to the GEO archive. The accession number is GSE61748. All tested genes sorted by fold changes are listed in Supplementary Data s04-s06. Pathway analysis was used to find out the significant pathway of the differential genes according to KEGG database. Fisher’s exact test was calculated to select the significant pathway, and the threshold of significance was defined by P-value and false discovery rate (FDR).53 GO analysis was performed to facilitate elucidating the biological implications of unique genes in the significant or representative profiles of the differentially expressed gene in the experiment. GO annotations were downloaded from NCBI (http://www. ncbi.nlm.nih.gov/), UniProt (http://www.uniprot.org/), and the GO (http://www. geneontology.org/). Fisher’s exact test was applied to identify the significant GO categories and FDR was used to correct the P-values. Construction of plasmids. Materials and Methods Ethi t t t Thi t Materials and Methods Ethic statement. This study was carried out in strict accordance with the Guidelines for the Care and Use of Animals of Northwest A&F University. All animal experimental procedures were approved by the Animal Care Commission of the College of Veterinary Medicine, Northwest A&F University. C57BL/6 mice were purchased from Xi’an Jiao-tong University, China. Five-week-old female athymic nude mice were purchased from The Fourth Military Medical University. Every effort was made to minimize animal pain, suffering, and distress, and reduce the number of animals used. Cell culture, transient transfection, and treatment. J1, R1, and D3 mESCs were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and grown adherent to plastic plates (Corning Costar, Cambridge, MA, USA) coated with matrigel (Geltrex, Thermo Fisher Scientific, San Jose, CA, USA) and maintained under a feeder-free and serum-free system. The ESC culture medium was composed of Knockout Dulbecco's modified Eagle's medium (Knockout DMEM, Thermo Fisher Scientific), 15% (v/v) Knockout Serum Replacement (Thermo Fisher Scientific), 1 × non-essential amino acids (Thermo Fisher Scientific), 100 μM β-mercaptoethanol (Millipore, Bedford, MA, USA), 2 mM glutamine (Thermo Fisher Scientific), 50 units/ml penicillin, 50 μg/ml streptomycin, and 1000 units/ml LIF (ESGRO, Millipore). The growth condition of mESCs was consistent throughout this study. Western blot analysis. Western blot analysis was performed as previously described.52 Blots were probed with 1/1000 rabbit anti-Oct4 (#2788), 1/1000 rabbit anti-MAP2 (#4542) (Cell Signaling Technology, Beverly, MA, USA), 1/500 rabbit anti- Foxj3 (#SAB2100844), 1/500 rabbit anti-Zbtb18 (#SAB2103436), 1/1000 mouse anti-Nestin (#MAB5326), 1/1000 rabbit anti-Brachyury (#B8436), 1/2000 mouse anti-β-actin (#A5441) (Sigma-Aldrich), 1/1000 goat anti-Flk1 (VEGF receptor 2, #AF644; R&D Systems, Minneapolis, MN, USA), 1/1000 mouse anti-Sox17 (#ab192453), 1/1000 rabbit anti-Foxa2 (#ab108422) (Abcam, Cambridge, MA, USA), and 1/1000 mouse anti-Tuj1 (neuronal class III β-tubulin, #MMS-435P; Covance, Princeton, NJ, USA). Immunoblots were revealed by autograph using SuperSignal west pico substrate (Thermo Fisher Scientific). The relative intensity of the protein bands was quantified using Image J software (NIH, Bethesda, MD, USA) and calculated by samples normalized to the controls. All data were presented as mean ± S.D. and derived from three independent experiments. Plasmids were transfected with Xfect Transfection Reagent (Clontech, Palo Alto, CA, USA) following the manufacturer’s instructions. The transfection efficiency was monitored by a backbone vector with an extra eGFP element and calculated by flow cytometry. The average transfection efficiency was ~ 75%–85%, and the representative result is shown in Supplementary Figure S7. Discussion Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional differentiation of ESCs was determined. TF, transcription factor Figure 8 Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional differentiation of ESCs was determined. TF, transcription factor Cell Death and Disease MiR-219 promotes neural differentiation H Wu et al 10 the mechanism of miRNA-mediated neural differentiation of ESCs, as well as the plasticity and dynamic nature of the gene regulatory networks during neural differentiation. However, the Foxj3 and Zbtb18 regulatory activities dependent on other transcription factors remain unclear. The specific mechanism of Foxj3 and Zbtb18 for determining the fate of neural differentiation and the functional roles of Foxj3 and Zbtb18 in multiple biological processes requires further investigation. the mechanism of miRNA-mediated neural differentiation of ESCs, as well as the plasticity and dynamic nature of the gene regulatory networks during neural differentiation. However, the Foxj3 and Zbtb18 regulatory activities dependent on other transcription factors remain unclear. The specific mechanism of Foxj3 and Zbtb18 for determining the fate of neural differentiation and the functional roles of Foxj3 and Zbtb18 in multiple biological processes requires further investigation. Luciferase assays. Luciferase assays were performed using the DLR Assay System (Promega) as previously described.52 In brief, ESCs were cotransfected with a luciferase reporter construct and internal control plasmid pRL-SV40 (Promega). Cells were lysed 24 h after transfection or treatment, and relative luciferase activities were measured by firefly luciferase luminescence divided by Renilla luciferase luminescence. Luciferase assays. Luciferase assays were performed using the DLR Assay System (Promega) as previously described.52 In brief, ESCs were cotransfected with a luciferase reporter construct and internal control plasmid pRL-SV40 (Promega). Cells were lysed 24 h after transfection or treatment, and relative luciferase activities were measured by firefly luciferase luminescence divided by Renilla luciferase luminescence. Real-time qRT-PCR. We used qRT-PCR to analyze miRNA expression. Total RNA samples (including small RNA molecules) were isolated using mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). Purified RNA was reverse-transcribed using a miScript II RT Kit (Qiagen, Hilden, Germany). Discussion The expression of mature miRNAs was quantified using a miScript SYBR Green PCR Kit, which contained 10× miScript Universal Primer (Qiagen), and was performed according to the manufacturer’s instructions. Quantization of U6 was performed to normalize miRNA expression levels. We also used qRT-PCR to analyze gene expression. Total RNA samples were isolated using Trizol reagent (Invitrogen). Purified RNA was reverse-transcribed using a SYBR PrimeScript RT–PCR Kit (Takara, Otsu, Shiga, Japan). The expression of mRNAs was quantified using a SYBR Premix ExTaq II Kit (Takara). Real-time PCR was performed on an ABI StepOnePlus PCR system (Applied Biosystems, Foster City, CA, USA), and results were normalized to β-actin mRNA levels. Data were analyzed using the 2  ΔΔCt method. Primer sequences used for qPCR are listed in Supplementary Tables S6 and S7. PUBLISHER’S NOTE Teratoma formation, H&E staining, and scoring method. The ESCs with stable expressions of Foxj3/Zbtb18 were pretreated with RA for 48 h, and injected to nude mice to generate teratomas. The differentiations of the three germ layers were analyzed by H&E staining. In brief, teratomas were fixed with 10% buffered formaldehyde for more than 24 h, embedded in paraffin, sectioned, and stained with H&E according to the standard procedure. 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These fragments were inserted into pCMV-HA (Clontech; verified in Supplementary Figure S8), pCDH-MCS-T2A- Puro-MSCV (System Biosciences, Palo Alto, CA, USA), and pCAG-EGFP (kindly provided by Dr Wei Zhang) vectors. The 3′-UTR of Foxj3 (1084 bp in length. Located at bases 2041–3124 refer to NM_172699.3; –34 to +1050 if consider base 2075 as the start site of UTR) and Zbtb18 (936 bp in length. Located at bases 2886–3821 refer to NM_001012330.1; +1141 to + 2076 if consider base 1746 as the start site of UTR) were amplified from J1 cDNA and inserted into the psicheck-2 vector (Promega, Madison, WI, USA) through standard molecular cloning methods and confirmed by sequencing. All the primers used for plasmid construction are listed in Supplementary Tables S4 and S5. Gene co-expression networks were presented to find the relations among genes. Gene co-expression networks were built according to the normalized expression values of genes selected from genes in significant GO terms. 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Cell Death and Disease is an open-access journal published by Nature Publishing Group. Conflict of Interest Conflict of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. 29. Di Padova M, Caretti G, Zhao P, Hoffman EP, Sartorelli V. MyoD acetylation influences temporal patterns of skeletal muscle gene expression. J Biol Chem 2007; 282: 37650–37659. Acknowledgements. We would like to thank Dai Chen, Mingfei Feng, and Lianle Bian of Novel Bioinformatics company for their technical assistance during the conduct of bioinformatics analysis; Jiaxing Lv and Jiong Chen for their contribution in plasmid construction, and Prof. Zhang of the Fourth Military Medical University for his assistance in nude mice feeding and H&E analysis. This work was funded by State Key Program of National Natural Science Foundation of China (No. 31530075) and China Postdoctoral Science Foundation Grant (No. 2016M590978). 30. 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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/ 43. Kashyap V, Gudas LJ. Epigenetic regulatory mechanisms distinguish retinoic acid-mediated transcriptional responses in stem cells and fibroblasts. J Biol Chem 2010; 285: 14534–14548. 44. Tang S, Huang G, Fan W, Chen Y, Ward JM, Xu X et al. SIRT1-mediated deacetylation of CRABPII regulates cellular retinoic acid signaling and modulates embryonic stem cell differentiation. Mol Cell 2014; 55: 843–855. 45. Kashyap V, Gudas LJ, Brenet F, Funk P, Viale A, Scandura JM. Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid. 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Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone
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ORIGINAL RESEARCH published: 01 June 2016 doi: 10.3389/fpls.2016.00713 Elena Gottardini 1, Antonella Cristofori 1, Elisa Pellegrini 2*, Nicola La Porta 1, 3, 4, Cristina Nali 2, Paolo Baldi 5 and Gaurav Sablok 1, 6 Elena Gottardini 1, Antonella Cristofori 1, Elisa Pellegrini 2*, Nicola La Porta 1, 3, 4, Cristina Nali 2, Paolo Baldi 5 and Gaurav Sablok 1, 6 1 Fondazione Edmund Mach, Sustainable Agro-Ecosystems and Bioresources Department, Research and Innovation Centre, Trento, Italy, 2 Department of Agriculture, Food and Environment, University of Pisa, Pisa, Italy, 3 MOUNTFOR Project Centre, European Forest Institute, Trento, Italy, 4 Consiglio Nazionale delle Ricerche, Istituto per la Valorizzazione del Legno e delle Specie Arboree, Florence, Italy, 5 Fondazione Edmund Mach, Genomics and Biology of Fruit Crops Department, Research and Innovation Centre, Trento, Italy, 6 Plant Functional Biology and Climate Change Cluster (C3), University of Technology Sydney, Sydney, NSW, Australia Edited by: Edited by: Mohammad Anwar Hossain, Bangladesh Agricultural University, Bangladesh Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O − 3 treatment (60 ppb, 5 h d 1 for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts Mohammad Anwar Hossain, Bangladesh Agricultural University, Bangladesh Reviewed by: Golam Jalal Ahammed, Zhejiang University, China Marcelo Falsarella Carazzolle, State University of Campinas, Brazil *Correspondence: Elisa Pellegrini elisa.pellegrini@for.unipi.it Specialty section: This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science Received: 18 December 2015 Accepted: 09 May 2016 Published: 01 June 2016 Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone Elena Gottardini 1, Antonella Cristofori 1, Elisa Pellegrini 2*, Nicola La Porta 1, 3, 4, Cristina Nali 2, Paolo Baldi 5 and Gaurav Sablok 1, 6 June 2016 | Volume 7 | Article 713 INTRODUCTION respect, it is important to categorize acute vs. chronic exposures, respectively in short- and long-time treatments (Miller, 2011). Ozone (O3) is a gas naturally present in both troposphere and stratosphere. Particularly, tropospheric O3 results from a series of complex photochemical reactions involving primary pollutants, such as nitrogen oxides (NOx), volatile organic compounds (VOC) and carbon monoxide (CO) mainly generated by human activities (Jenkin and Clemitshaw, 2000). O3-producing photochemical reactions are favored by high temperatures and elevated light intensities (Cristofanelli and Bonasoni, 2009). During summertime, the Mediterranean basin is characterized by specific meteorological conditions (i.e., sunny, hot, and dry climate) that enhance photochemical O3 formation (Millàn et al., 2000). At the mid-latitudes of the Northern Hemisphere, O3 concentrations have more than doubled over the last decades (Monks et al., 2015). Especially in Europe, a general trend toward a decline in peak concentrations has been observed, taking into account the implementation of European air pollution policies reducing precursor emissions. On the other hand, Dawnay and Mills (2009) documented a concomitant increase in O3 background concentrations, due to the rise in anthropogenic emissions on a global scale. The ambient O3 concentrations have a marked impact not only on human health (Yang and Omaye, 2009; Norval et al., 2011), but also on natural ecosystems, crop productivity (yield and quality), manufactures and works of art (Cass et al., 1989). For these reasons, it is important to understand the regulatory behavior of O3 induced stress in plants (e.g., Braun et al., 2014; Doring et al., 2014a; González-Fernández et al., 2014). Taking into account the literature, a bio-molecular approach might enable a better understanding of oxidative stress-plant interactions. Current knowledge concerning specific molecular alterations caused by O3 at the transcriptomic level is limited (Heath, 2008; Kanter et al., 2013) and has been primarily elucidated (i) in model plants, like Arabidopsis thaliana (Hirayama and Shinozaki, 2010) and Medicago truncatula (Puckette et al., 2009, 2012); (ii) in crops, like Pisum sativum (Savenstrand et al., 2000), Oryza sativa (Frei, 2015; Sarkar et al., 2015); and (iii) in herbaceous annual plants, like Centaurea jacea (Francini et al., 2008), Melissa officinalis (Doring et al., 2014b). Moreover, interactions occurring between O3 exposure and changes in the expression profile of several genes have been described in some woody species. Olbrich et al. INTRODUCTION (2010) studied transcriptional responses in juvenile Fagus sylvatica saplings fumigated with a double concentration of ambient O3 over 3 years by conducting microarray hybridization. Recently, the effects of O3 (twice ambient concentration) on F. sylvatica were investigated performing a large-scale protein analysis based on 2-D Fluorescence Difference Gel Electrophoresis (2-DE DIGE) (Kerner et al., 2014). Rizzo et al. (2007) identified the differential expression of genes induced by an episodic O3 treatment (150 ppb for 5 h) in two poplar clones with different O3-sensitivity, performing Suppression Subtractive Hybridization (SSH). Nathaniel et al. (2011) investigated the transcriptional and genetics O3 responsiveness (chronic and acute treatment) in two divergent Populus species. Using an inbred F2 mapping population derived from these two species, they mapped quantitative trait loci (QTLs) associated with O3 response, and examined segregation of the transcriptional response to O3 and co-localized genes showing divergent responses between tolerant and sensitive genotypes. Tuomainen et al. (1996) studied the acute O3-induced reactions at biochemical and transcriptomic levels in two Betula clones differing in O3-sensitivity, whereas Zinser et al. (2000) focalized on Pinus sylvestris. Similarly, Kontunen-Soppela et al. (2010) revealed the patterns of gene expression in Betula papyrifera leaves exposed to twice ambient O3 concentration using microarray analyses. After entering into the leaf via the open stomata, O3 interacts immediately with biological molecules (like bio-membranes and enzymes) and releases reactive oxygen species (ROS), thereby triggering an oxidative burst (Jaspers et al., 2005). Plants deploy several response mechanisms, some of which are universally conserved among species (Whaley et al., 2015). Through a signaling cascade (Vainonen and Kangasjärvi, 2015), O3 affects primarily biological processes involved in plant productivity, such as regulation of structural and chemical components of photosynthesis (Pellegrini et al., 2015), respiration and transpiration (Heath, 2008). The toxicology of this pollutant is complex: many factors such as species, provenance, genotype and leaf age together with environmental, nutritional, and health conditions, play a key role in determining the overall plant response (Manninen et al., 2009). Moreover, contrasting results may be caused by different O3 concentrations as well as by different spatial and temporal scales of this pollutant. Citation: Gottardini E, Cristofori A, Pellegrini E, La Porta N, Nali C, Baldi P and Sablok G (2016) Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone. Front. Plant Sci. 7:713. doi: 10.3389/fpls.2016.00713 June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org Gottardini et al. Ozone-Molecular Response of Wayfaring for several functional categories, including specific transcription factors (MYB and WRKY). This study demonstrates that a complex sequence of events takes place in the cells at intracellular and membrane level following O3 exposure and elucidates the effects of this oxidative stress on the transcriptional machinery of the non-model plant species V. lantana, with the final aim to provide the molecular supportive knowledge for the use of this plant as O3-bioindicator. Keywords: SSH, gene ontology, photosynthesis, detoxification, HSP20-like chaperone, PCR-selectTM, lipoxygenase activity Frontiers in Plant Science | www.frontiersin.org Cultural Practices, Plant Material, and Ozone Exposure One-year-old agamically reproduced saplings of V. lantana were grown for 1 month in plastic pots containing a mix of steam sterilized medium soil and peat (1:1) in a controlled environment facility (steady temperature of 20 ± 1◦C, relative humidity (RH) of 85 ± 5% and photon flux density at plant height of 530 µmol photon m−2 s−1 provided by incandescent lamps, following a 14 h photoperiod). A sub-sample of 18 uniform plants were selected when they were ca. 35 cm tall (ca. 30 fully expanded leaves), and were placed in a controlled fumigation environment facility under the same climatic conditions as the growth chamber. Nine plants were exposed to 60 ± 13 ppb of O3 (1 ppb = 1.96 µg m−3, at 20◦C and 101.325 kPa) for 45 consecutive days (5 h d−1, in form of a square wave between 9:00 a.m. and 2:00 p.m.). At the same time, nine control plants were exposed to charcoal-filtered air. The entire methodology was performed according to Lorenzini et al. (1994). Leaf samples (n = 5) were collected from three treated and three control plants after 15 days of fumigation (before the onset of foliar symptoms), immediately frozen in liquid nitrogen (N2) and kept at −80◦C for RNA extraction. INTRODUCTION In this In this study, we focused on wayfaring tree (Viburnum lantana L.), a common deciduous shrub species, widespread in most part of Europe, North Africa, North America and temperate Asia, and well-known for its sensitivity to O3 (Novak et al., Frontiers in Plant Science | www.frontiersin.org June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 2 Ozone-Molecular Response of Wayfaring Gottardini et al. was added. After 30 min on ice, the RNA was selectively pelleted by centrifugation (21,000 × g for 20 min at 4◦C) and re- suspended in 500 µl of SSTE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8), 1% SDS, 1 M NaCl], pre-heated at 65◦C. An equal volume of chloroform:isoamyl alcohol (24:1 v/v) was added, centrifuged (11,000 × g for 10 min at 4◦C) and the supernatant was transferred to a new tube and precipitated with one volume of cold isopropanol. The pellet was washed with ethanol (70%), air-dried and re-suspended in DEPC-treated water. Messenger RNA was isolated using GenEluteTM mRNA Miniprep kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. SSH was performed using PCR- selectTMcDNA subtraction kit (Clontech Laboratories, Mountain View, CA, USA) following the procedure described in the user manual. Forward and reverse subtractions were performed using control and O3-treated leaves and PCR products were subsequently sequenced using 454-pyrosequencing (GS FLX+ System, Roche Diagnostics GmbH, Penzberg, Germany). Raw reads obtained from the present study are recorded in the European Nucleotide Archive (ENA) under the project number PRJEB9317. 2003; Calatayud et al., 2010). Wayfaring tree sensitivity has been already assessed in terms of morphological (i.e. foliar symptoms) and physiolgical traits in order to evaluate its potential as bio- indicator (Gottardini et al., 2010, 2014a,b). Although a series of studies has been conducted on Viburnum spp. (Clement and Donoghue, 2012), this report represents the first attempt to assess the O3 sensitivity of this species at molecular level. Specifically, paired SSH and 454-pyrosequencing analyses were performed in plants exposed to near-ambient O3 concentrations in controlled environmental conditions. Data collected in this study may be useful to better understand results obtained in natural field conditions. Sequence Cleaning, Processing, and Functional Annotation Sequencing reads, obtained from the two libraries (induced and repressed), were cleaned, removing adapter and primer sequences and trimming low-quality ends. We further removed reads shorter than 100 bp and having an average Phred equivalent quality score lower than 15 bp. After read cleaning, homo-polymer stretches (polyA/T) were estimated and were subsequently masked using an in-house PERL script. Masking of the polyA/T was done to increase the sensitivity and specificity in the assembly. For the creation of the Ensembl transcriptome, all the cleaned reads of the induced and repressed libraries were concatenated and a single Ensembl transcriptome was built using MIRA (Chevreux et al., 2004) and CAP3, an overlap layout consensus assembly approach (Huang and Madan, 1999) with an overlap percentage identity cut-offof 97%, to avoid the formation of the spurious assemblies. Resulting contigs and singletons were clustered into representative set of unigenes for each library. All the repetitive reads that were falling into the mega-hub during the MIRA assembly were discarded so as to ensure the correctness and the accuracy of the assembled unigenes. Symptom Assessment Each marked leaf was scored in percent of O3-damaged surface (5%-classes) by in-hand examination with a 10x hand lens and symptoms identified as reported by Gottardini et al. (2014a). For each plant, the mean percentage of injured leaf area of the marked leaves per date was calculated. Following unigene assembly, unigenes in each library were subjected to BLASTx with an E-value cut offof 1E-5 against NCBI database available from http://www.ncbi.nlm.nih.gov. All the unigenes were translated into six possible translational frames using the sixpack package of the EMBOSS available from http:// emboss.sourceforge.net/ and the putative open-reading frames (ORF) were extracted using the Getorf of the EMBOSS package. All the translated frames were queried for the identification of the InterPro domains and the longest frame with no internal stop codon and with assigned InterPro domain was kept as an assigned functional domain to that respective unigene. FastAnnotator (Chen T. W. et al., 2012) and PLAZA version 2.5 (Van Bel et al., 2013) were run to identify functional annotations Read Generation and De novo Assembly for Plants Exposed to O3 Stress associated with the unigenes. Gene Ontology (GO) was derived for each unigenes, slimmed using the plant GO slim (available from http://www.geneontology.org) and classified according to biological and molecular functions and cellular localization. Transcription factors were identified using the PlantTFcat (Dai et al., 2013). p 3 A total of 43,815 and 13,610 high quality reads were generated in induced and repressed libraries, respectively (Table 1). Following sequencing, reads were filtered as described in the Materials and Methods section, resulting in a total of 38,800 and 12,495 clean reads (Table 1). The mean read length for the induced and repressed library was 319 ± 156.7 and 255 ± 107.4 bp, respectively (data not shown). 454-assembly using MIRA of the Ensembl transcriptome yielded a total of 1238 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (GC = 49.9%, Table 1). ORF predictions revealed a total of 161 (13.0%) sequences with a proper start codon and a total of 574 sequences (46.3%) with proper stop codons. Interestingly, a low number of unigenes (73, 5.9%) with predicted ORF contain frameshifts, which indicates a good quality of the assembly. Identified frameshifts were corrected using FrameDP (Gouzy et al., 2009). Expression Assessment using Read Mapping Back to Assembled Ensemble Transcriptome To evaluate the expression levels of the unigenes in induced/repressed libraries, we mapped the individual library reads back to the ensembl transcriptome and reads per kilobase per million (RPKM) was calculated as expression estimate: RPKM (A) = 1,000,000 × C × 1000)/(N × L), where A is defined as the expression of the unigene, C corresponds to the reads that align uniquely to the unigene, N refers to total number of reads that uniquely aligned to all genes, and L refers to the length of gene A. Transcript mapping was performed using BWA-SW algorithm, as implemented in Burrows-Wheeler Aligner [BWA, available from http://bio-bwa.sourceforge.net/; Li and Durbin (2009)]. Expression values were further analyzed to identify the transcripts whose expression was significantly up- or down- regulated during the O3 treatment, using the log2 fold change (RPKM induced/RPKM repressed), as previously described in Kanter et al. (2013). To identify the functionally and statistically enriched biological pathways and GOs in the up- and down- regulated unigenes, those showing up- and down-regulation in Ensembl unigenes were analyzed using KOBAS, with A. thaliana as a background dataset. All the identified biological pathways and GOs were statistically evaluated using the hypergeometric test/Fisher’s exact test followed by Benjamini and Hochberg FDR correction [P < 0.001 (Mao et al., 2005; Wu et al., 2006; Xie et al., 2011)]. Functional Classification for Plants Exposed to O3 Functional classification of the assembled unigenes indicates the putative functional changes occurring at gene level in plants subjected to O3. The Ensembl transcriptome was annotated by performing stringent BLASTx searches (E-value threshold, 1E-5) against NCBI and PLAZA version 2.5. Functional annotation of the Ensembl transcriptome (1,238 unigenes) revealed a total of 744 transcript sequences (60.1%), with an assigned GO category and a total of 772 transcript sequences (62.4%), with an assigned InterPro domain, respectively (Table 1 and Supplementary Table 1 and Supplementary Datasheet 1). The results of BLASTx (E-value threshold, 1E-5) searches against NCBI database and PLAZA version 2.5 (Van Bel et al., 2013) resembled those reported for whole genome expressed genes and tissue-specific cDNA extracts in other plant species (Legrand et al., 2007, 2010; Hao et al., 2015). Based on GO-slim annotations, Ensembl unigenes were classified into three ontological categories: cellular component, biological RNA Extraction and PCR-Select for Library Creation Total RNA was extracted from leaves using the protocol described by Gambino et al. (2008). Frozen leaves were ground to fine powder using a pre-chilled mortar and a pestle. Five milliliter of extraction buffer [2% CTAB, 2.5% PVP-40, 2 M NaCl, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0), and 2% β-mercaptoethanol, added just before use] were heated at 65◦C and added to 1 g of ground tissue. After 10 min of incubation at 65◦C, two independent extractions were performed, using chloroform:isoamyl alcohol (24:1 v/v). The supernatant was transferred to a new tube and LiCl (3 M final concentration) Frontiers in Plant Science | www.frontiersin.org June 2016 | Volume 7 | Article 713 3 Ozone-Molecular Response of Wayfaring Gottardini et al. Frontiers in Plant Science | www.frontiersin.org Expression Estimates and Functional Profiling of Up- and Down-Regulated Unigenes FIGURE 1 | Functional classification of Viburnum lantana ensembl unigenes within cellular component (A), biological process (B), and molecular function (C) categories. g Expression abundance for the unigenes was estimated by mapping the reads from the individual library to the assembled Ensemble transcriptome and was assessed as number of reads per RPKM. According to Kanter et al. (2013), prior to linking the expression estimates to biological functions, unigenes having a RPKM lower than 7 were discarded to avoid the false interpretation of the log2 fold change values. A total of 78 (6.3%) Ensembl unigenes showed a variation in expression values after O3-treatment. In particular, 41 ensembl unigenes showed an up- regulation average [log2 (RPKM induced/RPKM repressed) of 3.7 (2.1 SE) ranged between 0.4 and 8.7 (Table 2)] and the other 37 showed a down-regulation [log2 (RPKM induced/RPKM repressed) ranged between –0.1 and –8.7 (Table 3)]. Functional ontology and the assigned InterPro domain of the up- and down- regulated unigenes (30 and 29, respectively) are tabulated in the Supplementary Table 2. We observed induction of unigenes (EnsVib0416 and EnsVib0041), which are functionally associated to heat-shock proteins (IPR008978, HSP20-like chaperone; IPR002068, Heat shock protein Hsp20; Supplementary Table 2). The expression levels of HSPs (IPR008978 and IPR002068) in terms of RPKM for the induced library were 58,328.8 and 12,510.56 (vs. 140.4 and 212.3, for the repressed library respectively), which clearly indicate a strong induction of HSPs in O3-treated plants [log2 fold 8.7 and 5.9, respectively (Table 2)]. The observed results are in accordance with previous observations reported by Eckey-Kaltenbach et al. (1997) in Petroselinum crispum seedlings exposed to O3 (200 ppb, 10 h) and post-cultivated in pollutant-free air, using a Northern blot analysis. Recently, Al-Whaibi (2011) reviewed the role of HSPs demonstrating their function as molecular chaperones, regulating the (i) localization, (ii) degradation, (iii) accumulation, FIGURE 1 | Functional classification of Viburnum lantana ensembl unigenes within cellular component (A), biological process (B), and molecular function (C) categories. and (iv) folding of proteins during their synthesis. Taking into account these studies, HSPs can be considered as the first line of defense against O3 (Haslbeck and Vierling, 2015). Using a proteomic approach, Torres et al. (2007) documented that expression levels of HSPs were strongly increased in O3-stressed young leaves of maize (200 ppb, 3 h) and that their functions were correlated with glycolysis, photosynthesis, antioxidant-, and pathogen-related defense. Visible Foliar Symptoms y p After 15 days of O3 fumigation leaves did not show any visible symptoms. Thirty days from the beginning of the exposure [AOT40 = 3000 ppb h; AOT40: ozone Accumulated Over a Threshold of 40 ppb, sensu de Leeuw and van Zantwoort (1997)], fully expanded leaves from O3-fumigated plants showed several minute (Ø 1-2 mm) roundish dark-blackish necrosis located in the interveinal area of the adaxial surface. Symptoms were observed on all the examined leaves (n = 30) and plants (n = 6), with an injured leaf area per plant accounting for 4.2% (SE 1.4%; range 1–20%). At the end of the experiment (AOT40 4500 ppb h, 45 days from the beginning of exposure), the injured area was 16.7% (SE 2.47%; range 5–60%) of the total surface. Visible foliar injury has been used in many field experiments as an indicator of the response of V. lantana to O3 exposure (e.g., Gottardini et al., 2010). Fully expanded leaves showed symptoms similar to those previously reported in seedlings from OTC experiments (Novak et al., 2005, 2008) and in plants grown in natural conditions (Gottardini et al., 2014a,b). TABLE 1 | Summary of RNA sequencing and de novo assembly to construct the gene set of Viburnum lantana plants exposed to O3 treatment (60 ppb of O3, 5 h d−1 for 15 consecutive days). Induced Repressed Ensembl Initial sequencing reads 43815 13610 57425 Cleaned reads 38800 12495 51295 Unigenes 543 705 1238 Total length of Unigenes (bp) 137438 254585 389126 N50 stats (pb): 277 434 389 Total GC count (pb): 68866 126700 194216 GC (%): 50.1 49.8 49.9 GO categories 262 (48.3%) 481 (68.2%) 744 (60.1%) Functional protein domains 295 (54.3%) 476 (67.5%) 772 (62.4%) Number of the sequencing reads, clustering details, and the unigene details. GC, guanine-cytosine content; GO, gene ontology. TABLE 1 | Summary of RNA sequencing and de novo assembly to construct the gene set of Viburnum lantana plants exposed to O3 treatment (60 ppb of O3, 5 h d−1 for 15 consecutive days). Frontiers in Plant Science | www.frontiersin.org June 2016 | Volume 7 | Article 713 4 Ozone-Molecular Response of Wayfaring Gottardini et al. process, and molecular function (Figure 1). Within the cellular component category, 21 GO slims were identified including cell, membrane, thylakoid, and other apparatus (Figure 1A). Visible Foliar Symptoms Cell, cellular component, intracellular, and cytoplasm were the most represented slims in terms of number of genes (393, 393, 323, and 231, respectively). On the other hand, 42 GO slims were recognized within the biological process category, such as translation, reproduction, transport, photosynthesis, cell death, and other functions (Figure 1B). Among all these slims, metabolic and cellular processes were the most frequent ones (502 and 419 genes, respectively). Aside from GO cellular processes, the response to stress was the next most abundant GO slim (110 gene numbers) after biological and biosynthetic process (400 and 154, respectively). It comprised responses to different stresses, including abiotic and endogenous stimuli (93 and 46 genes, respectively, Figure 1B). Within the molecular function category, 24 GO slims were observed including protein binding, transcription regulator activity, carbohydrate binding, hydrolase activity, receptor activity, and other enzymatic function (Figure 1C). Among all of these slims, binding and catalytic activity appeared more frequently (415 and 380, respectively). FIGURE 1 | Functional classification of Viburnum lantana ensembl unigenes within cellular component (A), biological process (B), and molecular function (C) categories. Frontiers in Plant Science | www.frontiersin.org Expression Estimates and Functional Profiling of Up- and Down-Regulated Unigenes It is worthwhile to mention that HSPs (especially HSP20 superfamily) play a pivotal role in protecting the photosynthetic machinery against damage caused by photo-oxidative stress (Lee et al., 2000). Although this property has been demonstrated during heat stress, we can suppose that the induction of HSPs by O3 treatment could (i) increase the resistance of photosynthetic machinery to photoinhibition, and (ii) affect stress tolerance. June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 5 Ozone-Molecular Response of Wayfaring Ozone-Molecular Response of Wayfaring Gottardini et al. TABLE 2 | Expression log2(RPKM induced/RPKM repressed) of the up-regulated Ensembl unigenes having homologous gene families acco PLAZA version 2.5 and associated Interpro domains. Ensembl Unigene Homologous Gene Families Associated InterPro domains Log2 (RPKM induced/RP EnsVib0002 HOM000181 IPR001024 IPR008976 IPR001246 IPR000907 IPR013819 7.1 EnsVib0022 HOM000082 IPR009038 IPR000348 2.4 EnsVib0041 HOM000926 IPR008978 IPR002068 5.9 EnsVib0046 HOM005074 IPR008892 1.0 EnsVib0120 – – 0.7 EnsVib0128 HOM000463 IPR002912 3.6 EnsVib0140 HOM000058 IPR013128 IPR000668 IPR013201 2.2 EnsVib0155 HOM000058 IPR013128 IPR000668 IPR013201 1.4 EnsVib0159 HOM002664 IPR002020 IPR016142 IPR016141 5.5 EnsVib0185 HOM001028 IPR017936 IPR012335 IPR000866 IPR012336 0.4 EnsVib0225 – – 2.5 EnsVib0241 HOM000826 IPR012677 IPR000504 5.1 EnsVib0243 HOM000826 IPR012677 IPR000504 6.5 EnsVib0251 – – 5.6 EnsVib0267 HOM002675 IPR000772 IPR001574 IPR017989 IPR016139 IPR016138 IPR008997 5.3 EnsVib0271 – – 1.2 EnsVib0273 HOM000085 IPR012269 IPR000425 4.0 EnsVib0283 – – 2.5 EnsVib0308 – – 4.5 EnsVib0314 HOM003594 IPR004662 IPR011148 IPR001057 IPR001048 3.6 EnsVib0316 – – 1.0 EnsVib0332 – – 4.3 EnsVib0341 HOM000031 IPR000109 IPR016196 5.0 EnsVib0359 – – 4.7 EnsVib0376 HOM000016 IPR002213 5.9 EnsVib0383 HOM000438 IPR007608 4.5 EnsVib0387 HOM000533 IPR008580 4.7 EnsVib0402 – – 5.9 EnsVib0416 HOM000926 IPR008978 IPR002068 8.7 EnsVib0419 HOM000451 IPR005016 4.9 EnsVib0421 HOM002452 IPR003823 1.4 EnsVib0469 HOM000198 IPR012335 IPR002109 IPR012336 IPR014756 IPR003172 1.0 EnsVib0492 HOM004549 IPR011990 IPR006597 3.4 EnsVib0505 HOM000025 IPR003959 IPR005936 IPR003593 IPR000642 5.1 EnsVib0516 HOM000393 IPR012335 IPR010987 IPR004046 IPR012336 IPR004045 IPR017933 5.3 EnsVib0535 HOM001845 IPR008962 IPR002110 IPR000535 IPR020683 0.5 EnsVib0765 HOM005953 IPR014946 3.5 EnsVib0767 – – 3.3 EnsVib0812 HOM000507 IPR016040 IPR001891 IPR012301 IPR012302 1.5 EnsVib0830 HOM000080 IPR014778 IPR012287 IPR017930 IPR006447 IPR009057 4.9 EnsVib0869 HOM000033 IPR013525 IPR017871 IPR003593 IPR003439 1.5 RPKM, kilo base of exon model per million mapped reads. We observed a log2 fold up-regulation (RPKM (Maccarrone et al., 1992). Recent RNA-seq based tran TABLE 2 | Expression log2(RPKM induced/RPKM repressed) of the up-regulated Ensembl unigenes having homologous gene families according to PLAZA version 2.5 and associated Interpro domains. Expression Estimates and Functional Profiling of Up- and Down-Regulated Unigenes Ensembl Unigene Homologous Gene Families Associated InterPro domains Log2(RPKM induced/RPK EnsVib0016 HOM000635 IPR020568 IPR000754 IPR014721 −4.6 EnsVib0032 HOM005074 IPR008892 −1.3 EnsVib0035 HOM000181 IPR001024 IPR008976 IPR001246 IPR000907 IPR013819 −2.2 EnsVib0036 HOM000181 IPR001024 IPR008976 IPR001246 IPR000907 IPR013819 −8.7 EnsVib0039 HOM000950 IPR011032 IPR013149 IPR013154 IPR016040 IPR002085 IPR020843 −0.1 EnsVib0071 – – −1.6 EnsVib0080 HOM004197 IPR006311 IPR008797 −5.7 EnsVib0083 HOM000339 IPR020478 IPR010979 IPR001892 IPR001965 IPR011011 IPR017956 −3.2 EnsVib0085 HOM001274 IPR003959 −1.3 EnsVib0139 HOM000013 IPR018957 IPR001841 −0.1 EnsVib0148 HOM000250 IPR001395 −0.6 EnsVib0162 HOM000005 IPR001128 IPR017973 IPR002401 −6.0 EnsVib0178 HOM000181 IPR001024 IPR008976 IPR001246 IPR000907 IPR013819 −5.0 EnsVib0199 HOM001274 IPR003959 −5.4 EnsVib0212 HOM004197 IPR006311 IPR008797 −3.8 EnsVib0217 – – −0.2 EnsVib0244 HOM000648 IPR013785 IPR000741 −3.6 EnsVib0266 HOM001274 IPR003959 −4.4 EnsVib0284 HOM003875 IPR009346 −1.6 EnsVib0313 HOM000858 IPR000894 −5.3 EnsVib0435 HOM000051 IPR016040 IPR002347 IPR002198 −0.8 EnsVib0445 HOM005189 IPR017498 −6.1 EnsVib0462 HOM000934 IPR013845 IPR000876 IPR013843 IPR005824 IPR002942 −2.2 EnsVib0476 HOM001646 IPR020568 IPR000851 IPR013810 IPR005324 IPR014721 IPR018192 −1.1 EnsVib0499 – – −1.4 EnsVib0501 HOM001714 IPR006214 −4.5 EnsVib0509 – – −5.1 EnsVib0512 HOM002153 IPR008991 IPR018259 IPR001147 −8.5 EnsVib0518 HOM000034 IPR012334 IPR000070 IPR006501 IPR011050 −7.2 EnsVib0638 HOM002856 IPR006082 −1.2 EnsVib0688 HOM000502 IPR018957 IPR001650 IPR001841 IPR014021 IPR014001 IPR000330 −0.6 EnsVib0735 HOM003079 IPR003095 IPR015609 IPR001623 −2.6 EnsVib0746 HOM000011 IPR005123 −2.6 EnsVib0792 – – −2.6 EnsVib0899 – – −0.6 EnsVib0984 – – −1.3 EnsVib1062 – – −1.6 RPKM, kilo base of exon model per million mapped reads. TABLE 3 | Expression log2(RPKM induced/RPKM repressed) of the down-regulated Ensembl unigenes having homologous gene families according to PLAZA version 2.5 and associated Interpro domains. RPKM, kilo base of exon model per million mapped reads. A. thaliana ecotype Columbia (Col-0) plants exposed to O3 (300 ppb, 6 h) after 3 h from the beginning of the treatment. Antioxidant enzymatic activities were expected to rise during a situation that leaded to increased oxidative stress. Results indicate that GSH-dependent detoxification pathways were induced by O3. Specifically, the expression level of GSH in terms of RPKM for the induced library was 1,198.5 (vs. 312.6 for the repressed library), which suggests an involvement of disulphide bridges in redox-control process. This is in agreement with the results reported by D’Haese et al. (2006) for A. thaliana plants exposed to an episodic O3 treatment (150 ppb, 8 h). has been implicated in membrane alteration and could mediate the O3-effect. Expression Estimates and Functional Profiling of Up- and Down-Regulated Unigenes TABLE 2 | Expression log2(RPKM induced/RPKM repressed) of the up-regulated Ensembl unigenes having homologous gene families according to PLAZA version 2.5 and associated Interpro domains. Ensembl Unigene Homologous Gene Families Associated InterPro domains RPKM, kilo base of exon model per million mapped reads. We observed a log2 fold up-regulation (RPKM induced/RPKM repressed = 7.1) of a unigene (EnsVib0002) functionally associated to lipoxygenase activity (GO:0016165, Supplementary Table 2). This is supported by similar findings in Lens culinaris seedlings exposed to O3 flux after 30 min from the beginning of the treatment (Maccarrone et al., 1997). In soybean seedlings, O3 up-regulated the lipoxygenase gene and its activity, with a concomitant enhanced membrane lipid peroxidation (Maccarrone et al., 1992). Recent RNA-seq based transcriptomics indicated an increased expression of genes involved in lipid metabolic process in two soybean varieties exposed to an episodic O3 treatment [25–75 ppb, 4 h (Whaley et al., 2015)]. Plant lipoxygenases, which use molecular oxygen to produce hydroperoxides from unsaturated fatty acids, play a key role in (i) growth and development, (ii) senescence, and (iii) responses to biotic and abiotic stresses. In particular, lipoxygenase activity June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 6 Ozone-Molecular Response of Wayfaring Gottardini et al. TABLE 3 | Expression log2(RPKM induced/RPKM repressed) of the down-regulated Ensembl unigenes having homologous gene families ac PLAZA version 2.5 and associated Interpro domains. Frontiers in Plant Science | www.frontiersin.org Transcription Factors Thirty-four families of transcription factors were observed for Ensembl unigenes (Supplementary Table 3). Myeloblast (MYB)- factors were previously described to be associated with a diverse array of cellular responses, including plant secondary metabolism as well as biotic and abiotic tolerance (Kwon et al., 2013) and were observed in the induced library. MYB proteins responded at the transcriptional level to O3 stress in V. lantana (EnsVib0607_ORF+3, IPR001005, and IPR009057 domains). MYB transcription factors could have repressing effects on genes involved in the biosynthesis of phenylpropanoids (Bender and Fink, 1998), flavonoids (Borevitz et al., 2000), auxin and consequently have an effect on the maintenance of cell wall development, cuticle formation, and lipid metabolism. Few studies based on the evaluation of plant responses to drought, salt, and UV stress (Hemm et al., 2001; Golldack et al., 2014) have reported the functional role of MYBs. To identify the functional enriched terms and pathways and to approximate the coverage of sequenced and assembled unigenes, we compared the identified 78 unigenes, whose expression altered during the O3 stress in V. lantana to A. thaliana genome predicted coding sequences (CDS) using KOBAS (E-value threshold, 1E-08; Supplementary Figure 1). Interestingly, 45 out of 78 unigenes showed putative functional orthologs in A. thaliana (Table 4). GO enrichment was observed according to the observed functionally enriched linoleate 13S- lipoxygenase activity (GO:0016165, P = 1.86E-07, corrected P = 1.59E-05), which might indicate the conversion of the linoleate into 13-HPODE, responsible for the activation of the lipid peroxidation process. This suggests that O3 treatments may induce deleterious effects on (i) integrity, (ii) conformation, and (iii) transport capacity of membranes in V. lantana, as reported in other species (Yan et al., 2010; Pellegrini et al., 2011). Dynamics of gas exchange results strongly altered as confirmed by the enrichment of GO categories related to thylakoid (GO:0009579, GO:0009535, GO:0055035, GO:0044436, GO:0009534, GO:0031976). It confirms that O3 exposure, by free-radical production, induces alterations in the (i) In our study, WRKY-factors were found, which have been previously reported to be important components in the complex signaling processes during plant stress responses (Dong et al., 2003; Zhang et al., 2015). Far less information is available to understand the function of WRKY proteins in abiotic stress. Some studies demonstrated that the expression of many WRKY genes is greatly and rapidly induced in response to wounding, temperature, nutrient deficiency, drought, and salinity (Chen L. et al., 2012). Expression Estimates and Functional Profiling of Up- and Down-Regulated Unigenes It is worthwhile to mention that lipid oxidation is a double-edged event due to (i) its damaging effects on lipids and membrane components and (ii) a putative beneficial role in the signaling pathway [e.g., jasmonic acid formation, Vaultier and Jolivet (2015)]. An induction of unigenes (EnsVib0516) functionally associated with glutathione (GSH) was observed with GO GSH binding (GO:0043295), GSH transferase (GO:0004364), and GSH peroxidase activities (GO:0004602, Supplementary Table 2), according with the earlier reports by Tosti et al. (2006) in June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 7 Ozone-Molecular Response of Wayfaring Gottardini et al. A repression of unigenes (EnsVib0080 and EnsVib0212) functionally associated to photosynthetic process with GO: photosystem (GO:0009521) and photosynthesis (GO:0015979, Supplementary Table 2) was found. The expression levels of photosynthetic process in terms of RPKM for the induced library were 95.8 and 368.2 (vs. 4,909.0 and 5,013.0, for the repressed library respectively), clearly suggesting a decrease in photosynthetic performance in O3-treated plants [log2 (RPKM induced/RPKM repressed) = −5.8 and −3.8, respectively (Table 3)]. Similarly, Kontunen-Soppela et al. (2010) reported a decreased expression of photosynthesis- and carbon fixation- related genes in B. papyrifera plants exposed to O3 alone (2x ambient O3 concentration) or in combination with CO2 (target 550 ppm), during the growing season since 1998. A previous microarray analysis indicated that many genes involved in photosynthesis were down-regulated in Populus tremuloides plants (clone 216) subjected to chronic O3 fumigation (1.5x ambient O3 concentration for 5 consecutive years; Gupta et al., 2005). photosynthetic apparatus, (ii) content/pattern of thylakoid, and (iii) functional state of chloroplast membranes. Particularly, O3- derivative molecular species induce changes in gene expression responsible for rearrangements in the thylakoid architecture of V. lantana leaves limiting the damage of PSII activity (as a compensatory mechanism for the inhibiting photosynthetic effects) and, generally, counteracting the oxidative stress. Using RNA-seq, Liu et al. (2015) indicated the abundance and enrichment of the differentially expressed genes involved in the thylakoid, plastid part, chloroplast, and plastids envelope in Reaumuria soongorica leaves subjected to UV-B radiation. Additionally, previous immunological studies documented that O3 treatment could affect energy transfer processes by inducing alterations in thylakoid membrane proteins (Tognini et al., 1997; Ranieri et al., 2000). Following O3 treatment, a complex sequence of events takes place in the cells of V. Expression Estimates and Functional Profiling of Up- and Down-Regulated Unigenes lantana (at intracellular and membrane level), altering key biological processes [such as metabolism, protein fate (folding, modification, and destination) and transports] and molecular functions (catalytic and hydrolase activities). This suggests that plants react to O3 changing metabolic processes (for example lipid catabolism, sugar and amino acid metabolism) that are both used (i) directly, as alternative sources of energy, (nitrogen and carbon skeletons) and (ii) indirectly, as substrates of secondary metabolite modifications. According to Heath (2008), we can conclude that O3 induces deep changes in the expression of genes responsible for biochemical adjustments and metabolic shifts. Oxidation reactions would be expected to reduce net photosynthesis predisposing plants to the inhibition of PSII electron transport (Pellegrini, 2014) and possibly, accelerating the onset of cell senescence (Pellegrini et al., 2015). In the present study, impaired photosynthesis was seen as a down- regulation of PSII oxygen-evolving complex (OEC) PsbQ genes (regulators for the biogenesis of optically active PSII). According to Gururani et al. (2015), the down-regulation of OEC is an efficient and dynamic feedback mechanism to (i) reduce/regulate the generation of reactive oxygen radicals in PSII (favoring electron donation by non-water electron donors with a high rate constant) and (ii) to provide protection against photodamage in response to abiotic stress. In our case, the easy accessibility of non-water electrons from antioxidant molecules and the concomitant increase of GSH-related genes confirm that plants use a specific set of active mechanisms for ROS scavenging during O3 exposure. Transcription Factors WRKY proteins respond to O3 stress at the transcriptional level in V. lantana plants (EnsVib0769_ORF- 1, IPR003657 domain) and WRKY transcription factors could have inducing effects on genes involved in O3 perception/signal June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 8 Ozone-Molecular Response of Wayfaring Gottardini et al. TABLE 4 | Statistical analysis on functional homologs (GO categories) between Viburnum lantana and Arabidopsis thaliana ontology assignment and enrichment analysis using hypergeometric test/Fisher’s exact test and corrected values after Benjamini and Hochberg FDR correction. lysis using hypergeometric test/Fisher’s exact test and corrected values after Benjamini and Hochberg FDR correction. m Gene Ontology P-value Corrected P-value membrane GO:0034357 3.63E-11 2.02E-08 GO:0009579 3.94E-11 2.02E-08 koid membrane GO:0009535 1.65E-10 3.37E-08 membrane GO:0055035 1.73E-10 3.37E-08 iccation GO:0009269 1.79E-10 3.37E-08 GO:0044436 1.97E-10 3.37E-08 ane GO:0042651 3.28E-10 4.80E-08 koid GO:0009534 9.10E-10 1.04E-07 GO:0031976 9.10E-10 1.04E-07 mpartment GO:0031984 1.02E-09 1.04E-07 c process GO:0009106 1.62E-07 1.51E-05 oxygenase activity GO:0016165 1.86E-07 1.59E-05 bivore GO:0080027 4.34E-07 3.43E-05 process GO:0006546 6.30E-07 4.61E-05 no acid catabolic process GO:0009071 7.82E-07 5.34E-05 c process GO:0006544 9.61E-07 6.16E-05 no acid metabolic process GO:0009069 1.42E-06 7.68E-05 oenzyme metabolic process GO:0006733 1.42E-06 7.68E-05 n transport GO:0070838 2.16E-06 1.11E-04 c cation transport GO:0072511 2.34E-06 1.14E-04 ope GO:0009941 2.61E-06 1.21E-04 acid biosynthetic process GO:0006636 3.40E-06 1.45E-04 acid metabolic process GO:0033559 3.40E-06 1.45E-04 GO:0009526 4.11E-06 1.65E-04 meostasis GO:0030003 5.25E-06 2.07E-04 ostasis GO:0006873 7.37E-06 2.07E-04 activity GO:0016702 7.40E-06 2.07E-04 ma GO:0009570 8.42E-06 2.98E-04 etic process GO:0031408 8.85E-06 3.02E-04 homeostasis GO:0055082 1.02E-05 3.36E-04 nthetic process GO:0009108 1.07E-05 3.34E-04 GO:0009532 1.13E-05 3.46E-04 c process GO:0006766 1.22E-05 3.46E-04 c process GO:0031407 1.23E-05 3.46E-04 bolic process GO:0006732 1.25E-05 3.46E-04 metabolic process GO:0000096 1.72E-05 4.65E-04 asis GO:0019725 1.79E-05 4.70E-04 GO:0031975 1.96E-05 4.90E-04 pe GO:0031967 1.96E-05 4.90E-04 GO:0044434 2.02E-05 4.93E-04 perature stimulus GO:0009266 2.08E-05 4.95E-04 GO:0015979 2.23E-05 5.19E-04 id biosynthetic process GO:0008652 2.30E-05 5.24E-04 GO:0044435 2.66E-05 5.78E-04 sis GO:0055080 2.71E-05 5.78E-04 s with P < 0.001 are showed (45 on the total 78 found for Viburnum lantana). Only GO categories with P < 0.001 are showed (45 on the total 78 found for Viburnum lantana). DNA binding domain). Similar findings were reported by Tosti et al. (2006) in Col-0 Arabidopsis plants 3 h after the beginning of O3 treatment. SSH analysis indicated that WRKY genes may be involved in redox regulation in two poplar hybrid clones exposed DNA binding domain). Similar findings were reported by Tosti et al. REFERENCES Chen, L., Song, Y., Li, S., Zhang, L., Zou, C., and Yu, D. (2012). The role of WRKY transcription factors in plant abiotic stresses. Biochim. Biophys. Acta 1819, 120–128. doi: 10.1016/j.bbagrm.2011.09.002 Al-Whaibi, M. H. (2011). Plant heat-shock proteins: a mini review. J. King Saud Univ. Sci. 23, 139–150. doi: 10.1016/j.jksus.2010.06.022 Chen, T. W., Gan, R. C., Wu, T. H., Huang, P. J., Lee, C. Y., Chen, Y. Y., et al. (2012). FastAnnotator: an efficient transcript annotation web tool. BMC Genomics 13:S9. doi: 10.1186/1471-2164-13-S7-S9 Bender, J., and Fink, G. R. 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(2003) documented an over-representation of WRKY motifs in the promoter region of genes up-regulated by an episodic O3 exposure (350 ppb, 6 h) in Col-0 Arabidopsis plants. Xu et al. (2015) observed that four WRKY-transcriptional factors genes were highly induced by O3 treatment (350 ppb, 2 h) in Col-0 Arabidopsis plants. Furthermore, the expression gene profile after O3 fumigation was similar to that of tomato after Botrytis cinerea (a fungus) (Journot-Catalino et al., 2006) and Pseudomonas syringae (a bacterium) infections (Birkenbihl et al., 2012), suggesting that (i) O3 resembles a biotic elicitor and (ii) stress-regulated genes represent a general stress response. the putative characterization of identified unigenes in V. lantana and (ii) better understand the response to O3 exposure in a non-model species. SUPPLEMENTARY MATERIAL This is the first study on differentially expressed genes after O3 treatment in V. lantana plants. A large number of genes involved in signaling/transcription, stress/defense, and protein metabolism showed significant differences in expression of plants exposed to chronic O3 treatment, suggesting that complex molecular alterations occurred. By GO slims and pathways enrichment of the co-regulated genes, it could be demonstrated that following O3 exposure, a complex sequence of events takes place in the cells at intracellular and membrane level, altering a series of biological processes [such as metabolism, protein fate (folding, post-translational modification, and destination) and transport] and molecular functions (catalytic and hydrolase activities). Specifically, the down-regulation of genes associated to photosynthesis demonstrates the deleterious effects of O3. Up-regulation of genes involved in antioxidant-detoxification pathway and thylakoid-localized proteins may be an effective strategy of defense against O3. This research can be considered as an useful basis to (i) generate the functional resources for The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00713 Supplementary Figure 1 | Statistical pathway enrichment of the differentially expressed genes against Arabidopsis thaliana. Supplementary Figure 1 | Statistical pathway enrichment of the differentially expressed genes against Arabidopsis thaliana. Supplementary Table 1 | Table showing the GO and associated InterPro annotations with ensembl transcripts showing the up- and down-regulations during the ozone treatment. Supplementary Table 2 | Detailed classification of expression log2(RPKM induced/RPKM repressed) of the up- and down-regulated Ensembl unigenes having homologous gene families according to PLAZA version 2.5 and associated InterPro domains. Abbreviations: RPKM: kilo base of exon model per million mapped reads. Detailed Classification of the associated functional categories associated with the expressed transcripts in ozone treatment. Supplementary Table 3 | Table showing the family and associated InterPro domains of ensembl transcription factors. AUTHOR CONTRIBUTIONS The work presented here was carried out in collaboration among all authors. NL and CN defined the research theme and obtained funding. EG, AC, EP, PB, and GS designed methods, carried out laboratory experiments, and analyzed the data. AC, EP, and GS co-designed experiments, discussed analyses, interpreted the results, and wrote the paper. All authors have contributed to discuss the results and implications of the work and to comment on the manuscript at all stages before approvation. REFERENCES doi: 10.1105/tpc.106.044149 Eckey-Kaltenbach, H., Kiefer, E., Grosskopf, E., Ernst, D., and Sandermann, H. Jr. (1997). 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KOBAS server: a web-based platform for automated annotation and pathway identification. Nucleic Acids Res. 34, 720–724. doi: 10.1093/nar/gkl167 Novak, K., Skelly, J. M., Schaub, M., Kräuchi, N., Hug, C., Landolt, W., et al. (2003). Ozone air pollution and foliar injury development on native plants of Switzerland. Environ. Pollut. 125, 41–52. doi: 10.1016/S0269-7491(03)00085-X Xie, C., Mao, X., Huang, J., Ding, Y., Wu, J., Dong, S., et al. (2011). KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39, 316–322. doi: 10.1093/nar/gkr483 Olbrich, M., Gerstner, E., Bahnweg, G., Häberle, K.-H., Welzl, G., Heller, W., et al. (2010). Transcriptional signatures in leaves of adult European beech trees (Fagus sylvatica L.) in an experimentally enhanced free air ozone setting. Environ. Pollut. 158, 977–982. doi: 10.1016/j.envpol.2009.08.001 Xu, E., Vaahtera, L., and Brosché, M. (2015). 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Res. 674, 45–54. doi: 10.1016/j.mrgentox.2008.10.005 Pellegrini, E., Francini, A., Lorenzini, G., and Nali, C. (2015). Ecophysiological and antioxidant traits of Salvia officinalis under ozone stress. Environ. Sci. Pollut. Res. 22, 13083–13093. doi: 10.1007/s11356-015-4569-5 Zhang, L., Gu, L., Ringler, P., Smith, S., Rushton, P. J., and Shen, Q. J. (2015). Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells. Plant Sci. 236, 214–222. doi: 10.1016/j.plantsci.2015.04.014 Puckette, M., Iyer, N. J., Tanh, Y., Dai, X.-B., Zhao, P., and Mahalingam, R. (2012). REFERENCES W., and Jordán, M. J. (2015). Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea. Gene 558, 41–53. doi: 10.1016/j.gene.2014.12.043 Maccarrone, M., Veldink, G. A., Vliegenthart, F. G., and Agrò, A. F. (1997). Ozone stress modulates amine oxidase and lipoxygenase expression in lentil (Lens culinaris) seedlings. 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Safe. 115, 101–111. doi: 10.1016/j.ecoenv.2015.02.010 Manninen, S., Huttunen, S., Vanhatalo, M., Pakonen, T., and Hämäläinen, A. (2009). Inter- and intra-specific responses to elevated ozone and chamber climate in northern birches. Environ. Pollut. 157, 1679–1688. doi: 10.1016/j.envpol.2008.12.008 two tropical rice cultivars against ambient and elevated ozone. Ecotox. Environ. Safe. 115, 101–111. doi: 10.1016/j.ecoenv.2015.02.010 Savenstrand, H., Brosche, M., Angehagen, M., and Strid, A. (2000). Molecular markers for ozone stress isolated by suppression subtractive hybridization: specificity of gene expression and identification of a novel stress-regulated gene. Mao, X., Cai, T., Olyarchuk, J. G., and Wei, L. (2005). Automated genome annotation and pathway identification using the KEGG orthology (KO) as a controlled vocabulary. 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Plant 5, 187–204. doi: 10.1093/mp/ssr069 Zinser, C., Jungblut, T., Heller, W., Seidlitz, H. K., Schnitzler, J.-P., Ernst, D., et al. (2000). The effect of ozone in Scots pine (Pinus sylvestris L.): gene expression, biochemical changes and interactions with UV-B radiation. Plant Cell Environ. 23, 975–982. doi: 10.1046/j.1365-3040.2000.00613.x Puckette, M., Peal, L., Steele, J., Tang, Y., and Mahalingam, R. (2009). Ozone responsive genes in Medicago truncatula: analysis by suppression subtraction hybridization. J. Plant Physiol. 166, 1284–1295. doi: 10.1016/j.jplph.2009. 01.009 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ranieri, A., Serini, R., Castagna, A., Nali, C., Baldan, B., Lorenzini, G., et al. (2000). Differential sensitivity to ozone in two poplar clones. Analysis of thylakoid pigment-protein complexes. Physiol. Plant. 110, 181–188. doi: 10.1034/j.1399- 3054.2000.110206.x Copyright © 2016 Gottardini, Cristofori, Pellegrini, La Porta, Nali, Baldi and Sablok. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Rizzo, M., Bernardi, R., Salvini, M., Nali, C., Lorenzini, G., and Durante, M. (2007). Identification of differentially expressed genes induced by ozone stress in sensitive and tolerant poplar hybrids. J. Plant Physiol. 164, 945–949. doi: 10.1016/j.jplph.2006.07.012 Sarkar, A., Singh, A. A., Agrawal, S. B., Ahmad, A., and Rai, S. P. (2015). Cultivar specific variations in antioxidative defense system, genome and proteome of June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 12
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Review of: "Factors Influencing Access to Credit for Rural People in Ethiopia"
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Qeios, CC-BY 4.0 · Review, July 11, 2023 Review of: "Factors Influencing Access to Credit for Rural People in Ethiopia" Enyang Besong Susan Potential competing interests: No potential competing interests to declare. Thank you for inviting me to review the manuscrip 'Factors Influencing Access to Credit For Rural People in Ethopia". Below our my remarks and suggestions for the authors. The Abstract is well structured. The introduction in my opinion is too long and does not present the theoretical underpinnings and contributions of the paper . I will suggest the authors focus on highlighting the novelty of their project. looking at the manuscript it appears the authors are simply summarizing what has been done. In addition the authors suggest prior studies focus on agriculture, however they have made no effort to distinguish their work from those of prior researchers. The authors have not clearly established the research question(s) they seek to answer. so far they said they want to close the gap by ‘examining the important socio-economic, demographic, institutional, and other factors that influence access to credit for rural individuals in the Bilate Zuria district, located in the Sidama region of Ethiopia’. It is not clear if the gap is the lack of study in the Zuria district since the literature they present suggest rural credit constrians have been well documented in Africa at large and Ethopia in particular. The result and discussion section is too hard to read and understand. I will suggest the authors use sub-headings. From their conceptual framework, the indicators were grouped into four categories. Thus there should be four main headings and each variable in each category should have a sub-title. This will improve the structure and readability of the manuscript. Also, the authors should clearly state what column represent what variable in “Table 4” In the abstract, the authors mention policy implication. However, there are no policy recommendations in the conclusion. I suggest the authors highlight the theoretical and policy implications of their findings in the section titled conclusion. The citation style is not well presented in most instances e.g ‘Samuel Semma Waje, 2020’ I think should be “Waje 2020”. The authors should check and ensure their citation is standardize. There are also some typo errors which I suggest 'Grammarly" can quickly correct. Qeios ID: 7J2A0K · https://doi.org/10.32388/7J2A0K 1/1
https://openalex.org/W3092996383
https://link.springer.com/content/pdf/10.1007/s10109-020-00336-0.pdf
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Progress in the R ecosystem for representing and handling spatial data
Journal of geographical systems
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ERROR: type should be string, got "https://doi.org/10.1007/s10109-020-00336-0\nJournal of Geographical Systems (2021) 23:515–546 https://doi.org/10.1007/s10109-020-00336-0\nJournal of Geographical Systems (2021) 23:515–546 ORIGINAL ARTICLE Abstract Twenty years have passed since Bivand and Gebhardt (J Geogr Syst 2(3):307–317, \n2000. https​://doi.org/10.1007/PL000​11460​) indicated that there was a good match \nbetween the then nascent open-source R programming language and environment \nand the needs of researchers analysing spatial data. Recalling the development of \nclasses for spatial data presented in book form in Bivand et al. (Applied spatial data \nanalysis with R. Springer, New York, 2008, Applied spatial data analysis with R, \n2nd edn. Springer, New York, 2013), it is important to present the progress now \noccurring in representation of spatial data, and possible consequences for spatial \ndata handling and the statistical analysis of spatial data. Beyond this, it is imperative \nto discuss the relationships between R-spatial software and the larger open-source \ngeospatial software community on whose work R packages crucially depend. Keywords  Spatial data analysis · Open-source software · R programming language JEL Classification  C00 · C88 · R15 JEL Classification  C00 · C88 · R15 JEL Classification  C00 · C88 · R15 Progress in the R ecosystem for representing and handling \nspatial data Roger S. Bivand1 Received: 9 October 2019 / Accepted: 8 September 2020 \n© The Author(s) 2020\n/ Published online: 16 October 2020 1\t\nDepartment of Economics, Norwegian School of Economics, Helleveien 30, 5045 Bergen, \nNorway *\t Roger S. Bivand \n\t\nroger.bivand@nhh.no *\t Roger S. Bivand \n\t\nroger.bivand@nhh.no\n1\t\nDepartment of Economics, Norwegian School of Economics, Helleveien 30, 5045 Bergen, \nNorway 1  Introduction While Bivand and Gebhardt (2000) did provide an introduction to R as a statistical \nprogramming language and to why one might choose to use a scripted language like \nR (or Python), this article is both retrospective and prospective. It is possible that \nthose approaching the choice of tools for spatial analysis and for handling spatial \ndata will find the following less than inviting; in that case, perusal of early chapters \nof Lovelace et al. (2019) will provide useful context. Two further pointers include \nthe fact that R and most R add-on packages are open-source software and so with-\nout licence fees or other such restrictions. The second pointer is that all scripting 012341 456789)\n3 012341 456789)\n3 516 R. S. Bivand languages provide the structures needed for reproducible research, and open-source \nsoftware gives the interested researcher the means to run the scripts needed to repli-\ncate results without cost, given access to adequate hardware. Hence, an overview of \nthe development of the use of R for handling spatial data can cast light on how and \nwhy steps fashioning today’s software were taken. Of course, an overview of the use \nof R for analysing spatial data would also be tempting, but, with about 900 R pack-\nages using spatial data handling classes and objects, would far exceed the bounds of \na single article. The R statistical programming language and environment has been used for han-\ndling and analysing spatial data since its inception, partly building on its heritage \nfrom S and S-Plus. When the conceptualization of spatial data was introduced in \nthe sp package (Pebesma and Bivand 2005, 2020, on the Comprehensive R Archive \nNetwork (CRAN) since 2005), it was expected that some packages would adopt its \nclasses. Some years later, adoption rates had picked up strongly, as had use of the \nsp-based packages rgdal (Bivand et al. 2020, on CRAN since 2003) for input/output \nand rgeos (Bivand and Rundel 2020, on CRAN since 2011) for geometric manipula-\ntion of vector data. Packages depending on sp classes continue to require support as more mod-\nern data representations have been introduced in the sf (Pebesma 2018, 2020a, on \nCRAN since 2016) and stars (Pebesma 2020c, on CRAN since 2018) packages. 1 3 1  Introduction The sf package provides the data reading and writing, and geometry manipulation \nfunctionalities found in rgdal and rgeos, and an alternative class representation \nfor vector data based on the Simple Features standard (Herring 2011; ISO 2004). The stars package adds facilities for handling spatial and spatio-temporal raster and \nvector data, building in part on work with the spacetime package (Pebesma 2012, \n2020b, on CRAN since 2010) and on raster handling in the raster package (Hij-\nmans 2020a, on CRAN since 2010). The raster package will not be discussed in this \nreview, mostly because information in the Github “rspatial” organization (https​://\ngithu​b.com/rspat​ial) repositories suggests that development is in flux and that raster \nis becoming a new package called terra (Hijmans 2020b). Discussion here will con-\ncentrate on packages developed and maintained by the Github “r-spatial” organiza-\ntion (https​://githu​b.com/r-spati​al) of which I am a member. Newer visualization packages, such as tmap (Tennekes 2018, 2020, on CRAN \nsince 2014), mapview (Appelhans et al. 2020, on CRAN since 2015) and cartog-\nraphy (Giraud and Lambert 2016, 2017, 2020, on CRAN since 2015), give broader \nscope for data exploration and communication. Modelling and analysis packages \ndemonstrate the considerable range of implementations now available and are often \nsupported with additional code provided as supplementary material to journal arti-\ncles, for example in the Journal of Statistical Software spatial statistics special issue \n(Pebesma et al. 2015). The availability of software and scripts provides a helpful \nmechanism supporting reproducible research and hands-on reviewing in which \nreaders can read the code and scripts used in calculating the results presented in \npublished work (see, for example, Sawicka et al. 2018; Lovelace and Ellison 2018; \nEvangelista and Beskow 2018, in one issue of the R Journal). Some packages are used by others in turn forming dependency trees; because of \nthese dependencies, we can speak of an ecosystem. Class representations of data 1 3 Progress in the R ecosystem for representing and handling spatial… 517 are central, with the data frame conceptualization shaping much of the whole R \necosystem. For the modelling infrastructure to perform correctly, the relationships \nbetween objects containing data and the formula representations of models are cru-\ncial. Because both sp and sf provide similar interfaces for the data= arguments for \nmodel fitting functions by behaving as data.frame objects, transition from sp \nto sf representations is convenient by design. 1  Introduction The spdep package (Bivand 2020b, \non CRAN since 2002) for exploratory spatial data analysis and the new spatialreg \npackage (Bivand and Piras 2019, on CRAN since 2019) for spatial regression (split \nout of spdep) have been adapted to accommodate geometries held in sf classes, so \nboth approaches are viable. Other packages, such as mapview, tmap or stplanr \n(Lovelace et al. 2020) have been revised to permit use with both sp and sf objects. In \norder to retain backward compatibility, other central packages may choose to handle \nthe coexistence of sp and sf classes in the same way. Developers of new packages should choose to use sf and stars rather than sp, \nrgdal and rgeos (and terra rather than raster), but existing packages are free to \nadapt, or indeed to stay with sp, rgdal and rgeos, hoping that they may continue to \nbe maintained. Naturally, contributions to maintenance and development from those \nusing software on which one’s work depends are among the “prices” to be “paid” for \nopen-source software, so “hope” may involve commitment to take responsibility for \nmaintenance. If a maintainer is unable to continue in service, software like R add-on \npackages is termed “orphaned”, but may be adopted. This occurred very recently \nwith an R linear programming package lpSolve, which has been adopted by Gábor \nCsárdi to widespread relief and gratitude. Because there is no corporation tasked \nwith maintaining most R add-on packages, their future use has to depend on the user \ncommunity. One key reason why sf is much easier to maintain is that it was written using the \nRcpp package (Eddelbuettel et al. 2011, 2020; Eddelbuettel 2013; Eddelbuettel and \nBalamuta 2018, on CRAN since 2008) to interface C++ code in GDAL and now \nPROJ (both C++11), and GEOS code through the C API, whereas rgdal and rgeos \nhave more fragile handcrafted interfaces originally written for C99 and C++98. Maintaining Rcpp/C++11 interfaces is very much easier than older adaptations not \nas well understood by younger developers. However, the choice of C++ interfaces is \nnot necessarily robust (Kalibera 2019). Since there is a viable alternative to rgdal and rgeos, a fallback in the future for \nsp-dependent packages will be to use sf for reading and writing data, and geometry \nmanipulation, and to coerce to sp classes before passing to existing modelling code \nif sp classes are needed. 1  Introduction Maintainers of actively developed packages using sp vec-\ntor classes intensively and that are also impacted by changes in coordinate reference \nsystems (Sect. 4) are advised to consider transitioning to sf, as substantial revisions \nwill be needed anyway. In this review and prospect, the progress seen over the last 20  years will be \ndescribed, together with the unexpected consequences it engendered. The emer-\ngence of new technologies and standards has also led to the desirability of re-imple-\nmentation of data representations and analysis functionality, some of which is now \nin place and which will be described. Changes also impact the open-source librar-\nies on which core spatial functionality is built, leading to challenges in ensuring 1 3 3 3 518 R. S. Bivand backward compatibility. This will be shown using the example of coordinate refer-\nence systems. In terms of positionality, much of what follows will be presented from \nthe point of view of the author, documented as far as possible from email exchanges \nand similar sources. It is more than likely that other participants in the development \nof the R-spatial community may recall things differently, and of course, I acknowl-\nedge that this description is bound to be partial. 1  Typically plastic boxes containing static software licences attached to a computer port, often the paral-\nlel port otherwise used for printers in the pre-USB era. 2  \u0007Spatial data classes in the sp package CRAN listings show tripack (Renka and Geb-\nhardt 2020) and akima (Akima and Gebhardt 2020)—both with non-open-source 1 3 Progress in the R ecosystem for representing and handling spatial… 519 licences—available from August 1998 ported by Albrecht Gebhardt; ash and sgeo-\nstat (Majure and Gebhardt 2016) followed in April 1999. The spatial package was \navailable as part of MASS (the software supporting the four editions of Venables \nand Ripley 2002), also ported in part by Albrecht Gebhardt. In the earliest period, \nCRAN administrators helped practically with porting and publication. Albrecht and \nI presented an overview of possibilities of using R for research and teaching in spa-\ntial analysis and statistics in August 1998, subsequently published in this journal as \nBivand and Gebhardt (2000). Rowlingson and Diggle (1993) describe the S-PLUS version of splancs (Rowl-\ningson and Diggle 2017) for point pattern analysis. I had contacted Barry Rowl-\ningson in 1997, but only moved forward with porting as R’s package mechanism \nmatured. In September 1998, I wrote to him: “It wasn’t at all difficult to get things \nrunning, which I think is a result of your coding, thank you!” However, I added this \nspeculation: “An issue I have thought about a little is whether at some stage Albrecht \nand I wouldn’t integrate or harmonize the points and pairs objects in splancs, spa-\ntial and sgeostat—they aren’t the same, but for users maybe they ought to appear to \nbe so”. This concern with class representations for geographical data turned out to \nbe fruitful. A further step was to link GRASS and R, described in Bivand (2000), and fol-\nlowed up at several meetings and working closely with Markus Neteler. The inter-\nface has evolved, and its current status is presented by Lovelace et al. (2019, chap-\nter 9). A consequence of this work was that the CRAN team suggested that I attend a \nmeeting in Vienna in early 2001 to talk about the GRASS GIS interface. The meet-\ning gave unique insights into the dynamics of R development, and very valuable \ncontacts. Later the same year Luc Anselin and Serge Rey asked me to take part in a \nworkshop in Santa Barbara, which again led to many fruitful new contacts; my talk \neventually appeared as Bivand (2006), but the contacts made at the workshop were \nvery useful. 2  \u0007Spatial data classes in the sp package In the early and mid-1990s, those of us who were teaching courses in spatial analysis \nbeyond the direct application of geographical information systems (GIS) found the \npaucity of software limiting. In institutions with funding for site licences for GIS, it \nwas possible to write or share scripts for Arc/Info (in AML), ArcView (in Avenue) \nor later in Visual Basic for ArcGIS. If site licences and associated dongles1 used \nin the field were a problem (including problems for students involved in fieldwork \nin research projects), there were few alternatives, but opportunities were discussed \non mailing lists. One of these was the AI-Geostats listserve/mailing list started by \nGregoire Dubois in 1995; AI meant Arc/Info. Another community gathered around \nGRASS GIS and its transition to open-source development; it hosted, among other \nthings, src.contrib and src.garden directory trees with analysis tools (see \ncode stored in the https​://githu​b.com/OSGeo​/grass​-legac​y repository). From late 1996, the R programming language and environment began to be seen \nas an alternative for teaching and research involving spatial analysis by a few people \nincluding the author and Albrecht Gebhardt. R uses much of the syntax of S, then \navailable commercially as S-Plus, but was and remains free to install, use and extend \nunder the GNU General Public License (GPL). In addition, it could be installed \nportably across multiple operating systems, including Windows and Apple Mac OS. At about the same time, the S-Plus SpatialStats module was published (Kaluzny \net al. 1998), and a meeting occurred in Leicester in which many of those looking \nfor solutions took part. (My contribution was published in the meeting special issue \nBivand 1998.) Much of the porting of S code to R for spatial statistics was begun by Albrecht \nGebhardt as soon as the R package mechanism matured. The library() func-\ntion was upgraded in R 0.60 published in December 1997, and the Comprehensive \nR Archive Network was operating in January 1998. An exchange between Albre-\ncht Gebhardt and Thomas Lumley on the R-beta mailing list (https​://stat.ethz.ch/\npiper​mail/r-help/1997-Novem​ber/00188​2.html) shows that the package mecha-\nnism was not yet working predictably before 0.60 for contributed packages. Since \nteachers moving courses from S to R needed access to the S libraries previously \nused, porting was a crucial step. 2  \u0007Spatial data classes in the sp package Further progress during the intervening 4 years in the use of R in spa-\ntial econometrics was reported in Bivand (2002), building on Bivand and Gebhardt \n(2000), but preceding the release of the spdep package. During the second half of 2002, it seemed relevant to propose a spatial statis-\ntics paper session at the next Vienna meeting to be held in March 2003 (known as \nDistributed Statistical Computing (DSC) and led to useR! meetings), together with \na workshop to discuss classes for spatial data. I had reached out to Edzer Pebesma \nas an author of the stand-alone open-source program gstat (Pebesma and Wes-\nseling 1998); it turned out that he had just been approached to wrap the program for \nS-Plus. He saw the potential of the workshop immediately, and in November 2002 \nwrote in an email: “I wonder whether I should start writing S classes. I’m afraid \nI should”. Virgilio Gómez-Rubio had been developing two spatial packages, RAr-\ncInfo (Gómez-Rubio and López-Quílez 2005; Gómez-Rubio 2011) and DCluster \n(Gómez-Rubio et al. 2005, 2015), and was committed to participating. Although he \ncould not get to the workshop, Nicholas Lewin-Koh wrote in March 2003 that: “I \nwas looking over all the DSC material, especially the spatial stuff. I did notice, after \nlooking through peoples’ packages that there is a lot of duplication of effort. My \nsuggestion is that we set up a repository for spatial packages similar to the Biocon-\nductor mode, where we have a base spatial package that has S-4-based methods and 1 3 3 520 R. S. Bivand classes that are efficient and general.” Straight after the workshop, a collaborative \nrepository for the development of software using SourceForge was established, and \nthe R-sig-geo mailing list (still with over 3600 subscribers) was created to facilitate \ninteraction. classes that are efficient and general.” Straight after the workshop, a collaborative \nrepository for the development of software using SourceForge was established, and \nthe R-sig-geo mailing list (still with over 3600 subscribers) was created to facilitate \ninteraction. So the mandate for the development of the sp package emerged in discussions \nbetween interested contributors before, during and especially following the 2003 \nVienna workshop; most of us met at Pörtschach am Wörthersee in October 2003 \nat a meeting organized by Albrecht Gebhardt. 2  \u0007Spatial data classes in the sp package Coding meetings were organized by \nBarry Rowlingson in Lancaster in November 2004 and by Virgilio Gómez-Rubio \nin Valencia in May 2005, at both of which the class definitions and implementa-\ntions were stress-tested and often changed radically; the package was first published \non CRAN in April 2005. The underlying model adopted was for S4 (new style) \nclasses to be used, for \"Spatial\" objects, whether raster or vector, to behave like \n\"data.frame\" objects, and for visualization methods to make it easy to show the \nobjects. Package developers could choose whether they would use sp classes and \nmethods directly, or rather use those classes for functionality that they did not pro-\nvide themselves. The spatstat package (Baddeley and Turner 2005; Baddeley et al. 2015, 2020) was an early example of such object conversion (known as coercion in \nS and R) to and from sp classes and classes, with the coercion methods published in \nthe maptools package (Bivand and Lewin-Koh 2020).i Reading and writing ESRI Shapefiles had been possible using the maptools \npackage (Bivand and Lewin-Koh 2020) available from CRAN since August 2003, \nbut rgdal, on CRAN from November 2003, interfacing the external GDAL library \n(Warmerdam 2008) and first written by Tim Keitt, initially only supported accessing \nand reading raster data. Further code contributions by Barry Rowlingson for han-\ndling projections using the external PROJ.4 library and the vector drivers in the then \nOGR part of GDAL were folded into rgdal, permitting reading vector and raster \ndata into sp-objects and writing from sp-objects. For vector data, it became possible \nto project coordinates, and in addition to transform them where datum specifications \nwere available. Until 2019, the interfaces to GDAL and PROJ had been relatively \nstable, and upstream changes had not led to breaking changes for users of pack-\nages using sp classes or rgdal functionalities, although they have involved signifi-\ncant maintenance effort. The final part of the framework for spatial vector data han-\ndling was the addition of the rgeos package interfacing the external GEOS library \nin 2011, thanks to Colin Rundell’s 2010 Google Summer of Coding project. The \nrgeos package provided vector topological predicates and operations typically found \nin GIS such as intersection; note that by this time, both GDAL and GEOS used the \nSimple Features vector representation internally. By the publication of Bivand et al. 1 3 readxl\nwithr\nleaflet\ntidyr\nglue\nhtmlwidgets\nrjson\nstringi\nshiny\nrlang\ncrayon\nrvest\ncovr\nDT\ntibble\ndplyr\ntidyverse\nbroom\ntidyselect\nmaps\nhttr\nxml2\nassertthat\nhtmltools\nknitr\nrmarkdown\ndigest\nR6\nspelling\npurrr\nmagrittr\ncurl\nlubridate\nRCurl\nreadr\nsf\ntestthat\ndata.table\nXML\ndevtools\nrstudioapi\nkableExtra\nforeign\ntinytest\nroxygen2\nstringr\nyaml\njsonlite\nvdiffr\nshinyjs readxl\nwithr\nleaflet\ntidyr\nglue\nhtmlwidgets\nrjson\nstringi\nshiny\nrlang\ncrayon\nrvest\ncovr\nDT\ntibble\ndplyr\ntidyverse\nbroom\ntidyselect\nmaps\nhttr\nxml2\nassertthat\nhtmltools\nknitr\nrmarkdown\ndigest\nR6\nspelling\npurrr\nmagrittr\ncurl\nlubridate\nRCurl\nreadr\nsf\ntestthat\ndata.table\nXML\ndevtools\nrstudioapi\nkableExtra\nforeign\ntinytest\nroxygen2\nstringr\nyaml\njsonlite\nvdiffr\nshinyjs geosphere\npedometrics\ndeldir\nstabs\nakima\nspacetime\nmaptools\nspData\nFRK\nspam\nRStoolbox\nbiomod2graph4lg\nRandomFields\nspatialreg\ndismo\nINLA\nspatstat\nSSDM\nsp\ngeoR\nENMTools\nCARBayes\nGSIF\ngstat\nmuHVT\nBiodiversityR\nrnaturalearth\necospat\nfields\nspdep\nagricolae\nrgeos\nsplancs\nadehabitatLT\nrdwd\nrasterVis\ntree\nrgdal\ntrajectories\nspatialEco\ninlabru\nraster\nrworldmap\ngdistance\nmapdata\nRgoogleMaps\nCSTools\nplotKML\nsnowfall Fig. 1   Wordclouds of CRAN and BioConductor package dependencies, August 2020, left panel: cluster \n6 (pagerank range 0.002196–0.000070), right panel: cluster 2 (pagerank range 0.022419–0.000390, sf = \n0.000972) Fig. 1   Wordclouds of CRAN and BioConductor package dependencies, August 2020, left panel: cluster \n6 (pagerank range 0.002196–0.000070), right panel: cluster 2 (pagerank range 0.022419–0.000390, sf = \n0.000972) among the cumulated packages held on CRAN and those published by the Biocon-\nductor project. The left panel shows cluster 6, the spatial cluster with sp having a \npagerank of 0.002196, while the right panel shows cluster 2, which is dominated by \npackages developed by RStudio, a commercial company. The sf package is in cluster \n2, with a pagerank score of 0.000972, most likely in that cluster because it itself uses \nmany of the functionalities of RStudio packages. The two word clouds are scaled by \nthe largest pagerank of included packages, so the scales differ by almost an order of \nmagnitude. 2  https​://githu​b.com/rwinl​ib/gdal2​, https​://githu​b.com/rwinl​ib/gdal3​. 2  \u0007Spatial data classes in the sp package (2008), a few packages not written or main-\ntained by the book authors and their nearest collaborators had begun to use sp \nclasses. By the publication of the second edition (Bivand et al. 2013), we had seen \nthat the number of packages depending on sp, importing from and suggesting it (in \nCRAN terminology for levels of dependency) had grown strongly. In late 2014, de \nVries (2014) looked at CRAN package clusters from a page rank graph and found a \nclear spatial cluster that we had not expected. Figure 1 shows word clouds with char-\nacter sizes proportional to pagerank scores for two clusters found in August 2020 1 3 Progress in the R ecosystem for representing and handling spatial… 521 3  \u0007Spatial data classes in the sf and stars packages The raster package complemented sp for handling raster objects and their inter-\nactions with vector objects. It added to input/output using GDAL through rgdal, \nand better access to NetCDF files. It may be mentioned in passing that thanks to \nhelp from CRAN administrators and especially Brian Ripley, CRAN binary builds \nof rgdal for Windows and Apple Mac OSX became available from 2006, but with \na limited set of vector and raster drivers. Support from CRAN adminstrators and \nthe maintainers of the Github rwinlib/gdal2 and rwinlib/gdal3 repositories2 remains \ncentral to making packages available to users who are not able to install R source \npackages themselves, particularly linking to external libraries. Initially, raster was \nwritten in R using functionalities in sp and rgdal with rgeos coming later. It used a \nfeature of GDAL raster drivers permitting the successive reading of subsets of ras-\nters by row and column, allowing the processing of much larger objects than could \nbe held in memory. In addition, the concepts of bricks and stacks of rasters were \nintroduced, diverging somewhat from the sp treatment of raster bands as stacked \ncolumn vectors in a data frame. 1 3 522 R. S. Bivand As raster evolved, two other packages emerged raising issues with the ways in \nwhich spatial objects had been conceptualized in sp. The rgeos package used the C \napplication programming interface (API) to the C++ GEOS library, which is itself a \ntranslation of the Java Topology Suite (JTS). While the GDAL vector drivers did use \nthe standard Simple Features representation of vector geometries, it was not strongly \nenforced. This laxity now seems most closely associated with the use of ESRI Shape \nfiles as a de facto file standard for representation, in which some Simple Features \nare not consistently representable.3 Both JTS and GEOS required a Simple Features \ncompliant representation and led to the need for curious and fragile adaptations. For \nexample, these affected the representation of sp \"Polygons\" objects, which were \noriginally conceptualized after the Shapefile specification: ring direction determined \nwhether a ring was exterior or interior (a hole), but no guidance was given to show \nwhich exterior ring holes might belong to. As R provides a way to add a character \nstring comment to any object, such comments were added to each \"Polygons\" \nobject encoding the necessary information. 3  \u0007Spatial data classes in the sf and stars packages In this way, GEOS functionality could \nbe used, but the fragility of vector representation in sp was made obvious.f Another package affecting thinking about representation was spacetime, stacking \ncolumns for regular spatio-temporal objects with space varying faster than time. So \na single earth observation band observed repeatedly would be stored in a single col-\numn in a data frame, rather than in the arguably more robust form of a four-dimen-\nsional array, with the band taking one position on the final dimension. The second \nedition of Bivand et al. (2013) took up all of these issues in one way or another, but \nafter completing a spatial statistics special issue of the Journal of Statistical Soft-\nware (Pebesma et al. 2015), it was time to begin fresh implementations of classes for \nspatial data. 3  For recent examples see https​://githu​b.com/OSGeo​/gdal/issue​s/1787 and https​://githu​b.com/r-spati​al/\nsf/issue​s/1121. Progress in the R ecosystem for representing and handling spatial… 523 > df <- data.frame(a=letters[1:3], b=1:3)\n> df$c <- list(d=1, e=\"1\", f=TRUE)\n> str(df)\n’data.frame’: 3 obs. of\n3 variables:\n$ a: chr\n\"a\" \"b\" \"c\"\n$ b: int\n1 2 3\n$ c:List of 3\n..$ d: num 1\n..$ e: chr \"1\"\n..$ f: logi TRUE > df <- data.frame(a=letters[1:3], b=1:3)\n> df$c <- list(d=1, e=\"1\", f=TRUE)\n> str(df)\n’data.frame’: 3 obs. of\n3 variables:\n$ a: chr\n\"a\" \"b\" \"c\"\n$ b: int\n1 2 3\n$ c:List of 3\n..$ d: num 1\n..$ e: chr \"1\"\n..$ f: logi TRUE ’data.frame’: 3 obs. of\n3 variables: $ a: chr\n\"a\" \"b\" \"c\" $ b: int\n1 2 3 $ c:List of 3 ..$ d: num 1 ..$ e: chr \"1\" ..$ f: logi TRUE ..$ f: logi TRUE At useR! in 2016, list columns were declared “tidy”, using examples including \nthe difficulty of encoding polygon interior rings in non-list columns. The decision \nto accommodate “tidy” workflows as well as base-R workflows had already been \nmade, as at least some users only know how to use “tidy” workflows. Pebesma \n(2018) showed the status of the sf towards the end of 2017, with a geometry list \ncolumn containing R wrappers around objects adhering to Simple Features speci-\nfication definitions. Note also that from R 4.0.0, data.frame() does not con-\nvert character columns to factor as it did previously (Hornik 2020); column \n\"a\" is character in R 4 or later, and factor before R 4. 3.1  \u0007Simple Features in R It was clear that vector representations needed urgent attention, so the sf package \nwas begun, aiming to implement the most frequently used parts of the specification \n(ISO 2004; Kralidis 2008; Herring 2011). Development was supported by a grant \nfrom the then newly started R Consortium, which brings together R developers and \nindustry members. A key breakthrough came at the useR! 2016 conference, follow-\ning an earlier decision to re-base vector objects on data frames, rather than as in sp \nto embed a data frame inside a spatial object. Although data frame objects in S and \nR have always been able to take list columns as valid columns, such list columns \nwere not seen as “tidy” (Wickham 2014): 3 Progress in the R ecosystem for representing and handling spatial… > library(sf) The feature geometries are stored in numeric vectors, matrices or lists of \nmatrices and may also be subject to arithmetic operations. Features are held in \nthe \"XY\" class if two-dimensional, or \"XYZ\", \"XYM\" or \"XYZM\" if such coor-\ndinates are available (\"Z\" is usually treated as height and \"M\" as some meas-\nure, perhaps accuracy; both need to have specified units); all single features are \n\"sfg\" (Simple Features geometry) objects, with arithmetic and other operators: > pt1 <- st_point(c(1,3))\n> pt2 <- pt1 + 1\n> pt3 <- pt2 + 1\n> str(pt3)\n’XY’ num [1:2] 3 5 > pt1 <- st_point(c(1,3))\n> pt2 <- pt1 + 1\n> pt3 <- pt2 + 1\n> str(pt3)\n’XY’ num [1:2] 3 5 Geometries may be represented as “Well-Known Text” (WKT): > st_as_text(pt3)\n[1] \"POINT (3 5)\" > st_as_text(pt3)\n[1] \"POINT (3 5)\" > st_as_text(pt3)\n[1] \"POINT (3 5)\" > st_as_text(pt3)\n[1] \"POINT (3 5)\" or as “Well-Known Binary” (WKB) as in database “binary large objects” \n(BLOBs), resolving the problem of representation when working with GDAL vector \ndrivers and functions, and with GEOS predicates and topological operations: 1 3 3 524 R. S. Bivand _\n_\ny(p\n)\n[1] 01 01 00 00 00 00 00 00 00 00 00 08 40 00 00 00 00 00 00 14 40 A column of Simple Features geometries (\"sfc\") is constructed as a list of \"sfg\" \nobjects, which do not have to belong to the same Simple Features category; here, we \nassign the Web Mercator CRS to indicate that the points are projected to the plane, with \nposition measured in metres: > pt_sfc <- st_as_sfc(list(pt1, pt2, pt3), crs=3857)\n> str(pt_sfc)\nsfc_POINT of length 3; first list element:\n’XY’ num [1:2] 1 3 When sf was written, the units package was available (Pebesma et al. > library(sf) 2016, 2020) \nand could utilize the metric of the coordinates given in the declared CRS, so here inter-\npoint distances are measured in metres: > st_distance(pt_sfc)\nUnits: [m]\n[,1]\n[,2]\n[,3]\n[1,] 0.000000 1.414214 2.828427\n[2,] 1.414214 0.000000 1.414214\n[3,] 2.828427 1.414214 0.000000 [3,] 2.828427 1.414214 0.000000 If we re-specify the points as geographical coordinates in decimal degrees on the \nWGS84 ellipsoid, the distances will be given as metres, but measured over the ellipsoid: > pt_sfc1 <- st_as_sfc(list(pt1, pt2, pt3), crs=4326)\n> st_distance(pt_sfc1)\nUnits: [m]\n[,1]\n[,2]\n[,3]\n[1,]\n0.0 156759.1 313424.7\n[2,] 156759.1\n0.0 156665.6\n[3,] 313424.7 156665.6\n0.0 The most recent R Consortium grant covers the extension of analysis and data handling \nto global data representation; Pebesma and Dunnington (2020) present a roadmap for the \nuse of the s2 library for operations on geographical coordinates and its integration in sf. > mat <- matrix(0, 3, 3)\n> mat[1,2] <- mat[2,1] <- s2::s2_distance(st_as_text(pt1), st_as_text(pt2))\n> mat[1,3] <- mat[3,1] <- s2::s2_distance(st_as_text(pt1), st_as_text(pt3))\n> mat[2,3] <- mat[3,2] <- s2::s2_distance(st_as_text(pt2), st_as_text(pt3))\n> mat\n[,1]\n[,2]\n[,3]\n[1,]\n0.0 157106.0 314116.3\n[2,] 157106.0\n0.0 157010.4\n[3,] 314116.3 157010.4\n0.0 1 3 1 3 Progress in the R ecosystem for representing and handling spatial… 525 Finally, an \"sfc\" object, a geometry column, can be added to a data.frame \nobject using st_geometry(), which sets a number of attributes on the object and \ndefines it as also being an \"sf\" object (the \"agr\" attribute if populated shows \nhow observations on non-geometry columns should be understood): > st_geometry(df) <- pt_sfc\n> str(df)\nClasses ‘sf’ and ’data.frame’: 3 obs. of\n4 variables:\n$ a\n: chr\n\"a\" \"b\" \"c\"\n$ b\n: int\n1 2 3\n$ c\n:List of 3\n..$ d: num 1\n..$ e: chr \"1\"\n..$ f: logi TRUE\n$ geometry:sfc_POINT of length 3; first list element:\n’XY’ num\n1 3\n- attr(*, \"sf_column\")= chr \"geometry\"\n- attr(*, \"agr\")= Factor w/ 3 levels \"constant\",\"aggregate\",..: NA NA NA\n..- attr(*, \"names\")= chr [1:3] \"a\" \"b\" \"c\" Classes ‘sf’ and ’data.frame’: 3 obs. 4  util=\"ogr2ogr\", options=\"-nlt CONVERT_TO_LINEAR\".\n5  https​://www.ogc.org/stand​ards/geopa​ckage​. > library(sf) of\n4 variables: $ a\n: chr\n\"a\" \"b\" \"c\" :List of 3 c\n:List of 3 ..$ d: num 1 ..$ e: chr \"1\" ..$ f: logi TRUE - attr(*, \"agr\")= Factor w/ 3 levels \"constant\",\"aggregate\",..: NA NA NA\n..- attr(*, \"names\")= chr [1:3] \"a\" \"b\" \"c\" The sf package implements all of the Simple Features geometry categories, but \nsome geometries need be converted to be used inside R, with, for example, the gdal_\nutils() function to convert curve geometries in an input file to linear geometries.4 \nMany of the functions in the sf package begin with st_ as a reference to the same \nusage in PostGIS, where the letters were intended to symbolize “spatial type”.i The vector file creation and reading functionality found in rgdal is also avail-\nable in sf, with substantial improvements with regard to creating spatial database \ntables and reading from databases. Writing a GeoPackage5 is as easy or easier than \nin rgdal, as the layer= and driver= arguments can be inferred from the given \nfile name. List columns cannot be written, because R list columns are not bound to \nbe consistently of the same type. > tf <- tempfile(fileext=\".gpkg\")\n> st_write(df, dsn=tf, quiet=TRUE)\nWarning message:\nIn clean_columns(as.data.frame(obj), factorsAsCharacter) :\nDropping column(s) c of class(es) list > tf <- tempfile(fileext=\".gpkg\")\n> st_write(df, dsn=tf, quiet=TRUE)\nWarning message:\nIn clean_columns(as.data.frame(obj), factorsAsCharacter) :\nDropping column(s) c of class(es) list > tf <- tempfile(fileext=\".gpkg\")\n> st_write(df, dsn=tf, quiet=TRUE)\nWarning message:\nIn clean_columns(as.data.frame(obj), factorsAsCharacter) :\nDropping column(s) c of class(es) list The standard reading method is st_read, providing a similar functionality to \nthat in rgdal, with a number of differences related to character string encoding that \nwill cease to matter when users migrate to modern formats such as GeoPackage. 3 R. S. > library(sf) Bivand 526 > df1 <- st_read(dsn=tf, quiet=TRUE)\n> df1\nSimple feature collection with 3 features and 2 fields\ngeometry type:\nPOINT\ndimension:\nXY\nbbox:\nxmin: 1 ymin: 3 xmax: 3 ymax: 5\nprojected CRS:\nWGS 84 / Pseudo-Mercator\na b\ngeom\n1 a 1 POINT (1 3)\n2 b 2 POINT (2 4)\n3 c 3 POINT (3 5) In a “tidy” workflow, read_sf() can be used, returning a \"tibble-sf\" \ninheriting from a \"tbl_df\" object rather than from a data frame, and not con-\nverting character string columns into \"factor\" categorical variables by default \n(before R 4.0.0): > read_sf(dsn=tf) %>% dplyr::filter(a == \"c\")\nSimple feature collection with 1 feature and 2 fields\ngeometry type:\nPOINT\ndimension:\nXY\nbbox:\nxmin: 3 ymin: 5 xmax: 3 ymax: 5\nprojected CRS:\nWGS 84 / Pseudo-Mercator\n# A tibble: 1 x 3\na\nb\ngeom\n* <chr> <int> <POINT [m]>\n1 c\n3\n(3 5) sf also integrates GEOS topological predicates and operations into the same \nframework, replacing the rgeos package for access to GEOS functionality. The pre-\ncision and scale defaults differ between sf and rgeos slightly; both remain fragile \nwith respect to invalid geometries, of which there are many in circulation. From \nGEOS 3.8, both rgeos and sf offer functions to create valid geometries from invalid \nones; prior to GEOS 3.8, a function in lwgeom was used. Most recently, measure-\nments and operations on non-planar geometries have been moved from lwgeom to s2 \n(Dunnington et al. 2020; Pebesma and Dunnington 2020). Native support for units \nin sf objects carries through to measurements on outputs of topological operations: > buf_df1 <- st_buffer(df1, dist=0.3)\n> st_area(buf_df1)\nUnits: [m^2]\n[1] 0.2826142 0.2826142 0.2826142\n> plot(st_geometry(buf_df1))\n> plot(st_geometry(df1), add=TRUE, pch=4) 1 3 1 3 3 Progress in the R ecosystem for representing and handling spatial… 527 Fig. 2   Three points and three buffers plotted with geometry-specific plot methods for \"sfc\" objects Fig. 2   Three points and three buffers plotted with geometry-specific plot methods for \"sfc\" objects The sf package provides simple base graphics plotting methods. Those for just \nthe \"sfc\" column retrieved by st_geometry() do not take over the layout of \nthe graphics device, but for the \"sf\" object, they do, showing multiple non-geom-\netry columns in separate displays. Figure 2 shows a simple plot of the three buffer \npolygons overplotted with the three points. 3.2  \u0007Raster representations Like sf, the stars package for scalable, spatio-temporal tidy arrays was supported \nby an R Consortium grant. Spatio-temporal arrays were seen as an alternative way \nof representing multivariate spatio-temporal data from the choices made in the spa-\ncetime package, where a two-dimensional data frame contained stacked observation \npositions in space within stacked time points or intervals. The proposed arrays might \ncollapse to a raster layer if only one variable was chosen for one time point or inter-\nval. More important, the development of the package was extended to accommodate \na backend for earth data processing in which the data are retrieved and re-sampled \nas needed from servers, most often cloud-based servers. In most cases, these would \nbe raster geometries, but the array representation also handles irregular geometries \nthrough time. The R Consortium support was chiefly used to let contributors meet to \nmake progress on concepts and implementation. This example only covers a multiband raster taken from a Landsat 7 view of a small \npart of the Brazilian coast. In the first part, a GeoTIFF file is read into memory, using \nthree array dimensions, two in planar space, the third across six bands: 1 3 1 3 R. S. Bivand 528 > library(stars)\n> fn <- system.file(\"tif/L7_ETMs.tif\", package = \"stars\")\n> L7 <- read_stars(fn)\n> L7\nstars object with 3 dimensions and 1 attribute\nattribute(s): stars object with 3 dimensions and 1 attribute\nattribute(s): L7_ETMs.tif\nMin. :\n1.00\n1st Qu.: 54.00\nMedian : 69.00\nMean\n: 68.91\n3rd Qu.: 86.00\nMax. :255.00\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb L7_ETMs.tif\nMin. :\n1.00\n1st Qu.: 54.00\nMedian : 69.00\nMean\n: 68.91\n3rd Qu.: 86.00\nMax. :255.00\n( ) dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... 3.2  \u0007Raster representations FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb The bands can be operated on arithmetically, for example to generate a new object \ncontaining values of the normalized difference vegetation index through a function \napplied across the x and y spatial dimensions, using the st_apply abstraction: > ndvi <- function(x) (x[4] - x[3])/(x[4] + x[3])\n> (s2.ndvi <- st_apply(L7, c(\"x\", \"y\"), ndvi))\nstars object with 2 dimensions and 1 attribute\nattribute(s): > (s2.ndvi <- st_apply(L7, c(\"x\", \"y\"), ndvi))\nstars object with 2 dimensions and 1 attribute\nattribute(s):\nndvi\nMin. :-0.75342\n1st Qu.:-0.20301\nMedian :-0.06870\nMean\n:-0.06432\n3rd Qu.: 0.18667\nMax. : 0.58667\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] ( )\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] The same file can also be accessed using the proxy mechanism, which creates a link \nto the external entity, here a file: > L7p <- read_stars(fn, proxy=TRUE)\n> L7p\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif\n[1] \"[...]/L7_ETMs.tif\"\ndimension(s): > L7p <- read_stars(fn, proxy=TRUE)\n> L7p\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif\n[1] \"[...]/L7_ETMs.tif\" [1] \"[...]/L7_ETMs.tif\" dimension(s): ( )\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... 3.2  \u0007Raster representations FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb s\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\n1 352 9120761\n28 5 UTM Z\n25\nS\nh\nH\nFALSE\nNULL [ ] a\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf s\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x] x\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb 3 Progress in the R ecosystem for representing and handling spatial… 529 The same function can also be applied across the same two spatial dimensions \nof the array, but no calculation is carried out until the data are needed and the out-\nput resolution known, with the command needed to create the output stored in the \nobject: > (L7p.ndvi = st_apply(L7p, c(\"x\", \"y\"), ndvi))\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif\n[1] \"[...]/L7_ETMs.tif\" > (L7p.ndvi = st_apply(L7p, c(\"x\", \"y\"), ndvi)) > (L7p.ndvi = st_apply(L7p, c(\"x\", \"y\"), ndvi)\nstars_proxy object with 1 attribute in file:\n$L7 ETMs tif p\npp y\np,\n,\ny\n,\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif [1] \"[...]/L7_ETMs.tif\" dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... 3.2  \u0007Raster representations FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb\ncall list:\n[[1]]\nst_apply(X = X, MARGIN = c(\"x\", \"y\"), FUN = ndvi) dimension(s): dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb\ncall list:\n[[1]]\nst_apply(X = X, MARGIN = c(\"x\", \"y\"), FUN = ndvi) a\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf 1]]\n_apply(X = X, MARGIN = c(\"x\", \"y\"), FUN = ndvi) The array object can also be split, here on the band dimension, to yield a repre-\nsentation as six rasters in list form: > (x6 <- split(L7, \"band\"))\nstars object with 2 dimensions and 6 attributes\nattribute(s): stars object with 2 dimensions and 6 attributes\nattribute(s):\nX1\nX2\nX3\nX4\nMin. : 47.00\nMin. : 32.00\nMin. : 21.00\nMin. :\n9.00\n1st Qu.: 67.00\n1st Qu.: 55.00\n1st Qu.: 49.00\n1st Qu.: 52.00\nMedian : 78.00\nMedian : 66.00\nMedian : 63.00\nMedian : 63.00\nMean\n: 79.15\nMean\n: 67.57\nMean\n: 64.36\nMean\n: 59.24\n3rd Qu.: 89.00\n3rd Qu.: 79.00\n3rd Qu.: 77.00\n3rd Qu.: 75.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nX5\nX6\nMin. :\n1.00\nMin. :\n1.00\n1st Qu.: 63.00\n1st Qu.: 32.00\nMedian : 89.00\nMedian : 60.00\nMean\n: 83.18\nMean\n: 59.98\n3rd Qu.:112.00\n3rd Qu.: 88.00\nMax. :255.00\nMax. :255.00\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] X1\nX2\nX3\nX4\nMin. : 47.00\nMin. : 32.00\nMin. : 21.00\nMin. 3.2  \u0007Raster representations :\n9.00\n1st Qu.: 67.00\n1st Qu.: 55.00\n1st Qu.: 49.00\n1st Qu.: 52.00\nMedian : 78.00\nMedian : 66.00\nMedian : 63.00\nMedian : 63.00\nMean\n: 79.15\nMean\n: 67.57\nMean\n: 64.36\nMean\n: 59.24\n3rd Qu.: 89.00\n3rd Qu.: 79.00\n3rd Qu.: 77.00\n3rd Qu.: 75.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nX5\nX6\nMin. :\n1.00\nMin. :\n1.00\n1st Qu.: 63.00\n1st Qu.: 32.00\nMedian : 89.00\nMedian : 60.00\nMean\n: 83.18\nMean\n: 59.98\n3rd Qu.:112.00\n3rd Qu.: 88.00\nMax. :255.00\nMax. :255.00\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] These rasters may also be subjected to arithmetical operations, and as may be \nseen, explicit arithmetic on the six rasters has the same outcome as applying the \nsame calculation to the three-dimensional array: 1 3 530 R. S. Bivand > x6$mean <- (x6[[1]] + x6[[2]] + x6[[3]] + x6[[4]] + x6[[5]] + x6[[6]])/6\n> xm <- st_apply(L7, c(\"x\", \"y\"), mean)\n> all.equal(xm[[1]], x6$mean)\n[1] TRUE > x6$mean <- (x6[[1]] + x6[[2]] + x6[[3]] + x6[[4]] + x6[[5]] + x6[[6]])/6\n> xm <- st_apply(L7, c(\"x\", \"y\"), mean)\n> all.equal(xm[[1]], x6$mean)\n[1] TRUE The extension to a gridded temporal dimension or to irregular spatial and tem-\nporal entities is not particularly difficult, but it remains to document the possibili-\nties of the package more fully. 3.3  \u0007Visualization The classInt package (Bivand 2020a) for finding thematic mapping class inter-\nvals is used directly in plot methods in sf and stars, and in the tmap (Tennekes \n2018, 2020) and cartography (Giraud and Lambert 2016, 2017, 2020) packages. Lapa et al. (2001) (Leprosy surveillance in Olinda, Brazil, using spatial analysis \ntechniques) made available the underlying data set of Olinda census tracts (setor) \nin the Corrego Alegre 1970-72 / UTM zone 25S projection (EPSG:22525); we \nwill use the data set and the deprivation variable to point to visualization alterna-\ntives now available. > olinda <- st_read(\"olinda.gpkg\", quiet=TRUE) The style= argument gives the choice of method used for finding the number \nof classes specified, with \"fisher\" being a natural breaks method, and \"bclust\" \nbagged clustering from the e1071 package (Meyer et al. 2019): > library(classInt) > library(classInt)\n> cI_fisher <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"fisher\")\n> set.seed(1)\n> cI_bclust <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"bclust\")\nCommittee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1)\nComputing Hierarchical Clustering > cI_bclust <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"bclust\")\nCommittee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1)\nComputing Hierarchical Clustering > cI_bclust <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"bclust\")\nCommittee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1) Committee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1)\nComputing Hierarchical Clustering For a long time, the RColorBrewer package (Neuwirth 2014) colour palettes \nwere the obvious choice for thematic cartography, but more recently other packages, \nsuch as rcartocolor (Nowosad 2019), have become available, often as supersets of \nthe RColorBrewer palettes: > pal <- rcartocolor::carto_pal(7, \"SunsetDark\")\n> plot(cI_fisher, pal, xlab=\"DEPRIV\", ylab=\"\")\n> plot(cI_bclust, pal, xlab=\"DEPRIV\", ylab=\"\") > pal <- rcartocolor::carto_pal(7, \"SunsetDark\") > plot(cI_fisher, pal, xlab=\"DEPRIV\", ylab=\"\")\n> plot(cI_bclust, pal, xlab=\"DEPRIV\", ylab=\"\") Figure 3 shows plots of the two class interval schemes, with obvious differences \nbetween the two styles. Both styles are attempting to balance low within-class vari-\nance and high between-class variance. 1 3 1 3 Progress in the R ecosystem for representing and handling spatial… 531 0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\nfisher\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\nbclust\nFig. 3   Deprivation by census tract in Olinda, Brazil; empirical cumulative distribution function and class \nintervals for two class intervals: left panel: natural breaks; right panel: bagged clustering Fig. 3   Deprivation by census tract in Olinda, Brazil; empirical cumulative distribution function and class \nintervals for two class intervals: left panel: natural breaks; right panel: bagged clustering Figure 4 shows maps of the same variable with the same class intervals chosen \ninternally using classInt functionality, with the same palette. The syntax is a little \ndifferent, and here, the sf plot method default key has been turned off to permit \nside-by-side display. This method is really best at providing glimpses of the data, \nrather than at creating complete figures, in contrast to the richer functions in cartog-\nraphy; both use base graphics. Fig. > plot(olinda[,\"DEPRIV\"], nbreaks=7, breaks=\"fisher\", pal=pal, key.pos=NULL,\n+\nmain=\"\")\n> library(cartography)\n> choroLayer(olinda, var=\"DEPRIV\", method=\"fisher-jenks\", nclass=7, col=pal,\n+\nlegend.values.rnd=3) > olinda <- st_read(\"olinda.gpkg\", quiet=TRUE) 6   Thematic maps with ggplot2: left panel: default map of a continuous variable; right panel: grati-\ncule removed and palette modified Fig. 7   Thematic maps with ggplot2, natural breaks class intervals: left panel: natural breaks class inter-\nvals; right panel: bagged clustering class intervals Fig. 7   Thematic maps with ggplot2, natural breaks class intervals: left panel: natural breaks class inter-\nvals; right panel: bagged clustering class intervals We can also display the bagged clustering class intervals beside the natural \nbreaks map, again using ggplot2; Figure 7 shows the maps, but because a con-\ntinuous scale is still enforced, all that changes is the position of the breaks on \nthe key. > g3 <- g1 + theme_void() + scale_fill_gradientn(colours=pal,\n+\nbreaks=round(cI_bclust$brks, 3)) > g3 <- g1 + theme_void() + scale_fill_gradientn(colours=pal,\n+\nbreaks=round(cI_bclust$brks, 3)) > olinda <- st_read(\"olinda.gpkg\", quiet=TRUE) 4   Deprivation by census tract in Olinda, Brazil, natural breaks class intervals; left panel: sf plot \nmethod; right panel: cartography choropleth map Fig. 4   Deprivation by census tract in Olinda, Brazil, natural breaks class intervals; left panel: sf plot \nmethod; right panel: cartography choropleth map 3 532 R. S. Bivand Fig. 5   tmap output, natural breaks class intervals; left panel: object o1 without boundaries; right panel: \nobject o2 with boundaries Fig. 5   tmap output, natural breaks class intervals; left panel: object o1 without boundaries; right panel: \nobject o2 with boundaries Figure 5 shows how tmap functions use grid graphics to permit graphics objects to \nbe updated and then plotted using tmap_arrange(). Once again, the same class \nintervals are chosen internally using classInt, with the same palette. The left panel is \nupdated on the right to add thin boundaries between census tracts. tmap also offers \nsmall multiples of facets, for example thematic maps of the same variable observed at \nsuccessive time periods using the same class intervals. An extensive discussion of the \nuse of tmap is provided by Lovelace et al. (2019). > library(tmap)\n> o1 <- tm_shape(olinda) + tm_fill(\"DEPRIV\", style=\"fisher\", n=7, palette=pal)\n> o2 <- o1 + tm_borders(lwd=0.8) > library(tmap)\n> o1 <- tm_shape(olinda) + tm_fill(\"DEPRIV\", style=\"fisher\", n=7, palette=pal)\n> o2 <- o1 + tm_borders(lwd=0.8) > library(tmap) The ggplot2 package (Wickham et al. 2020) provides the geom_sf() facil-\nity for mapping sf objects: > library(ggplot2)\n> g1 <- ggplot(olinda) + geom_sf(aes(fill=DEPRIV))\n> g2 <- g1 + theme_void() + scale_fill_gradientn(colours=pal,\n+\nbreaks=round(cI_fisher$brks, 3)) This approach also builds on grid graphics. It is possible to set a theme that \ndrops the arguably unnecessary graticule, but there is a lot of intervention \nrequired to get a simple map. To get proper class intervals involves even more \nwork, because the package takes specific, not general, positions on how graphics \nare observed. ColorBrewer, for example, eschews continuous colour scales based \non cognitive research, but ggplot2 enforces them by default for continuous vari-\nables. Figure 6 shows the default choice of palette, updated to remove the unnec-\nessary graticule and to use the user-specified palette and class intervals: 1 3 1 3 3 Progress in the R ecosystem for representing and handling spatial… 533 Fig. 6   Thematic maps with ggplot2: left panel: default map of a continuous variable; right panel: grati-\ncule removed and palette modified Fig. 3.4  \u0007Reverse dependencies of the sp and sf packages R packages can possess forward or reverse dependencies. Forward or upstream \ndependencies are typically on R itself, a small number of packages whose func-\ntionalities are used in the package in question (by loading and attaching the pack-\nage (dependencies) or just loading the namespace of the package (imports)), and \npossibly external software libraries. Reverse or downstream dependencies are \npackages that themselves use the package in question by loading and attaching \nit, only loading its namespace or using it on demand (suggests). sp and sf were \nwritten carefully to minimize forward dependencies, with sp only depending on \nand importing packages included in every R distribution by default and sf adding \nCRAN contributed packages Rcpp and units required to build the package and 1 3 3 534 R. S. Bivand classInt for class intervals, DBI for interfacing spatial databases and magrittr \nfor piped operations, where none of these extra forward dependencies draws in \nmany other packages. classInt for class intervals, DBI for interfacing spatial databases and magrittr \nfor piped operations, where none of these extra forward dependencies draws in \nmany other packages. In sp, the compiled code (written in C) is self-contained and is made available to \nother packages, chiefly rgdal and rgeos, to link to their compiled code. rgdal links \nto sp and to the external libraries PROJ and GDAL. GDAL itself links to PROJ and \ncan link to GEOS and many other libraries needed for specific drivers. The external \nsoftware versions used may be reported using *_extSoftVersion, here using \nthe :: operator to avoid attaching the packages being queried: > rgdal::rgdal_extSoftVersion()\nGDAL GDAL_with_GEOS\nPROJ\nsp\n\"3.1.2\"\n\"TRUE\"\n\"7.1.0\"\n\"1.4-4\" The versions vary between platforms and by the installation method used; as \npackage maintainer, I often run with pre-release or latest versions of external soft-\nware to attempt to detect and mitigate changes before they impact users’ workflows. For rgdal, the versions of GDAL, PROJ and sp are reported, together with a test \nshowing whether GDAL was built linking to GEOS, something that affects the \nbehaviour of some drivers. The report for rgeos is simpler, only listing the versions \nof GEOS itself and sp. 3.4  \u0007Reverse dependencies of the sp and sf packages > rgeos::rgeos_extSoftVersion()\nGEOS\nsp\n\"3.8.1\" \"1.4-4\" In the case of sf, and because it brings together access to the GDAL and GEOS \nexternal libraries through Simple Features representation for vector objects, the \nexternal software versions supported are the union of those seen above, omitting \nlinkage to a separate package defining classes for objects. In addition, it reports \nwhich API is used for PROJ, either proj.h or not (the earlier proj_api.h). > sf_extSoftVersion()\nGEOS\nGDAL\nproj.4 GDAL_with_GEOS\nUSE_PROJ_H\n\"3.8.1\"\n\"3.1.2\"\n\"7.1.0\"\n\"true\"\n\"true\" Table 1 shows the structure of reverse dependency counts for sp and sf. Recursive \ndependencies traverse through the whole CRAN dependency tree; the first column \nof the table shows counts of “depends” and “imports” dependencies counted across \nthe whole tree. These split into 1285 only involving sp, 232 involving both pack-\nages and 65 only involving sf. If we additionally include “suggests” dependencies, \nboth packages may be used at least indirectly by all CRAN packages. The two right \ncolumns show the same counts, but only for packages’ first-order dependencies on \nsp, sf or both. We can note that of these for “depends” and “imports” dependencies, \n459 only involve sp, 63 involve both packages and 121 only involve sf. In the first 1 3 Progress in the R ecosystem for representing and handling spatial… Progress in the R ecosystem for representing and handling spatial…\nTable 1   Reverse dependency \ncounts for sp and sf, August \n2020, for recursive and non-\nrecursive reverse dependencies \ntaken as “Depends” and \n“Imports” only, and with \n“Suggests”\nRecursive\nRecursive \nw/suggests\nNot recursive\nNot recursive \nw/suggests\nSum sp\n1517\n16,619\n522\n629\nSum sf\n297\n16,619\n184\n277\nOnly sp\n1285\n0\n459\n513\nOnly sf\n65\n0\n121\n161\nBoth\n232\n16,619\n63\n116\n535 535 column, the number of packages only depending on sf is less than when we ignore \nrecursive dependencies in the third column, which is packages using sf that also use \nsp. The number of packages only using sf is encouraging, given that it first entered \nCRAN in October 2016. It is also encouraging that a fair number of these packages use both sp and sf, \nshowing existing packages are preserving legacy workflows, but also opening up for \nmore modern object representations. It takes time and effort to communicate the \ndesirability of migrating from sp representations to sf and probably stars. 3.4  \u0007Reverse dependencies of the sp and sf packages Although \nkeeping the R code running is feasible, including compiled code not using external \nsoftware, migration to sf is advisable. 4  \u0007Upstream software dependencies of the R‑spatial ecosystem When changes occur in upstream external software, R packages using these libraries \noften need to adapt, but package maintainers try very hard to shield users from any \nnegative consequences, so that legacy workflows continue to provide the same or \nat least similar results from the same data. The code shown in Bivand et al. (2008, \n(2013) is almost all run nightly on a platform with updated R packages and external \nsoftware. This does not necessarily trap all differences (figures are not compared), \nbut is helpful in detecting impacts of changes in packages or external software. It is \nalso very helpful that CRAN servers using the released and development versions of \nR, and with different versions of external software, also run nightly checks. Again, \nsometimes changes are only noticed by users, but quite often checks run by main-\ntainers and by CRAN alert us to impending challenges. Tracking the development \nmailing lists of the external software communities, all open source, can also show \nhow thinking is evolving. However, sometimes code tidying in external software \ncan have unexpected consequences, breaking not sf or sp with rgdal or rgeos, but a \npackage further downstream. Bivand (2014) discusses open-source geospatial soft-\nware stacks more generally, but here we will consider ongoing changes in PROJ and \nlinked changes in GDAL. We will use the example of the location of the Broad Street pump in Soho, London, \nrelated to the 1854 Cholera epidemic and Dr John Snow’s intervention Brody et al. 1 3 536 R. S. Bivand (2000), distributed with sf (from version 0.8–1). Although it was known that changes in \nupstream software would impact workflows, the extent of those impacts became clear \nusing a standard example following upgrading to PROJ 6 and GDAL 3 in 2019: (2000), distributed with sf (from version 0.8–1). Although it was known that changes in \nupstream software would impact workflows, the extent of those impacts became clear \nusing a standard example following upgrading to PROJ 6 and GDAL 3 in 2019: > bp_file <- system.file(\"gpkg/b_pump.gpkg\", package=\"sf\")\n> b_pump_sf <- st_read(bp_file, quiet=TRUE) > bp_file <- system.file(\"gpkg/b_pump.gpkg\", package=\"sf\")\n> b_pump_sf <- st_read(bp_file, quiet=TRUE) Before R packages upgraded the way coordinate reference systems were represented \nin early 2020, our Proj4 string representation suffered degradation. Taking the Proj4 \nstring defined in PROJ 5 for the British National Grid, we can see a +datum=OSGB36 \nkey–value pair. 4  \u0007Upstream software dependencies of the R‑spatial ecosystem But when processing this input with PROJ 6 and GDAL 3, this key is \nremoved. Checking, we can see that reading the input string appears to work, but the \noutput for the Proj4 string drops the +datum=OSGB36 key–value pair, introducing \ninstead the ellipse implied by that datum: > proj5 <- paste0(\"+proj=tmerc +lat_0=49 +lon_0=-2 +k=0.9996012717\",\n+\n\" +x_0=400000 +y_0=-100000 +datum=OSGB36 +units=m +no_defs\")\n> legacy <- st_crs(proj5)\n> proj6 <- legacy$proj4string\n> proj5_parts <- unlist(strsplit(proj5, \" \"))\n> proj6_parts <- unlist(strsplit(proj6, \" \"))\n> proj5_parts[!is.element(proj5_parts, proj6_parts)]\n[1] \"+datum=OSGB36\"\n> proj6_parts[!is.element(proj6_parts, proj5_parts)]\n[1] \"+ellps=airy\" > proj5 <- paste0(\"+proj=tmerc +lat_0=49 +lon_0=-2 +k=0.9996012717\n+\n\" +x_0=400000 +y_0=-100000 +datum=OSGB36 +units=m +no_defs\")\n> legacy <- st_crs(proj5)\n> proj6 <- legacy$proj4string\n> proj5_parts <- unlist(strsplit(proj5, \" \"))\n> proj6_parts <- unlist(strsplit(proj6, \" \"))\n> proj5_parts[!is.element(proj5_parts, proj6_parts)]\n[1] \"+datum=OSGB36\"\n> proj6_parts[!is.element(proj6_parts, proj5_parts)]\n[1] \"+ellps=airy\" We can emulate the problem seen following the release in May 2019 of GDAL 3.0.0 \nusing PROJ 6, by inserting the degraded Proj4 string into the Broad Street pump object. The coordinate reference system representation is now ignorant of the proper datum \nspecification: > b_pump_sf1 <- b_pump_sf > b_pump_sf1 <- b_pump_sf _p\np_\n_p\np_\n> st_crs(b_pump_sf1) <- st_crs(st_crs(b_pump_sf1)$proj4string) p\np\np\np\n> st_crs(b_pump_sf1) <- st_crs(st_crs(b_pump_sf1)$proj4string) > st_crs(b_pump_sf1) <- st_crs(st_crs(b_pump_sf1)$proj4string) Why does this matter? For visualization on a web map, for example using the \nmapview package, the projected geometries are transformed to the same WGS84 \nellipse and datum (EPSG:4326) that were used in PROJ 4 as a transformation hub. In leaflet, these are projected to Web Mercator (EPSG:3857). In mapview(), the \nsf::st_transform() function is used, so we will emulate this step before hand-\ning on the geometries for display. 4  \u0007Upstream software dependencies of the R‑spatial ecosystem The projected British National Grid point location is \nunchanged: 1 3 1 3 Progress in the R ecosystem for rep\n> all.equal(st_coordinate\n+\nst_coordinate\n[1] TRUE\nHowever, because the \nresentation of its coordin\nformed identically to WG\nFigure 8:\n> b_pump_sf_ll <- st_tran\n> b_pump_sf1_ll <- st_tra\n> st_distance(b_pump_sf_l\nUnits: [m]\n[,1]\n[1,] 125.0578 Progress in the R ecosystem for representing and handling spatial… 537 > all.equal(st_coordinates(st_geometry(b_pump_sf)),\n+\nst_coordinates(st_geometry(b_pump_sf1)))\n[1] TRUE However, because the one of the objects now has a degraded Proj4 string rep-\nresentation of its coordinate reference system, the output points, apparently trans-\nformed identically to WGS84, are now some distance apart, as is also shown in \nFigure 8: > b_pump_sf_ll <- st_transform(b_pump_sf, 4326)\n> b_pump_sf1_ll <- st_transform(b_pump_sf1, 4326)\n> st_distance(b_pump_sf_ll, b_pump_sf1_ll)\nUnits: [m]\n[,1]\n[1,] 125.0578 > b_pump_sf_ll <- st_transform(b_pump_sf, 4326) [,1]\n[1,] 125.0578 Once PROJ 6 and GDAL 3 had stabilized in the summer of 2019, we iden-\ntified the underlying threat as lying in the advertised degradation of GDAL’s \nexportToProj4() function. When reading raster and vector files, the coordinate ref-\nerence system representation using Proj4 strings would often be degraded, so that fur-\nther transformation within R (also using GDAL/PROJ functionality) would be at risk of \nmuch greater inaccuracy than with PROJ 5 and GDAL 2. Since then, sf, sp with rgdal Fig. 8   Displays made using mapview displays of the Broad Street pump, with the green point within 2 m \nof the pump location, and the red point in Ingestre Place because of the loss of the datum specification Fig. 8   Displays made using mapview displays of the Broad Street pump, with the green point within 2 m \nof the pump location, and the red point in Ingestre Place because of the loss of the datum specification Fig. 8   Displays made using mapview displays of the Broad Street pump, with the green point within 2 m \nof the pump location, and the red point in Ingestre Place because of the loss of the datum specification 1 3 3 538 R. S. 7  https​://cran.r-proje​ct.org/web/packa​ges/rgdal​/vigne​ttes/CRS_proje​ction​s_trans​forma​tions​.html. 6  https​://www.r-spati​al.org/r/2020/03/17/wkt.html.\n7 8  State of https​://githu​b.com/rsbiv​and/sp of 18 August 2020 or later. 4  \u0007Upstream software dependencies of the R‑spatial ecosystem Bivand and raster have adopted the 2019 version of the “Well-Known Text” coordinate refer-\nence system representation WKT2-2019 (ISO 2019) instead of Proj4 strings to contain \ncoordinate reference system definitions.6 Accommodations have also been provided so \nthat the S3 class \"crs\" objects used in objects defined in sf, and the formal S4 class \n\"CRS\" objects used objects defined in sp and raster, can continue to attempt to support \nProj4 strings in addition, while other package maintainers and workflow users catch up.7 l\nFollowing an extended campaign of checking about 900 reverse dependencies \n(packages depending on sp, rgdal and others) and dozens of github issues, most of \nthe consequences of the switch to WKT2 among packages have now been addressed. Most recently (late August 2020), 115 packages have been offered rebuilt stored \nobjects that had included \"CRS\" objects without WKT2 definitions. i\nThis approach has ensured that spatial objects, whether created within R, read in from \nexternal data sources or read as stored objects, all have WKT2 string representations of \ntheir coordinate reference systems, and for backward compatibility can represent these in \naddition as Proj4 strings. Operations on objects should carry forward the new represen-\ntations, which should be written out to external data formats correctly. There is a minor \ndivergence between sf and sp (and thus rgdal): In sf, the axis order of the CRS is pre-\nserved as instantiated, but objects do not have their axes swapped to accord with authori-\nties unless sf::st_axis_order() is set TRUE. This can appear odd, because \nalthough the representation records a northings–eastings axis order, data are treated as \neastings–northings in plotting, variogram construction and so on: > st_crs(\"EPSG:4326\")\nCoordinate Reference System:\nUser input: EPSG:4326\nwkt:\nGEOGCRS[\"WGS 84\",\nDATUM[\"World Geodetic System 1984\",\nELLIPSOID[\"WGS 84\",6378137,298.257223563,\nLENGTHUNIT[\"metre\",1]]],\nPRIMEM[\"Greenwich\",0,\nANGLEUNIT[\"degree\",0.0174532925199433]],\nCS[ellipsoidal,2],\nAXIS[\"geodetic latitude (Lat)\",north,\nORDER[1],\nANGLEUNIT[\"degree\",0.0174532925199433]],\nAXIS[\"geodetic longitude (Lon)\",east,\nORDER[2],\nANGLEUNIT[\"degree\",0.0174532925199433]], ANGLEUNIT[\"degree\",0.0174532925199433]], ANGLEUNIT[\"degree\",0.0174532925199433]] g\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] g\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] USAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] USAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] ID[\"EPSG\",4326]] In sp/rgdal, attempts are made to ensure that axis order is in the form termed \nGIS, traditional or visualization that is always eastings–northings: 1 3 Progress in the R ecosystem for representing and handling spatial… > library(sp)\n> cat(wkt(CRS(\"EPSG:4326\")))\nGEOGCRS[\"WGS 84\",\nDATUM[\"World Geodetic System 1984\",\nELLIPSOID[\"WGS 84\",6378137,298.257223563,\nLENGTHUNIT[\"metre\",1]],\nID[\"EPSG\",6326]],\nPRIMEM[\"Greenwich\",0,\nANGLEUNIT[\"degree\",0.0174532925199433],\nID[\"EPSG\",8901]],\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nAXIS[\"latitude\",north,\nORDER[2],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]]] > library(sp)\n> cat(wkt(CRS(\"EPSG:4326\")))\nGEOGCRS[\"WGS 84\",\nDATUM[\"World Geodetic System 1984\",\nELLIPSOID[\"WGS 84\",6378137,298.257223563,\nLENGTHUNIT[\"metre\",1]],\nID[\"EPSG\",6326]],\nPRIMEM[\"Greenwich\",0,\nANGLEUNIT[\"degree\",0.0174532925199433],\nID[\"EPSG\",8901]],\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nAXIS[\"latitude\",north,\nORDER[2],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]]] Progress in the R ecosystem for representing and handling spatial… 539 9  nl is used to represent the verbatim new line character in the processed article. USAGE[ USAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]]] The probability of confusion increases when coercing from sf to sp and vice \nversa, with the representations most often remaining unchanged.8 > sf_from_sp <- st_crs(CRS(\"EPSG:4326\"))\n> o <- strsplit(sf_from_sp$wkt, nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl))\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nAXIS[\"latitude\",north,\nORDER[2], > sf_from_sp <- st_crs(CRS(\"EPSG:4326\"))\n> o <- strsplit(sf_from_sp$wkt, nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl))\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nAXIS[\"latitude\",north,\nORDER[2], > sf_from_sp <- st_crs(CRS(\"EPSG:4326\")) o <- strsplit(sf_from_sp$wkt, nl)[[1]] p\np\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl)) (p\n( [g\np(\n|\n|\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nAXIS[\"latitude\",north,\nORDER[2], > sp_from_sf <- as(st_crs(\"EPSG:4326\"), \"CRS\")\n> o <- strsplit(wkt(sp_from_sf), nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl))\nCS[ellipsoidal,2],\nAXIS[\"geodetic latitude (Lat)\",north,\nORDER[1],\nAXIS[\"geodetic longitude (Lon)\",east,\nORDER[2], > sp_from_sf <- as(st_crs(\"EPSG:4326\"), \"CRS\") > sp_from_sf <- as(st_crs(\"EPSG:4326\"), \"CRS\")\n> o <- strsplit(wkt(sp_from_sf), nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl)\nCS[ellipsoidal,2],\nAXIS[\"geodetic latitude (Lat)\",north,\nORDER[1],\nAXIS[\"geodetic longitude (Lon)\",east,\nORDER[2], > o <- strsplit(wkt(sp_from_sf), nl)[[1]] p\np\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl)) Both of these coercions are using the same underlying PROJ and GDAL versions, \nand the same PROJ metadata. Once work in progress is completed, coercions should \nrespect the setting of sf::st_axis_order(). 1 3 540 R. S. Bivand It may useful for users to know of other differences between sf and sp/rgdal. Transformation in sf uses code in GDAL, which in turn uses functions in PROJ; in \nsp/rgdal, PROJ is used directly for transformation. In order to demonstrate more of \nwhat is happening, let us coerce these sf objects to sp (they are both planar with an \nx–y axis order): > b_pump_sp <- as(b_pump_sf, \"Spatial\")\n> b_pump_sp1 <- as(b_pump_sf1, \"Spatial\") > b_pump_sp <- as(b_pump_sf, \"Spatial\")\n> b_pump_sp1 <- as(b_pump_sf1, \"Spatial\") We will also set up a temporary directory for use with the on-demand grid down-\nload functionality in PROJ 7; this must be done before rgdal is loaded: > td <- tempfile()\n> dir.create(td)\n> Sys.setenv(\"PROJ_USER_WRITABLE_DIRECTORY\"=td)\n> library(rgdal) > td <- tempfile()\n> dir.create(td)\n> Sys.setenv(\"PROJ_USER_WRITABLE_DIRECTORY\"=td)\n> library(rgdal) > td <- tempfile()\n> dir.create(td)\n> Sys.setenv(\"PROJ_USER_WRITABLE_DIRECTORY\"=td)\n> library(rgdal) > td <- tempfile() > td <- tempfile()\n> dir.create(td)\n( > dir.create(td) > library(rgdal) In sf, areas of interest need to be given by the users, while in transformation and \nprojection in rgdal, these are calculated from the object being projected or trans-\nformed. The provision of areas of interest is intended to reduce the number of candi-\ndate coordinate operations found by PROJ. USAGE[ > WKT <- wkt(b_pump_sp)\n> o <- list_coordOps(WKT, \"EPSG:4326\")\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 8 7 1\n> aoi0 <- project(t(unclass(bbox(b_pump_sp))), WKT, inv=TRUE)\n> aoi <- c(t(aoi0 + c(-0.1, +0.1)))\n> o_aoi <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o_aoi), nrow(o_aoi[o_aoi$instantiable,]), sum(o_aoi$number_grids))\n[1] 5 4 1 rgdal::list_coordOps() accesses the PROJ metadata database to \nsearch through candidate coordinate operations, ranking them by accuracy, \nreturning a data frame of operations. When an area of interest is provided, candi-\ndates falling outside it are dropped. Coordinate operations that cannot be instanti-\nated because of missing grids are also listed. We can see here that when an area \nof interest is not given, 8 candidate operations are found when the WKT string \ncontains datum information. Of these, 7 may be instantiated, with 1 needing a \ngrid (here, the operation that cannot be instantiated). Three operations cease to be \ncandidates if we use an area of interest. In sp/rgdal, the coordinate operation last used is returned and can be retrieved \nusing rgdal::get_last_coordOp(); coordinate operations are repre-\nsented as pipelines (Knudsen and Evers 2017; Evers and Knudsen 2017), intro-\nduced in PROJ 5 and using the PROJ key–value pair notation:9 1 3 1 3 Progress in the R ecosystem for representing and handling spatial… 541 > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\")\n> cat(strwrap(get_last_coordOp()), sep=nl)\n+proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601\n+x_0=400000 +y_0=-100000 +ellps=airy +step +proj=push +v_3 +step +proj=cart\n+ellps=airy +step +proj=helmert +x=446.448 +y=-125.157 +z=542.06 +rx=0.15\n+ry=0.247 +rz=0.842 +s=-20.489 +convention=position_vector +step +inv\n+proj=cart +ellps=WGS84 +step +proj=pop +v_3 +step +proj=unitconvert\n+xy_in=rad +xy_out=deg > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\")\n> cat(strwrap(get_last_coordOp()), sep=nl)\n+proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601\n+x_0=400000 +y_0=-100000 +ellps=airy +step +proj=push +v_3 +step +proj=cart\n+ellps=airy +step +proj=helmert +x=446.448 +y=-125.157 +z=542.06 +rx=0.15\n+ry=0.247 +rz=0.842 +s=-20.489 +convention=position_vector +step +inv\n+proj=cart +ellps=WGS84 +step +proj=pop +v_3 +step +proj=unitconvert\n+xy_in=rad +xy_out=deg > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\") > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\") > b_pump_sp_ll <- spTransform(b_pump_sp, \"EP\n> cat(strwrap(get_last_coordOp()), sep=nl) p\np\np\np\np\np\np\n> cat(strwrap(get_last_coordOp()), sep=nl) +proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601\n+x_0=400000 +y_0=-100000 +ellps=airy +step +proj=push +v_3 +step +proj=cart\n+ellps=airy +step +proj=helmert +x=446.448 +y=-125.157 +z=542.06 +rx=0.15\n+ry=0.247 +rz=0.842 +s=-20.489 +convention=position_vector +step +inv\n+proj=cart +ellps=WGS84 +step +proj=pop +v_3 +step +proj=unitconvert\n+xy_in=rad +xy_out=deg Here, we can see that an inverse projection from the specified Transverse Mer-\ncator projection is made to geographical coordinates, followed by a seven-param-\neter Helmert transformation to WGS84 ellipsoid and datum. 10  https​://cdn.proj.org. > enable_proj_CDN()\n[1] \"Using: /tmp/Rtmp5keMtw/file1c4592b3b37c2\"\n> list.files(td)\ncharacter(0) > o <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 5 5 1 > o <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 5 5 1 > o <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 5 5 1 > b_pump_sp_llg <- spTransform(b_pump_sp, \"EPSG:4326\") > cat(strwrap(get_last_coordOp()), sep=nl) +proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601 +x_0=400000 +y_0=-100000 +ellps=airy +step +proj=hgridshift +grids=uk_os_OSTN15_NTv2_OSGBtoETRS.tif +step +proj=unitconvert +xy_in=rad\n+xy_out=deg Now the downloaded grid is cached in the database in the designated CDN direc-\ntory and may be used for other transformations using the same operation. > list.files(td)\n[1] \"cache.db\"\n> file.size(file.path(td, list.files(td)[1]))\n[1] 319488\n> disable_proj_CDN() > list.files(td)\n[1] \"cache.db\"\n> file.size(file.path(td, list.files(td)[1]))\n[1] 319488\n> disable_proj_CDN() Once again, the distance between the point transformed from the sf object as read \nfrom file and the point with the degraded coordinate reference system emulating the \neffect of the change in behaviour of GDAL’s exportToProj4() in GDAL 6 and \nlater is about 125 m. Using the CDN shifts the output point by 1.7 m. For confirma-\ntion, the output transformed coordinates for the sp and sf objects using the Helmert \ntransformation are the same. > c(spDists(b_pump_sp1_ll, b_pump_sp_ll),\n+\nspDists(b_pump_sp_llg, b_pump_sp_ll))*1000\n[1] 125.057683\n1.751474\n> all.equal(unname(coordinates(b_pump_sp_ll)),\n+\nunname(st_coordinates(st_geometry(b_pump_sf_ll))))\n[1] TRUE > c(spDists(b_pump_sp1_ll, b_pump_sp_ll),\n+\nspDists(b_pump_sp_llg, b_pump_sp_ll))*1000\n[1] 125.057683\n1.751474\n> all.equal(unname(coordinates(b_pump_sp_ll)),\n+\nunname(st_coordinates(st_geometry(b_pump_sf_ll))))\n[1] TRUE > c(spDists(b_pump_sp1_ll, b_pump_sp_ll),\n+\nspDists(b_pump_sp_llg, b_pump_sp_ll))*1000\n[1] 125.057683\n1.751474\n> all.equal(unname(coordinates(b_pump_sp_ll)),\n+\nunname(st_coordinates(st_geometry(b_pump_sf_ll))))\n[1] TRUE Although it appears that most of the consequences of the change in representa-\ntion of coordinate reference systems from Proj4 to WKT2 strings have now been \naddressed, we still see signs on the mailing list and on Twitter that users, natu-\nrally directing their attention to their analytical or visualization work, may still \nbe confused. The extent of the spatial cluster of R packages is so great that it will \nundoubtedly take time before the dust settles. However, we trust that the operation \nof upgrading representations is now largely complete. Multiple warnings issued in \nsp workflows, now noisily drawing attention to possible degradations in workflows, \nwill by default be muted when sp 1.5 and rgdal 1.6 are released. USAGE[ The parameters are \ncontained in the best instantiable coordinate operation retrieved from the PROJ \ndatabase. > o <- list_coordOps(wkt(b_pump_sp1), \"EPSG:4326\", area_of_interest=aoi)\n> cat(nrow(o), o$ballpark, nl)\n1 TRUE > b_pump_sp1_ll <- spTransform(b_pump_sp1, \"EPSG:4326\") > cat(strwrap(get_last_coordOp()), sep=nl) Going on to the case of the degraded representation, only 1 operation is found, \nwith only ballpark accuracy. With our emulation of the dropping of +datum= sup-\nport in GDAL’s exportToProj4(), we see that the coordinate operation pipe-\nline only contains the inverse projection step, accounting for the observed shift of \nthe Broad Street pump to Ingestre Place. Finally, sp/rgdal may use the provision of on-demand downloading of transfor-\nmation grids to provide more accuracy (CDN, from PROJ 7).10 Before finding and \nchoosing to use a coordinate operation using an on-demand downloaded grid, the \ndesignated directory is empty: > enable_proj_CDN()\n[1] \"Using: /tmp/Rtmp5keMtw/file1c4592b3b37c2\"\n> list.files(td)\ncharacter(0) Using the CDN, all the candidate operations are instantiable, and the pipeline \nnow shows a horizontal grid transformation rather than a Helmert transformation. 1 3 R. S. Bivand 542 5  \u0007Outlook As has already been mentioned, either most sp-based workflows continue to func-\ntion using sf objects or changes have been made in packages like spdep and its mod-\nelling counterpart spatialreg to permit scripts and packages using these packages \nto continue to function. Some workflows require more attention than others, but 1 3 Progress in the R ecosystem for representing and handling spatial… 543 the transition to sf from sp, rgdal and rgeos should be unproblematic. Shifting to \nnew visualization packages like tmap, cartography and mapview should also be \nrelatively easy, and use of sf and the new visualization packages should certainly \nbecome standard for new research and teaching. It may take a little longer for stars \nto find its place, but work here is continuing, and will stabilize before long. Similar \nremarks apply to transition from raster to terra. The key challenges for handling spatial data using R concern the upstream \nsoftware libraries in the open-source geospatial software stack, not just PROJ as \ndescribed above or changes in validity requirements in GEOS and other libraries, \nbut also the opportunities opened up by access to cloud-based earth observation data \nstreams. Because incoming data may take forms as yet not provided for, even where \nGDAL drivers become available, changes in object representations may become \nnecessary. In particular, this relates to spatio-temporal data, where trajectory data \nare especially demanding. These data representation challenges are actually oppor-\ntunities for users and developers to continue cooperating to contribute to making the \nR ecosystem for handling and analysing spatial and spatio-temporal data even more \ncapable and performant. Finally, please note that a CODECHECK certificate for this \npaper is available at https​://doi.org/10.5281/zenod​o.40038​48. CODECHECK is an \nopen-science initiative to facilitate sharing of computer programs and results pre-\nsented in scientific publications (see https​://codec​heck.org.uk/) Funding  Open Access funding provided by Norwegian School of Economics. Funding  Open Access funding provided by Norwegian School of Economics. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, \nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as \nyou give appropriate credit to the original author(s) and the source, provide a link to the Creative Com-\nmons licence, and indicate if changes were made. The images or other third party material in this article \nare included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. 5  \u0007Outlook If material is not included in the article’s Creative Commons licence and your intended use is \nnot permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission \ndirectly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​\nses/by/4.0/. nding  Open Access funding provided by Norwegian School of Economics. References https\nCRAN.R-proje​ct.org/packa​ge=rgeos​, R package version 0.5-3 Bivand R, Pebesma E, Gomez-Rubio V (2008) Applied spatial data analysis with R. Springer, New Y Bivand R, Pebesma E, Gomez-Rubio V (2008) Applied spatial data analysis with R. Springer, New Yor\nBivand R, Pebesma E, Gomez-Rubio V (2013) Applied spatial data analysis with R, 2nd edn. 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References https​://do\norg/10.32614​/RJ-2016-061 Pebesma E, Mailund T, Kalinowski T (2020) units: measurement units for R vectors. https​://CRAN.R-\nproje​ct.org/packa​ge=units​, r package version 0.6-7 p\nj\ng p\ng\np\ng\nRenka RJ, Gebhardt A (2020) tripack: triangulation of irregularly spaced data. https​://CRAN.R-proje​\n/\nk\ni\nk\nk\ni\n1 3 9 1 Renka RJ, Gebhardt A (2020) tripack: triangulation of ir\nct.org/packa​ge=tripa​ck, R package version 1.3-9.1 enka RJ, Gebhardt A (2020) tripack: triangulation of irregularly spaced data. https​://CRAN.R-proje​\nct.org/packa​ge=tripa​ck, R package version 1.3-9.1 (\n)\np\ng\nct.org/packa​ge=tripa​ck, R package version 1.3-9.1 ct.org/packa​ge=tripa​ck, R package version 1.3-9.1 Rowlingson B, Diggle PJ (1993) Splancs: spatial point pattern analysis code in S-Plus. 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Springer, Berlin, pp 87–104 Wickham H (2014) Tidy data. J Stat Softw 59(10):1–23. https​://doi.org/10.18637​/jss.v059.i10 Wickham H, Chang W, Henry L, Pedersen TL, Takahashi K, Wilke C, Woo K, Yutani H (2020) ggplot2: \ncreate elegant data visualisations using the grammar of graphics. https​://CRAN.R-proje​ct.org/packa​\nge=ggplo​t2, R package version 3.3.2 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published \nmaps and institutional affiliations. Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published \nmaps and institutional affiliations. 1 3 1 3 1 3"
https://openalex.org/W2338185363
https://publikationen.bibliothek.kit.edu/1000055744/4284342
English
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Infrared Spectroscopy of Bilberry Extract Water-in-Oil Emulsions: Sensing the Water-Oil Interface
Biosensors
2,016
cc-by
8,846
Infrared Spectroscopy of Bilberry Extract Water-in-Oil Emulsions: Sensing the Water-Oil Interface 2 Erlangen Graduate School in Advanced Optical Technologies, University of Erlangen-Nuremberg, Erlangen 91052, Germany 3 School of Engineering, University of Aberdeen, Aberdeen AB24 3UE, UK 4 Section I: Food Process Engineering, Institute of Process Engineering in Life Sciences, Karlsruhe Institute of Technology, Karlsruhe 76131, Germany; heike.schuchmann@kit.edu 5 Present affiliation: BASF SE, Ludwigshafen 67056, Germany; kerstin.frank@basf.com * Correspondence: jkiefer@uni-bremen.de; Tel.: +49-421-218-64777 3 School of Engineering, University of Aberdeen, Aberdeen AB24 3UE, UK 4 Section I: Food Process Engineering, Institute of Process Engineering in Life Sciences, Karlsruhe Institute of Technology, Karlsruhe 76131, Germany; heike.schuchmann@kit.edu 5 Present affiliation: BASF SE, Ludwigshafen 67056, Germany; kerstin.frank@basf.com * Correspondence: jkiefer@uni-bremen.de; Tel.: +49-421-218-64777 3 School of Engineering, University of Aberdeen, Aberdeen AB24 3UE, UK 4 Section I: Food Process Engineering, Institute of Process Engineering in Life Sciences, Karlsruhe Institute of Technology, Karlsruhe 76131, Germany; heike.schuchmann@kit.edu 5 Present affiliation: BASF SE, Ludwigshafen 67056, Germany; kerstin.frank@basf.com * Correspondence: jkiefer@uni-bremen.de; Tel.: +49-421-218-64777 Academic Editor: Torsten Frosch Received: 16 February 2016; Accepted: 8 April 2016; Published: 14 April 2016 Received: 16 February 2016; Accepted: 8 April 2016; Published: 14 April 2016 Abstract: Water-in-oil (w/o) emulsions are of great interest in many areas of the life sciences, including food technology, bioprocess engineering, and pharmaceuticals. Such emulsions are complex multi-component systems and the molecular mechanisms which lead to a stable emulsion are yet to be fully understood. In this work, attenuated total reflection (ATR) infrared (IR) spectroscopy is applied to a series of w/o emulsions of an aqueous anthocyanin-rich bilberry extract dispersed in a medium chain triglyceride (MCT) oil phase. The content of the emulsifier polyglycerin-polyricinoleat (PGPR) has been varied systematically in order to investigate whether or not its concentration has an impact on the molecular stabilization mechanisms. The molecular stabilization is accessed by a careful analysis of the IR spectrum, where changes in the vibrational frequencies and signal strengths indicate alterations of the molecular environment at the water/oil interface. The results suggest that adding emulsifier in excess of 1% by weight does not lead to an enhanced stabilization of the emulsion. Keywords: MCT oil; hydrogen bonding; interfacial layer; vibrational spectroscopy; functional food; anthocyanin biosensors biosensors biosensors biosensors 1. Introduction p p p A few years ago, our group extended the ATR-FTIR approach to the analysis of water in the interfacial layer of water-in-oil (w/o) emulsions [10]. Making use of the difference in absorption between the oil and the water phase in a selected range of the spectrum allowed the nature of the interfacial molecular interactions to be analyzed. The OH vibrational stretching modes were utilized as sensors for the molecular environment. It was found that the emulsifier weakens the hydrogen bonding network in the interfacial water layer leading to a stabilized water droplet and a decreased tendency of water droplets to coalesce. In the present work, we apply the above ATR-FTIR method to study the effect of varying the emulsifier content on the molecular stabilization mechanism. The samples under investigation are of interest in the area of food technology and food chemistry: an emulsion where an aqueous anthocyanin solution in terms of a bilberry extract is dispersed in a medium chain triglyceride (MCT) oil stabilized with polyglycerine-polyricinoleat (PGPR) as emulsifier. Anthocyanin compounds are among the most important hydrophilic plant pigments [11,12]. They are considered as food additives owing to their beneficial anti-oxidant, anti-carcinogenic as well as immune modulating effects [12–15]. Unfortunately, anthocyanins exhibit relatively low chemical stability. Environmental stresses such as pH-values above 4, thermal stress, and the presence of oxygen or certain enzymes accelerate their degradation [16,17]. This poses a challenge in the processing of anthocyanin-containing foods. One possibility to overcome this problem and to protect the bioactive ingredients against environmental stresses is the formulation of multiple emulsions, e.g., a water-in-oil-in-water (w/o/w) system. In such a multiphase system, the sensitive molecules are incorporated into aqueous droplets with a stable chemical composition and pH value for an optimal anthocyanin stability [18,19]. The water droplets are then dispersed in a continuous oil phase. In a further step, this w/o emulsion is dispersed in another aqueous phase. It is needless to say that the formulation of a stable w/o/w emulsion requires both experience and a good understanding of the interfacial phenomena. Despite the effort in the production, such multiple emulsions can offer benefits beyond the mere protection of bioactive ingredients. 1. Introduction Emulsions are multiphase systems comprising of two or more liquids that are immiscible with each other. Typically, the dispersed phase in the form of small droplets is evenly distributed in the continuous phase. To stabilize the emulsion and to avoid macroscopic phase separation, emulsifiers in terms of surface-active substances (surfactants) are admixed. These surfactants are normally soluble, predominantly in one phase, but they exhibit functional groups that can interact with the other phase at the interfacial boundary. On the one hand, the emulsifier represents an additive, which ensures emulsion stability. On the other hand, its concentration should be limited to a minimum in order to save costs and to avoid undesirable effects. For example, in the area of food technology, the emulsifier may alter the taste when its concentration reaches a certain threshold. Hence, in order to optimize emulsion systems there is a need to understand the mechanisms at the interface between the dispersed and the continuous phase. This is the key to develop strategies for selecting the best emulsifier for a given system and minimize the required concentration. An important step towards understanding the molecular mechanisms is the development of suitable analytical techniques offering high sensitivity for the interactions at the interface. Vibrational Biosensors 2016, 6, 13; doi:10.3390/bios6020013 www.mdpi.com/journal/biosensors www.mdpi.com/journal/biosensors 2 of 11 Biosensors 2016, 6, 13 spectroscopic methods have proven their capability for studying intra- and intermolecular interactions in pure substances and mixtures [1–3]. They have also been successfully applied to emulsions. Jorgensen et al. [4] used transmission-mode FTIR spectroscopy to study structural changes of proteins in w/o emulsions. The same method was employed by Zhou et al. [5] and Valero et al. [6] to study water structures in AOT/alkane/water micro-emulsions of different composition. An attempt to distinguish between interfacial and bulk water IR signals in AOT reverse micelles was made by Sechler et al. [7]. They carried out a detailed analysis of the spectra recorded in emulsions with a systematically varied ratio between interfacial and bulk water by varying the droplet diameters. Kemsley et al. [8] used attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to quantitatively analyze the fat content of cream samples. Nickolov et al. [9] applied the same technique to investigate water structures in a nanoparticle synthesis which was realized in emulsified droplets acting as micro-reactors. They focused on structural changes inside the droplets during the precipitation process. 2.2. ATR Spectroscopy The measurements were carried out on a Bruker Vertex v70 FTIR spectrometer equipped with a diamond ATR accessory. The number of reflections at the surface is 1, and the reflection angle is 45˝. Spectra were recorded taking 16 scans with a nominal resolution of 1 cm´1. All spectra were corrected for water vapor and carbon dioxide interference. In order to check the reproducibility of the measurement, three spectra of each sample were recorded and subtracted from each other pairwise. This subtraction resulted in statistical noise across the entire spectrum. From this, we concluded that statistical errors in the data can be considered to be negligible. To carry out a measurement, a droplet of the emulsion was placed on the diamond crystal and covered with a cap. The basics of studying w/o emulsions with ATR-IR spectroscopy were described in detail in [10] and Figure 1 shows the situation near the crystal surface. The surface of the ATR crystal is predominantly wetted by the oil phase as no significant contribution from bulk water was observed in the emulsion spectra. On the left hand side of the drawing in Figure 1, the schematic intensity distribution of the evanescent field is plotted as a function of distance from the surface. It represents a single-exponential decay in the absence of absorbing molecules. When a wavelength is considered at which significant absorption in the oil phase takes place, the penetration depth is reduced starting at the surface. On the other hand, when the wavelength of interest is absorbed in the water phase only, the initial intensity decay at the surface remains the same as in the figure, but at the w/o interface the decay constant changes abruptly and the decay is enhanced inside the droplet. As a simplified picture, this can be considered as the product of two exponential functions, one taking into account the medium without absorption and one taking into account the absorption effects via the Beer-Lambert law. In our previous work [10] it was shown that the resulting thickness of the interfacial water layer that is probed is only a few hundred nanometers. This means that the signals are dominated by the interfacial water rather than from bulk water. Moreover, the probed thickness in terms of its 1/e value is independent of the distance of a droplet from the surface. 2.1. Sample Preparation Anthocyanin-enriched w/o emulsions were prepared as follows. Aqueous phase: For the preparation of the aqueous anthocyanin solutions, 0.05% (w/w) anthocyanin-rich bilberry extract (Kaden Biochemical GmbH, Hamburg, Germany) was dissolved in distilled water under constant stirring for 30 min via a magnetic stirrer at room temperature. Insoluble solid extract particles were separated by filtration (Sartorius Stedim biotech Germany, Filter Discs, Grade 388). Oil phase: The emulsifier-containing oil-phase was prepared by dissolving 1, 5, or 10 wt % polyglycerin-polyricinoleat (PGPR 4150, Palsgaard®, Juelsminde, Denmark) in MCT-oil (Medium Chain Triglyceride, Schuman und Sohn GmbH, Germany). The samples were stirred for 1 h via a propeller mixer at 40 ˝C. Emulsion: The w/o emulsions were then prepared via a high pressure homogenizer (MF 110 EH-30, Microfluidics®, Westwood, MA, USA) operated at 1000 bar pressure difference, where the water phase (30 wt %) was emulsified in the surrounding oil phase (70 wt %). The diameter of the water droplets in the emulsion was about 2 µm (Sauter mean diameter). 1. Introduction For example, during the digestive process, the multiple emulsion structure of several cladding layers composed of a triglyceride phase and several emulsifiers and further stabilizing ingredients can enable a triggered release of the encapsulated molecules at different locations in the gastrointestinal tract. Previous work has shown that oil droplets can sustain stomach conditions [20,21]. This makes them a perfect shell for anthocyanin protection in the gastric passage. It was also shown that the protective effect of the capsule structure and a possible targeted release of the encapsulated matter at the effective location strongly depends on the emulsion microstructure [22]. The present study focuses on the initial w/o emulsion system, as the interface between the aqueous anthocyanin solution and the oil is crucial for the behavior from a functional food point of view. The aim of this work is to investigate the effect of the emulsifier concentration on the molecular stabilization mechanism. For this purpose, w/o emulsions are studied and the emulsifier concentration in the oil phase is varied between 1% and 10% by weight, which represents the practically relevant 3 of 11 Biosensors 2016, 6, 13 range. The samples are analyzed using ATR-FTIR spectroscopy, for which we have shown previously that the main signals originate from only a thin water layer (below 0.5 µm) at the w/o interface [10]. In addition, we present a vibrational spectroscopic analysis of the individual components of the emulsion system. 3.1.1. Bilberry Extract Solution 3.1.1. Bilberry Extract Solution 3.1.1. Bilberry Extract Solution The IR spectrum of the aqueous solution of the bilberry extract is shown in Figure 2. Due to the very small content of bilberry extract, the spectrum is virtually identical to the spectrum of pure water. It is dominated by broad bands, where the most prominent features are the OH bending mode at 1636 cm−1 and the broad OH stretching band ranging from 2800 to 3700 cm−1. Our focus will be on the stretching band, the shape of which is an indicator of the hydrogen-bonding environment. In other words, we utilize water as a molecular sensor for studying the effects of the more complicated oil and emulsifier molecules. The IR spectrum of the aqueous solution of the bilberry extract is shown in Figure 2. Due to the very small content of bilberry extract, the spectrum is virtually identical to the spectrum of pure water. It is dominated by broad bands, where the most prominent features are the OH bending mode at 1636 cm´1 and the broad OH stretching band ranging from 2800 to 3700 cm´1. Our focus will be on the stretching band, the shape of which is an indicator of the hydrogen-bonding environment. In other words, we utilize water as a molecular sensor for studying the effects of the more complicated oil and emulsifier molecules. The IR spectrum of the aqueous solution of the bilberry extract is shown in Figure 2. Due to the very small content of bilberry extract, the spectrum is virtually identical to the spectrum of pure water. It is dominated by broad bands, where the most prominent features are the OH bending mode at 1636 cm−1 and the broad OH stretching band ranging from 2800 to 3700 cm−1. Our focus will be on the stretching band, the shape of which is an indicator of the hydrogen-bonding environment. In other words, we utilize water as a molecular sensor for studying the effects of the more complicated oil and emulsifier molecules. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Fo a detailed a aly i the OH ba d e o ded i the bilbe y e t a t a d the e ul io a Figure 2. IR spectrum of aqueous bilberry extract solution. 3. Results and Discussion h ll 3. Results and Discussion 3. Results and Discussion This section will present the results and their interpretation. We start with the vibrational analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational analysis of the individual components of the emulsions and then proceed to the emulsion samples. 3.1. Vibrational Analysis of the Individual Components 3.1. Vibrational Analysis of the Individual Components 3.1. Vibrational Analysis of the Individual Components 3.1. Vibrational Analysis of the Individual Components 3.1. Vibrational Analysis of the Individual Components 3.1. Vibrational Analysis of the Individual Components 2.2. ATR Spectroscopy The intensity distribution of the evanescent field is illustrated as a function of distance from the surface. 2.2. ATR Spectroscopy Therefore, heterogeneities in the emulsion (e.g., in terms of droplet concentration) will not affect the measurement. However, it must be kept in mind that this is only true for the spectral regions where water and not the oil or the emulsifier absorb. Consequently, we focus on the OH stretching region in our analysis. 4 of 11 4 f 11 Biosensors 2016, 6, 13 osensors 2016, 6, 13 4 of osensors 2016, 6, 13 4 of Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is illustrated as a function of distance from the surface. osensors 2016, 6, 13 4 o Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where the crystal is in contact with the emulsion sample. 3.1.1. Bilberry Extract Solution 3.1.1. Bilberry Extract Solution 3.1.1. Bilberry Extract Solution The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. F d t il d l i th OH b d d d i th bilb t t d th l i Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. 5 of 11 Biosensors 2016, 6, 13 Biosensors 2016, 6, 13 For a detailed analysis, the OH bands recorded in the bilberry extract and the emulsions are de-convolved. The de-convolved spectrum will be presented in Section 3.2, but the results for the aqueous bilberry extract are discussed in the following. The OH stretching band was fitted by a sum of six individual Gaussian profiles using a least squares fit algorithm. The fitting parameters were the line position, the line width, and the line intensity. The number of profiles was fixed to allow an interpretation in line with Schmidt and Miki [23] and our previous work [10,24,25]. The results of the fitting procedure are summarized in Table 1. The center wavenumbers slightly differ from our earlier emulsion study [10], but it must be noted that the present spectra were recorded on a different instrument. The lines at 3625 and 2950 cm´1 together exhibit only about 5% of the total OH band intensity and are therefore not considered in the following discussion. The low wavenumber component at 3117 cm´1 can be assigned to tetra-coordinated, fully hydrogen-bonded water. The higher wavenumber components indicate water, which is not fully coordinated and hence exhibits stronger covalent bonds vibrating at higher frequency [3]. The 3230 cm´1 mode can be assigned to symmetrically hydrogen-bonded water, the 3375 cm´1 mode to asymmetrically hydrogen-bonded water, and the 3522 cm´1 mode to weakly hydrogen-bonded water. Table 1. Fitting parameters of the aqueous bilberry extract solution spectrum. 3.1.1. Bilberry Extract Solution 3.1.1. Bilberry Extract Solution 3.1.1. Bilberry Extract Solution Center frequency, peak width, and the percentage intensity with respect to the overall band by peak area are given for the six individual Gaussian profiles. Table 1. Fitting parameters of the aqueous bilberry extract solution spectrum. Center frequency, peak width, and the percentage intensity with respect to the overall band by peak area are given for the six individual Gaussian profiles. Profile Center Wavenumber in cm´1 Width in cm´1 Intensity in % peak 1 3625 94 2.24 peak 2 3522 155 12.48 peak 3 3375 188 39.27 peak 4 3230 172 29.07 peak 5 3117 200 14.14 peak 6 2950 200 2.80 1 2 Emulsifier 3.1.2. Emulsifier 3.1.2. Emulsifier The chemical structure of polyglycerin-polyricinoleat (PGPR) is depicted in Figure 3a. It emphasizes the backbone of polyglycerin and the substituents, R, represent H atoms, or a ricinoleic acid moiety or a polyricinoleic acid moiety. The structure of ricinoleic acid is shown in Figure 3b. It is an unsaturated omega-9 fatty acid and it is the only naturally occurring fatty acid exhibiting a hydroxyl group. This hydroxyl group allows the formation of polyricinoleic acid macromolecules via esterification. Figure 3c shows the 3D sketch of diricinoleic acid as an example. The emulsifier PGPR is strongly lipophilic and virtually not soluble in water at all. However, as it possesses several ether groups and a few OH groups with permanent dipoles it can interact with the water phase and stabilize the droplets by weakening the water-water hydrogen bonds and hence lowering the propensity to coalesce with other droplets. Figure 4 displays the IR spectrum of PGPR. The most predominant feature is the C=O stretching mode at 1732 cm´1. Further distinct peaks can be found in the CH stretching region of the spectrum around 3000 cm´1. Strong peaks are located 2854, 2924, and 2954 (shoulder) cm´1, as well as a weaker peak at 3011 cm´1. They can be assigned to symmetric and anti-symmetric stretching of CH2 and CH3 groups. The OH stretching appears as a broad band between 3100 and 3600 cm´1. At the low wavenumber wing, the OH stretching band overlaps with the CH stretching peaks, and hence care must be taken in the interpretation of this part of the spectrum. In the fingerprint region, a multitude of mainly CC stretching and CH bending modes is present resulting in a very dense structure of overlapping lines. A detailed analysis of this part of the spectrum would require the aid of computational methods and is beyond the scope of the present study. 6 of 11 Biosensors 2016, 6, 13 Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. 3.1.2. Emulsifier Biosensors 2016, 6, 13 6 of 11 Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; Biosensors 2016, 6, 13 6 of 11 Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. 3.1.3. MCT Oil and Oil Phase The oil used is a medium chain triglyceride i e a triglyceride typically with aliphatic C6 to C12 ( ) ( ) Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. rum of the PGPR emulsifier. The small peak around 2300 cm−1 mond crystal involved in the ATR measurement. 3.1.3. 3.1.2. Emulsifier MCT Oil and Oil Phase The oil used is a medium chain triglyceride i e a triglyceride typically with aliphatic C6 to C12 Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. fatty acid chains. Figure 5 sh 3.1.3. MCT Oil and Oil Phase The oil used is a mediu 3.1.3. MCT Oil and Oil Phase The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH bending modes is present resulting in a very dense structure of overlapping lines. Figure 5 IR spectrum of the medium chain triglyceride (MCT) oil The small peak around 2300 cm−1 Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. i I f h d h l d l h ll k d , p y y g shows the IR spectra of the mixtures that were used as continuous phase in the emulsions. They contained 1%, 5%, or 10%wt PGPR. The spectra are very similar as a consequence. The pure oil spectrum is shown as a reference. Distinct changes with the varied emulsifier concentration can be seen at a closer look only. For this purpose, a zoom in of the oil C=O peak around 1744 cm−1 is shown in Figure 6 as well. It becomes clear that the peak absorbance is reduced systematically with increasing emulsifier content. Moreover, aside from the change in the peak strength, the zoomed spectrum indicates a small shift as the oil peak at 1744 cm−1 decreases and, at the same time, the PGPR peak at 1732 cm−1 increases. This results in the appearance of an isosbestic point at 1735 cm−1, see inset in Figure 6. As aforesaid, the PGPR and oil spectra are very similar due to the structural similarity. Figure 6 shows the IR spectra of the mixtures that were used as continuous phase in the emulsions. They contained 1%, 5%, or 10%wt PGPR. The spectra are very similar as a consequence. The pure oil spectrum is shown as a reference. Distinct changes with the varied emulsifier concentration can be seen at a closer look only. For this purpose, a zoom in of the oil C=O peak around 1744 cm´1 is shown in Figure 6 as well. It becomes clear that the peak absorbance is reduced systematically with increasing emulsifier content. fatty acid chains. Figure 5 sh 3.1.3. MCT Oil and Oil Phase The oil used is a mediu 3.1.3. MCT Oil and Oil Phase Moreover, aside from the change in the peak strength, the zoomed spectrum indicates a small shift as the oil peak at 1744 cm´1 decreases and, at the same time, the PGPR peak at 1732 cm´1 increases. This results in the appearance of an isosbestic point at 1735 cm´1, see inset in Figure 6. is an experimental artifact of the diamond crystal involved in the ATR measurement. As aforesaid, the PGPR and oil spectra are very similar due to the structural similarity. Figure 6 shows the IR spectra of the mixtures that were used as continuous phase in the emulsions. They contained 1%, 5%, or 10%wt PGPR. The spectra are very similar as a consequence. The pure oil spectrum is shown as a reference. Distinct changes with the varied emulsifier concentration can be seen at a closer look only. For this purpose, a zoom in of the oil C=O peak around 1744 cm−1 is shown in Figure 6 as well. It becomes clear that the peak absorbance is reduced systematically with increasing emulsifier content. Moreover, aside from the change in the peak strength, the zoomed spectrum indicates a small shift as the oil peak at 1744 cm−1 decreases and, at the same time, the PGPR peak at 1732 cm−1 increases. This results in the appearance of an isosbestic point at 1735 cm−1, see inset in Figure 6. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. fatty acid chains. Figure 5 sh 3.1.3. MCT Oil and Oil Phase The oil used is a mediu 3.1.3. MCT Oil and Oil Phase Further distinct peaks can be found in the CH stretching region at 2854, 2923, and 2956 (shoulder) cm−1 as well as a weaker peak at 3007 cm−1 Their positions are almost identical C=O stretching mode, but it is located at 1744 cm´1. In other words, it is blue-shifted by 12 cm´1 with respect to the band at 1732 cm´1 in PGPR. This indicates that the C=O bond in the oils is stronger than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923, and 2956 (shoulder) cm´1, as well as a weaker peak at 3007 cm´1. Their positions are almost identical compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH bending modes is present resulting in a very dense structure of overlapping lines. to the emulsifier spectrum discussed in preceding section. The most predominant feature is again the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1 with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923, and 2956 (shoulder) cm−1, as well as a weaker peak at 3007 cm−1. Their positions are almost identical compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH bending modes is present resulting in a very dense structure of overlapping lines. Biosensors 2016, 6, 13 7 of 11 to the emulsifier spectrum discussed in preceding section. The most predominant feature is again the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1 with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923, and 2956 (shoulder) cm−1 as well as a weaker peak at 3007 cm−1 Their positions are almost identical Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. fatty acid chains. Figure 5 sh 3.1.3. MCT Oil and Oil Phase The oil used is a mediu 3.1.3. MCT Oil and Oil Phase g y , , g y yp y p fatty acid chains. Figure 5 shows the corresponding IR spectrum. The overall appearance is similar The oil used is a medium chain triglyceride, i.e., a triglyceride typically with aliphatic C6 to C12 fatty acid chains. Figure 5 shows the corresponding IR spectrum. The overall appearance is similar to the emulsifier spectrum discussed in preceding section. The most predominant feature is again the 7 of 11 11 Biosensors 2016, 6, 13 Biosensors 2016, 6, 13 C=O stretching mode, but it is located at 1744 cm´1. In other words, it is blue-shifted by 12 cm´1 with respect to the band at 1732 cm´1 in PGPR. This indicates that the C=O bond in the oils is stronger than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923, and 2956 (shoulder) cm´1, as well as a weaker peak at 3007 cm´1. Their positions are almost identical compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH bending modes is present resulting in a very dense structure of overlapping lines. to the emulsifier spectrum discussed in preceding section. The most predominant feature is again the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1 with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923, and 2956 (shoulder) cm−1, as well as a weaker peak at 3007 cm−1. Their positions are almost identical compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH bending modes is present resulting in a very dense structure of overlapping lines. Biosensors 2016, 6, 13 7 of 11 to the emulsifier spectrum discussed in preceding section. The most predominant feature is again the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1 with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger than in the emulsifier. 3.2. Spectroscopy of Emulsions 3.2. Spectroscopy of Emulsions The de-convolved OH bands of the aqueous bilberry extract solution and the three emulsions with varied emulsifier content are displayed in Figure 7. As described in Section 3.1.1, six individual Gaussian profiles have been used to fit the band. In each panel of Figure 7, the thick red line shows the experimental data, the thin brown line represents the sum of the Gaussian profiles, which are displayed in various colors individually. In order to get an overview the Tables 2–4 compare the center frequencies, the peak widths, and the percentage contribution of the individual Gaussian profiles. The de-convolved OH bands of the aqueous bilberry extract solution and the three emulsions with varied emulsifier content are displayed in Figure 7. As described in Section 3.1.1, six individual Gaussian profiles have been used to fit the band. In each panel of Figure 7, the thick red line shows the experimental data, the thin brown line represents the sum of the Gaussian profiles, which are displayed in various colors individually. In order to get an overview the Tables 2–4 compare the center frequencies, the peak widths, and the percentage contribution of the individual Gaussian profiles. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR; (d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR; (d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR; (d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR; (d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR; (d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. fatty acid chains. Figure 5 sh 3.1.3. MCT Oil and Oil Phase The oil used is a mediu 3.1.3. MCT Oil and Oil Phase The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm´1. a of the oil phases containing different amounts of emulsifier. % PGPR. The zoomed in region shows the C=O stretching pe i i t l tif t f th di d t l i around 2300 cm is an experimental artifact of the diamond crystal involved in the ATR measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak around 2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm´1. 8 of 11 8 of 11 Biosensors 2016, 6, 13 Bi 2016 6 13 3.2. Spectroscopy of Emulsions 3.2. Spectroscopy of Emulsions (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR; (d) Emulsion with 10% PGPR. Table 2. Center frequencies in cm−1 of the six individual Gaussian profiles obtained from fitting the infrared OH bands of the aqueous solution and the three emulsions. Table 2. Center frequencies in cm´1 of the six individual Gaussian profiles obtained from fitting the infrared OH bands of the aqueous solution and the three emulsions. Table 2. Center frequencies in cm−1 of the six individual Gaussian profiles obtained from fitting the infrared OH bands of the aqueous solution and the three emulsions. Table 2. Center frequencies in cm´1 of the six individual Gaussian profiles obtained from fitting the infrared OH bands of the aqueous solution and the three emulsions. Profile Aqueous Solution Emulsion with 1% PGPR Emulsion with 5% PGPR Emulsion with 10% PGPR peak 1 3625 3625 3625 3624 peak 2 3522 3544 3544 3542 peak 3 3375 3416 3416 3414 peak 4 3230 3291 3290 3290 peak 5 3117 3198 3198 3198 peak 6 2950 3025 3025 3025 Profile Aqueous Solution Emulsion with 1% PGPR Emulsion with 5% PGPR Emulsion with 10% PGPR peak 1 3625 3625 3625 3624 peak 2 3522 3544 3544 3542 peak 3 3375 3416 3416 3414 peak 4 3230 3291 3290 3290 peak 5 3117 3198 3198 3198 peak 6 2950 3025 3025 3025 9 of 11 Biosensors 2016, 6, 13 Table 3. Peak widths in cm´1 of the six individual Gaussian profiles obtained from fitting the infrared OH bands of the aqueous solution and the three emulsions. Profile Aqueous Solution Emulsion with 1% PGPR Emulsion with 5% PGPR Emulsion with 10% PGPR peak 1 94 94 94 94 peak 2 155 155 155 155 peak 3 188 188 188 188 peak 4 172 172 172 172 peak 5 200 195 195 195 peak 6 200 160 160 160 Table 4. Percentage intensity with respect to the overall band by peak area of the six individual Gaussian profiles obtained from fitting the infrared OH bands of the aqueous solution and the three emulsions. 3.2. Spectroscopy of Emulsions 3.2. Spectroscopy of Emulsions Profile Aqueous Solution Emulsion with 1% PGPR Emulsion with 5% PGPR Emulsion with 10% PGPR peak 1 2.24 2.68 2.64 2.61 peak 2 12.48 13.23 12.61 12.76 peak 3 39.27 41.34 42.61 41.90 peak 4 29.07 18.06 17.80 17.41 peak 5 14.14 20.14 19.85 20.00 peak 6 2.80 4.55 4.49 5.32 In the emulsions, the OH band and its contributions represented by the Gaussian profiles change significantly, which is in agreement with our previous study [10]. Aside from the peak at 3625 cm´1, which exhibits very low intensity in all cases, the peaks are blue-shifted in the emulsions with respect to the aqueous solution, see Table 2. This means that the corresponding covalent OH bonds are strengthened, which in turn indicates that the entire hydrogen-bonding network in the probed water layer at the interface is weakened by the interaction with emulsifier molecules. The peak widths in Table 3 reveal no or negligible differences between the aqueous solution and the emulsions. The same is true in terms of the percentage contribution to the overall band for most of the peaks, see Table 4. Only Peaks 4 and 5, corresponding to the profiles centered at 3230 and 3117 cm´1 respectively in the aqueous solution show a significant alteration. The peak at 3230 cm´1, which was assigned to symmetrically hydrogen-bonded water, is reduced in the emulsion by approximately one third with respect the aqueous system. On the other hand, the 3117 cm´1 peak, which was assigned to tetra-coordinated water, gains about 50% at the same time. This indicates that the fraction of fully hydrogen-bonded water molecules increases in the emulsion. However, it must be kept in mind that the frequency of the peak is blue-shifted with respect to the aqueous solution case. Hence, the data indicate that the network of these fully hydrogen-bonded molecules is significantly weaker than in bulk water. An interesting finding arises from the data in the Tables 2–4 as the changes of the spectroscopic parameters with the variation of the emulsifier concentration can be considered negligible. This indicates that the molecular stabilization at the w/o interface is rather independent of the emulsifier concentration. In other words, adding emulsifier in excess of 1% seems to not lead to an improved emulsion stability. 3.2. Spectroscopy of Emulsions 3.2. Spectroscopy of Emulsions From an engineering and an economic point of few, this finding is rather important as it indicates that a low amount of PGPR is sufficient to stabilize the w/o emulsion. References 1. Kiefer, J.; Noack, K.; Bartelmess, J.; Walter, C.; Dörnenburg, H.; Leipertz, A. Vibrational structure of the polyunsaturated fatty acids eicosapentaenoic acid and arachidonic acid studied by infrared spectroscopy. J. Mol. Struct. 2010, 965, 121–124. [CrossRef] 1. Kiefer, J.; Noack, K.; Bartelmess, J.; Walter, C.; Dörnenburg, H.; Leipertz, A. Vibrational structure of the polyunsaturated fatty acids eicosapentaenoic acid and arachidonic acid studied by infrared spectroscopy. J. Mol. Struct. 2010, 965, 121–124. [CrossRef] 2. Noack, K.; Leipertz, A.; Kiefer, J. Molecular interactions and macroscopic effects in binary mixtures of an imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45–53. [CrossRef] 3. Joseph, J.; Jemmis, E.D. Red-, blue-, or no-shift in hydrogen bonds: A unified explanation. J. Am. Chem. Soc. 2007, 129, 4620–4632. [CrossRef] [PubMed] 2. Noack, K.; Leipertz, A.; Kiefer, J. Molecular interactions and macroscopic effects in binary mixtures of an imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45–53. [CrossRef] 2. Noack, K.; Leipertz, A.; Kiefer, J. Molecular interactions and macroscopic effects in binary mixtures of an imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45–53. [CrossRef] 3. Joseph, J.; Jemmis, E.D. Red-, blue-, or no-shift in hydrogen bonds: A unified explanation. J. Am. Chem. Soc. 2007 129 4620 4632 [C R f] [P bM d] imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45 53. [CrossRef] 3. Joseph, J.; Jemmis, E.D. Red-, blue-, or no-shift in hydrogen bonds: A unified explanation. J. Am. Chem. Soc. 2007, 129, 4620–4632. [CrossRef] [PubMed] . Jorgensen, L.; Van de Weert, M.; Vermehren, C.; Bjerregaard, S.; Frokjaer, S. Probing structural change proteins incorprated into water-in-oil emulsions. J. Pharm. Sci. 2004, 93, 1847–1859. [CrossRef] [PubMe . Zhou, G.W.; Li, G.Z.; Chen, W.J. Fourier-transform infrared investigation on water states and conformations of Aerosol-OT in reverse microemulsions. Langmuir 2002, 18, 4566–4571. [CrossRef] conformations of Aerosol OT in reverse microemulsions. Langmuir 2002, 18, 4566 4571. [CrossRef] 6. Valero, M.; Sanchez, F.; Gomez-Herrera, C.; Lopez-Cornejo, P. Study of water solubilized in AOT/n-decane/water microemulsions. Chem. Phys. 2008, 345, 65–72. [CrossRef] 7. Sechler, T.D.; DelSole, E.M.; Deak, J.C. Measuring properties of interfacial and bulk water regions in a reverse micelle with IR spectroscopy: A volumetric analysis of the inhomogeneously broadened OH band. J. Colloid Interface Sci. 2010, 346, 391–397. [CrossRef] [PubMed] 8. Kemsley, E.K.; Appleton, G.P.; Wilson, R.H. Abbreviations The following abbreviations are used in this manuscript: The following abbreviations are used in this manuscript: PGPR: PolyGlycerine-PolyRicinoleat MCT: Medium Chain Triglyceride IR: InfraRed FTIR: Fourier-Transform InfraRed ATR: Attenuated Total Reflection PGPR: PolyGlycerine-PolyRicinoleat MCT: Medium Chain Triglyceride IR: InfraRed FTIR: Fourier-Transform InfraRed ATR: Attenuated Total Reflection 4. Conclusions In the present work, the vibrational spectra of an aqueous bilberry extract solution, a polyglycerine-polyricinoleat emulsifier and a medium chain triglyceride oil were studied by IR spectroscopy. Based on the vibrational analysis, the IR spectra of a series of water-in-oil emulsions 10 of 11 Biosensors 2016, 6, 13 comprising of the above components were studied. The emulsifier concentration was varied systematically. The OH stretching vibration band was utilized as a sensor for the molecular interactions at the water-oil interface leading to a stabilization of the emulsion. A detailed analysis of the OH band revealed that changing the emulsifier content has negligible effects on the stabilization of the emulsion. This is at least true for the concentration range studied, i.e., 1%–10%wt. However, in all emulsion spectra, the individual contributions to the OH stretching band are significantly blue-shifted. In other words, the hydrogen-bonding network in the water phase is weakened as a result of the presence of the emulsifier. Overall, we can conclude that ATR-IR spectroscopy is a useful tool for studying w/o emulsions. In particular, the OH stretching region of water in the spectrum represents a highly sensitive means of sensing changes in the molecular environment. Acknowledgments: This research project was supported by the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn), the AiF and the Ministry of Economics and Technology, AiF-Project No. 15612 N. Furthermore, we would like to thank Kaden Biochemical GmbH, Germany, for supplying us with bilberry extract, Palsgaard®Denmark for supplying us with PGPR 4150. Author Contributions: Johannes Kiefer and Heike P. Schuchmann conceived and designed the experiments; Kerstin Frank and Florian M. Zehentbauer performed the experiments; Johannes Kiefer, Florian M. Zehentbauer, and Kerstin Frank analyzed the data; all authors contributed to the interpretation of the data; all authors contributed to the preparation of the manuscript with Johannes Kiefer putting together the first draft. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Quantitative analysis of emulsions using attenuated total reflectance (ATR). Spectrochim. Acta 1994, 50A, 1235–1242. [CrossRef] 9. Nickolov, Z.S.; Paruchuri, V.; Shah, D.O.; Miller, J.D. FTIR-ATR studies of water structure in reverse micelles during the synthesis of oxalate precursor nanoparticles. Colloids Surf. A 2004, 232, 93–99. [CrossRef] 9. Nickolov, Z.S.; Paruchuri, V.; Shah, D.O.; Miller, J.D. FTIR-ATR studies of water structure in reverse micelles during the synthesis of oxalate precursor nanoparticles. Colloids Surf. A 2004, 232, 93–99. [CrossRef] 11 of 11 Biosensors 2016, 6, 13 10. Kiefer, J.; Frank, K.; Schuchmann, H.P. Attenuated total reflection infrared (ATR-IR) spectroscopy of a water-in-oil emulsion. Appl. Spectrosc. 2011, 65, 1024–1028. [CrossRef] [PubMed] 11. Newsome, A.G.; Culver, C.A.; van Breemen, R.B. Nature’s Palette: The Search for Natural Blue Colorants. J. Agric. Food Chem. 2014, 62, 6498–6511. [CrossRef] [PubMed] 12. Tsuda, T. Anthocyanins as Functional Food Factors—Chemistry, Nutrition and Health Promotion. Food Sci. Technol. Res. 2012, 18, 315–324. [CrossRef] 13. Prior, R.L.; Cao, G.H.; Martin, A.; Sofic, E.; McEwen, J.; O'Brien, C.; Lischner, N.; Ehlenfeldt, M.; Kalt, W.; Krewer, G.; et al. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. J. Agric. Food Chem. 1998, 46, 2686–2693. [CrossRef] 14. Cooke, D.; Schwarz, M.; Boocock, D.; Winterhalter, P.; Steward, W.P.; Gescher, A.J.; Marczylo, T.H. Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on ademona development in the Apc(Min) mouse model of intestinal carcinogenesis—Relationship with tissue anthocyanin levels. Int. J. Cancer 2006, 119, 2213–2220. [CrossRef] [PubMed] 15. Kong, J.M.; Chia, L.S.; Goh, N.K.; Chia, T.F.; Brouillard, R. Analysis and biological activities of anthocyanins. Phytochemistry 2003, 64, 923–933. [CrossRef] 16. Nielsen, I.L.F.; Haren, G.R.; Magnussen, E.L.; Dragsted, L.O.; Rasmussen, S.E. Quantification of anthocyanins in commercial black currant juices by simple high-performance liquid chromatography. Investigation of their pH stability and antioxidative potency. J. Agric. Food Chem. 2003, 51, 5861–5866. [CrossRef] [PubMed] 17. Hubbermann, E.M. Functional properties of anthocyanin concentrates and the influence of physicochemical parameters and food additives on the color and stability of isolated anthocyanins in food. PhD Dissertation, Christian-Albrecht-Universität zu Kiel, Kiel, Germany, 2005. 18. Frank, K.; Köhler, K.; Schuchmann, H.P. Formulation of labile hydrophilic ingredients in multiple emulsions: Influence of the formulation's composition on the emulsion’s stability and on the stability of entrapped bioactives. J. Dispers. Sci. Technol. 2011, 32, 1–6. [CrossRef] 19. References Baum, M.; Schantz, M.; Leick, S.; Berg, S.; Betz, M.; Frank, K.; Rehage, H.; Schwarz, K.; Kulozik, U.; Schuchmann, H.P.; et al. Is the antioxidative effectiveness of a bilberry extract influenced by encapsulation? J. Sci. Food Agric. 2014, 94, 2301–2307. [CrossRef] [PubMed] 20. Ax, K. Emulsionen und Liposomen als Trägersysteme für Carotinoide. PhD Dissertation, Uni Karlsruhe, Karlsruhe, Germany, 2004. 21. Ribeiro, H.S.; Schuchmann, H.P.; Engel, R.; Briviba, K.; Walz, E. Encapsulation of Caroteniods and Vitamins. In Encapsulation Technologies for Food Active Ingredients and Food Processing; Zuidam, N.J., Nedovic, V.A., Eds.; Springer: Heidelberg, Germany, 2009. 22. Palzer, S. Food structures for nutrition, health and wellness. Trends Food Sci. Technol. 2009, 20, 194–200. [CrossRef] 23. Schmidt, D.A.; Miki, K. Structural correlations in liquid water: A new interpretation of IR spectroscopy. J. Phys. Chem. A 2007, 111, 10119–10122. [CrossRef] [PubMed] 24. Wallace, V.M.; Dhumal, N.R.; Zehentbauer, F.M.; Kim, H.J.; Kiefer, J. Revisiting the Aqueous Solutions of Dimethyl Sulfoxide by Spectroscopy in the Mid- and Near-Infrared: Experiments and Car-Parrinello Simulations. J. Phys. Chem. B 2015, 119, 14780–14789. [CrossRef] [PubMed] J y [ ] [ ] 25. Kiefer, J.; Grabow, J.; Kurland, H.-D.; Müller, F.A. Characterization of Nanoparticles by Solvent Infrared Spectroscopy. Anal. Chem. 2015, 87, 12313–12317. [CrossRef] [PubMed] © 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/).
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Mauro Lombardi, University of Florence, Italy, mauro.lombardi@unifi.it, 0000-0002-3234-7039 FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup_best_practice) Mauro Lombardi, Introduzione, pp. 9-13, © 2021 Author(s), CC BY 4.0 International, DOI 10.36253/978-88- 5518-310-9.02, in Mauro Lombardi, Transizione ecologica e universo fisico-cibernetico. Soggetti, strategie, lavoro, © 2021 Author(s), content CC BY 4.0 International, metadata CC0 1.0 Universal, published by Firenze University Press (www.fupress.com), ISSN 2704-5919 (online), ISBN 978-88-5518-310-9 (PDF), DOI 10.36253/978-88-5518-310-9 Introduzione Stiamo vivendo in un’epoca molto particolare della storia dell’Umanità, che ha creato un potenziale tecnico-scientifico senza pari, grazie al quale è possibi- le elaborare la rappresentazione digitale dei processi dalla nano-scala alla scala globale. Il Pianeta è iperconnesso da dispositivi che elaborano flussi informati- vi generati da entità individuali e collettive, da esseri viventi e non. Individui e collettività possono esprimere emozioni e argomentazioni in una sorta di ‘Ba- bele organizzata’ e senza limiti, che nella vulgata è il web. I confini tra settori produttivi e attività economico-sociali sono diventati permeabili e confusi, nel senso che per ogni attività e output (materiali e immateriali) è sempre più spes- so necessario esplorare molteplici domini conoscitivi, quindi essere inseriti in strutture interattive, tendenzialmente multi-scala. In uno scenario di questo tipo l’innovazione tecnico-scientifica mostra un’accelerazione, che autorevoli studiosi stimano esponenziale, quindi capace di innescare mutamenti generalizzati nei modelli mentali, nei comportamenti strategici ed operativi, nelle competenze, nei sistemi di credenze e di valori su cui si fondano le società. A tutto questo bi- sogna poi aggiungere che flussi globali di informazioni e persone cambiano le relazioni tra le culture e gli assetti geo-politici con esiti imprevedibili. gt g p p L’insieme dei fenomeni e dei processi sinteticamente indicati delineano, per- tanto, un nuovo scenario, contraddistinto da alcune peculiarità: mutamenti ge- neralizzati e profondi, crisi di interi assetti tecno-economici, emergere di nuovi oligopoli globali (chiamati moligopoli al capitolo 3, par. 3), spinte irresistibili a cambiare la visioni individuali e collettive. TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Su queste basi non deve sorprendere che Centri di Ricerca internazionali e la maggioranza degli studiosi di molte discipline mettano al centro della riflessione due aspetti cruciali: 1) i mutamenti in atto nei sotto-sistemi, che compongono il sistema globale interconnesso; 2) le molte difficoltà con cui si devono misurare i processi decisionali a qualsiasi livello e in ogni ambito. i p q g Ciò che sta accadendo, inoltre, pone l’intera Umanità di fronte a sfide epocali: 1) l’evento pandemico mette in discussione le strutture sanitarie praticamente in quasi tutti i Paesi del mondo. 2) È concreto il rischio che la contrazione eco- nomico-produttiva, conseguente al lockdown dei primi mesi del 2020 e a quello in atto all’inizio del 2021, si trasformi in un periodo recessivo del quale non è possibile stimare durata e intensità. Introduzione Da ciò consegue il diffondersi di incertezze e ansietà, individuale e collettiva, per i timori di perdere il posto di lavoro e le aspettative di una diminuzione del proprio reddito. 3) La dinamica tecno-eco- nomica è sempre più spinta verso l’aumento della potenza computazionale e l’e- laborazione di sistemi di software (Intelligenza Artificiale e Machine Learning, algoritmi di editing genomico, modelli di simulazione ingegneristica e sociale). I risultati sono non di rado imprevedibili sul piano delle attività socio-economi- che, tecnico-scientifiche e sanitarie. 4) Si impone la necessità di cambiare stili di vita, modelli di produzione e consumo, meccanismi e modalità di relazioni sociali ed economiche, circuiti di feedback che aumentano incertezza e ansietà, mentre i processi economico-produttivi divengono sempre meno comprensibili, alla luce dei numerosi fattori causali che interagiscono nel determinare dinami- che fuori controllo. 5) La numerosità di fattori e processi che interagiscono sia a livello di nano-scala che sul piano globale incrementa il grado di complessità dell’evoluzione socio-economica, a cui si aggiunge – come si illustrerà nel capi- tolo 1 – la stretta compenetrazione tra le innumerevoli componenti sistemiche del ‘Sistema-Terra’. Dalle interconnessioni globali e dalla complessità evolutiva scaturiscono sfi- de epocali per l’Umanità e l’intero Pianeta: effetti dell’evento pandemico ancora fuori controllo; sfruttamento eccessivo delle risorse naturali non riproducibili, tra cui le cosiddette ‘terre rare’, essenziali per l’evoluzione della potenza compu- tazionale e dell’Intelligenza Artificiale (d’ora in poi IA); variazioni climatiche indotte da una serie di comportamenti alla lunga distruttivi (crescita di grandi insediamenti urbani, deforestazione, colture intensive), inquinamento marino (plastiche, sostanze chimiche, luce artificiale urbana che altera i ritmi circadia- ni di specie ittiche, National Geographic 2020); alterazione dell’Oceano e della criosfera a causa dei cambiamenti climatici e delle emissioni di CO2 in continuo aumento (NOAA 2021). Le implicazioni sull’evoluzione del Pianeta Terra, da un lato, e dall’altro le trasformazioni radicali e velocemente diffuse a livello globale sono sempre più al centro delle analisi dei maggiori centri di ricerca internazionali. È infatti quasi unanime la consapevolezza che l’evento pandemico sia una prova ulteriore che l’intera Umanità e il Pianeta Terra siano vicini ad un tipping point, ovvero che stiano vivendo una critical transition, caratterizzata dalla presenza di almeno quattro crisi congiunte: climatica, energetica, pandemica, economico-produttiva 10 INTRODUZIONE (vedi Cap. 3). Esse sono il risultato dell’evoluzione verso un mondo iperconnesso (Ubiquitous computing, Ubiquitous connectivity) e caratterizzato dalla creazione di strutture interattive globali. Introduzione Tutto ciò ha determinato processi di feedback positivi, con un incremento della complessità sistemica a livello locale e globa- le e il diffondersi di livelli crescenti di incertezza e di potenziale instabilità. In tale quadro i processi decisionali di imprese e individui – oltre che di organiz- zazioni e apparati istituzionali – devono misurarsi con sfide epocali e quindi occorre delineare nuovi strumenti di analisi e metodologie operative, al fine di orientarsi in uno scenario di evoluzione complessa e multidimensionale, acce- lerata dall’incremento della potenza computazionale disponibile per chiunque. In uno scenario globale investito da fenomeni e processi di tale portata è lo- gico che manchino coordinate generali e criteri applicativi per nuove regole di azione. È d’altra parte evidente come le sfide e i rischi siano così rilevanti da dover indurre a ripensare sistemi di pensiero consolidati e schemi operativi, allo scopo di agire in modo da evitare esiti distruttivi. L’alternativa all’inerzia e al deleterio arroccamento nel modo di pensare standard, non più appropriato, è effettuare uno sforzo serio e sistematico di riflessione, che parta dalla ricerca di strumenti e metodi che aiutino a comprendere i mutamenti in atto. Ciò può avvenire se, grazie ad un open mindset, si cerca di elaborare e adottare schemi teorici, sulla ba- se dei quali analizzare e descrivere in modo pregnante intuizioni razionali circa nuove tendenze tecno-economiche. Lo scopo deve essere quello di intravedere, data l’impossibilità di prevederli, trend morfologici e strutturali emergenti nelle interconnessioni che si sviluppano nel Sistema-Terra, al fine di progettare inter- venti correttivi ed evitare eventi catastrofici o gravemente distruttivi. ti gt Alla luce delle considerazioni iniziali, tre sono i punti fondamentali da assu- mere per orientare l’analisi e la riflessione: 1) bisogna assegnare centralità alla conoscenza dei sistemi complessi, in merito ai quali è doveroso creare e diffon- dere cultura tecnico-scientifica, manageriale ed etico-filosofica, dal momento che la posta in gioco appare sempre più il futuro dell’Umanità e degli esseri vi- venti, insieme a quello della Terra. 2) La prospettiva sistemica richiede un pro- fondo cambiamento dell’orizzonte temporale del quadro teorico e dei processi decisionali: è cruciale, infatti, la capacità di guardare al medio-lungo periodo, essenziale per prendere decisioni per disegni operativi nel breve coerenti con le visioni di lungo termine. 3) La natura sistemica globale e l’orizzonte temporale prolungato portano con sé il problema di come coordinare micro- e macro-com- portamenti (individuali e collettivi). Introduzione Ciò significa che occorrono lo sviluppo e la diffusione di una mentalità strategica, che sia in grado di realizzare processi e tendenze a varia scala in direzioni non deleterie. tfi Si tratta naturalmente di questioni di elevata difficoltà sia sul piano teorico che pratico, con implicazioni multidimensionali: tecnico-scientifiche, economi- che, sociali, politiche, istituzionali, geo-politiche. La piena consapevolezza della posta in gioco può essere un ottimo punto di partenza per cercare di elaborare strumenti e meccanismi atti a sintonizzare micro e macro-decisioni con traiet- torie evolutive meno dannose per tutti. Si tratta poi di pensare a dispositivi di varia natura, che consentano di verificare puntualmente se la progettazione in- 11 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO novativa assecondi o meno direttrici verso una dinamica sostenibile sul piano economico e ambientale. L’ambito in cui si muove la nostra analisi è prevalentemente economico, più precisamente quello delle attività umane finalizzate alla soddisfazione di bisogni, che nell’orizzonte odierno sembrano assumere sempre più natura immateriale, anche se alcune parti del mondo devono ancora fare i conti con la preponderanza di necessità materiali largamente insoddisfatte. Questo libro tenta di risponde- re alle esigenze indicate seguendo un preciso itinerario analitico e progettuale, proponendo al contempo strumenti teorici ed operativi. t p p p p Il lavoro è pertanto così articolato. Il Capitolo 1 introduce il concetto di universo fisico-cibernetico e le sfide per l’umanità che da esso scaturiscono. Il Capitolo 2 si concentra sull’analisi di alcune peculiarità dell’era odierna: com- plessità, incertezza e ansietà, conseguenti alla Grande Accelerazione che segna il passaggio dall’Olocene all’Antropocene, era in cui la ‘forza tellurica’ delle at- tività umane sembra più potente delle forze geofisiche ed astronomiche. Queste ultime per secoli hanno influenzato l’evoluzione sulla Terra, mentre oggi gli ef- fetti dei comportamenti umani producono effetti globali apparentemente privi di controllo. Come agire per riprenderne il controllo? Bisogna imparare molto dalla Natura e dalla sua evoluzione, dalla quale trarre una serie di lezioni essenziali: incessante capacità di apprendimento, adattatività, continua sperimentazione.t p ppt p Nel Capitolo 3 la trattazione approfondisce quella che molte discipline de- finiscono ‘transizione critica’ nell’evoluzione del Sistema-Terra e della storia umana, in quanto emergono segnali di uno scenario caratterizzato da una crisi sistemica globale. Viene argomentato come tale crisi si manifesti in un mondo divenuto ‘universo fisico-digitale’. Introduzione Una fase di transizione critica richiede il ri- pensamento complessivo della società in cui viviamo, per cui centri di ricerca e società di consulenza internazionali tematizzano modelli di riorganizzazione generale di imprese e attività, con l’introduzione di varie definizioni sintetiche: «The Great Reset» (World Economic Forum); «dal capitalismo degli sharehol- ders al capitalismo degli stakeholders» (Boston Consulting Group); «long term capitalism» (McKinsey Global Institute). t L’apparato teorico e la concettualizzazione operativa consentono nel Capito- lo 4 di avviare un itinerario strategico-cognitivo, che inizia con l’analisi delle sfide globali dell’era odierna, tra cui i cambiamenti dei processi economico-produttivi e i mutamenti tendenziali nel mondo del lavoro, delle competenze, delle strategie appropriate per creare occupazione, misurandosi in primis la questione cruciale dei prossimi decenni: il rapporto tra Intelligenza umana Intelligenza Artificia- le (par. 6). Per fronteggiare le sfide globali nel Capitolo 5 vengono proposti uno schema generale (Frame) e una metodologia appropriata, la cui formulazione sintetica è l’adaptive strategic learning. ftt Nel Capitolo 6 viene effettuato un approfondimento delle traiettorie tecno- economiche, rispetto alle quali viene esplicitato in modo specifico per ciascuna di esse uno schema generale di intervento strategico, basato su precisi elemen- ti: Identificazione delle missions, individuazione degli obiettivi e degli attori coinvolti, indicatori ai fini della valutazione ex-ante ed ex-post. Il ruolo degli at- 12 INTRODUZIONE tori individuali e collettivi viene approfondito nel Capitolo 7, insieme agli stru- menti tecnici ed operativi considerati idonei per valutare le politiche dirette a innescare e favorire processi innovativi all’altezza delle sfide globali, preceden- temente indicate. Sempre in questo capitolo si mette alla prova sul piano empi- rico lo schema teorico introdotto nei capitoli precedenti e arricchito da concetti desunti da Ackoff (1972) e Gharajedaghi (2011) con l’interpretazione di come alcune regioni italiane, considerate molto dinamiche dalla letteratura sull’ana- lisi dei sistemi locali, hanno reagito alla dinamica tecno-economica pre-Covid.ir g p Il Capitolo 8, infine, propone un frame di design thinking, funzionale al ripen- samento degli indicatori adatti per progettare e valutare le politiche dell’inno- vazione, in modo che gli interventi strategici siano coerenti con gli elementi del frame tecnico-scientifico esposto nel corso di tutto il libro e le cui linee essen- ziali sono richiamate nelle Conclusioni. NOAA, US Department of Commerce. 2021. How much oxygen comes from the ocean? At least of Earth’s oxygen comes from the Ackoff, R.L., e F.E. Emery. 1972. On Purposeful Systems. Chicago: Aldine-Atherton. Gharajedaghi, J. 2011. Systems Thinking: Managing Chaos and Complexity. Amsterdam: Elsevier. National Geographic. 2020. Marine pollution, explained. <https://www. nationalgeographic.com/environment/oceans/critical-issues-marine-pollution/> (2021-03-10). NOAA, US Department of Commerce. 2021. How much oxygen comes from the ocean? At least of Earth’s oxygen comes from the Bibliografia Ackoff, R.L., e F.E. Emery. 1972. On Purposeful Systems. Chicago: Aldine-Atherton. Gharajedaghi, J. 2011. Systems Thinking: Managing Chaos and Complexity. Amsterdam: Elsevier. National Geographic. 2020. Marine pollution, explained. <https://www. nationalgeographic.com/environment/oceans/critical-issues-marine-pollution/> (2021-03-10). NOAA, US Department of Commerce. 2021. How much oxygen comes from the ocean? At least of Earth’s oxygen comes from the Ackoff, R.L., e F.E. Emery. 1972. On Purposeful Systems. Chicago: Aldine-Atherton. Gharajedaghi, J. 2011. Systems Thinking: Managing Chaos and Complexity. Amsterdam: Elsevier. 13 13
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Production of schizophyllan from distiller’s dried grains with solubles by diverse strains of Schizophyllum commune
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© 2013 Sutivisedsak et al.; licensee Springer. 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. Production of schizophyllan from distiller’s dried grains with solubles by diverse strains of Schizophyllum commune Nongnuch Sutivisedsak, Timothy D Leathers* and Neil PJ Price * Correspondence: Tim.Leathers@ars.usda.gov Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA Open Access Open Access Abstract Eleven diverse strains of Schizophyllan commune were examined for production of the biopolymer schizophyllan from agricultural biomass. Strains were grown in malt extract (ME) basal medium containing 1% (w/v) distiller’s dried grains with solubles (DDGS), an abundant coproduct of fuel ethanol production by the dry grind process. Ten of 11 strains tested produced more than 2 g schizophyllan/L. Two strains, ATCC 20165 and CBS 266.60, produced more than 10 g schizophyllan/L. Schizophyllan from these strains was similar to commercial product in terms of solution viscosity, molecular weight, and surface tension properties, suggesting that they would be equivalent in biomaterial applications. Keywords: Distiller’s dried grains with solubles; DDGS; Schizophyllum commune; Schizophyllan schizophyllan for biomaterials applications. Schizo- phyllan could fit into the integrated biorefinery con- cept as a value-added bioproduct from biomass. One readily available type of agricultural biomass is dis- tiller’s dried grains with solubles (DDGS), an abun- dant coproduct of fuel ethanol production from corn by the dry-grind process (Rosentrater et al. 2012). We recently demonstrated the efficient utilization of DDGS for schizophyllan production by S. commune commercial production strain ATCC 38548 (Sutivisedsak et al. 2013). Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 a SpringerOpen Journal a SpringerOpen Journal Background g Schizophyllan is a homoglucan with a β-1,3-linked backbone and single β-1,6-linked glucose side chains at every third residue (Figure 1), produced by the fun- gus Schizophyllum commune (Rau 1999; Rau 2002). Schizophyllan is commercially produced for use in anti- cancer therapies and as a bioactive cosmetics ingredient (Rau 2002). However, schizophyllan also has unique physical properties of high viscosity, film formation, and thermal stability that suggest bulk biomaterials applica- tions. Schizophyllan can form oxygen-impermeable films for food preservation (Schulz et al. 1992), and it has been tested for use in enhanced petroleum recovery (Rau et al. 1992b). Despite the fact that S. commune is an ubiquitous mushroom found world-wide, commercial production and most research studies have involved a single strain, ATCC 38548 (Prokop et al. 1992; Rau et al. 1992a; Sanroman and Nunez 1993; Shu and Hsu 2011). Few additional strains have been studied for schizophyllan production, such as NRCM and CGMCC 5.113 (Kumari et al. 2008; Li et al. 2011). In the current study we compare 11 diverse strains of S. commune for their ability to produce schizophyllan in medium contai- ning DDGS. Although glucose is used in conventional pro- duction of schizophyllan, S. commune can utilize a number of sugars and agricultural residues for poly- saccharide production (Steiner et al. 1987; Leathers et al. 2006; Gao and Zhou 2008; Kumari et al. 2008; Shu and Hsu 2011). Inexpensive agricultural bio- mass residues could be appropriate for production of Sutivisedsak et al. SpringerPlus 2013, 2:476 Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Page 2 of 6 O OH OH HO HO O OH O HO O O OH O HO HO O O OH O HO HO n Schizophyllan repeat unit Figure 1 Chemical structure of the schizophyllan repeat unit. Schizophyllan repeat unit Figure 1 Chemical structure of the schizophyllan repeat unit. Figure 1 Chemical structure of the schizophyllan repeat unit. Results and discussion schizophyllan from agricultural biomass appears to be a common trait among strains of S. commune. Two of these strains, ATCC 20165 and CBS 266.60, produced more than 10 g schizophyllan/L (Table 2). These yields are comparable to those obtained from commercial production strain ATCC 38548 (Sutivisedsak et al. 2013). Schizophyllan preparations from strains ATCC 20165 and CBS 266.60 were further examined for so- lution viscosity, molecular weight, and surface tension properties. Production of schizophyllan by diverse strains of Schizophyllum commune A set of 11 S. commune strains was chosen for this study, representing diverse isolation sources and sites (Table 1). Only non-clinical isolates were used because the goal was to identify strains with potential for industrial pro- duction. Ten of these strains produced more than 2 g schizophyllan/L in ME basal medium containing 1% (w/v) DDGS (Table 2). Thus, the capacity to produce Table 1 Strains of Schizophyllum commune used in this study Strain Substrate of isolation Depositors Country Designation Synonym ATCC 20165 Kyowa Ferm. Ind. Co., Ltd. 6 F2 ATCC 26262 CA Raper 699 ATCC 26890 Y Koltin 700 ATCC 28095 Loblolly pine log ER Toole R-8 ATCC 44200 J.G.H. Wessels, Jun 1981 USA 4-39 CBS 341.81 CBS 199.27 Conserved stem, Hevea A. van Luijk, Koloniaal Inst., May 1927 MUCL 1008 CBS 249.69 In sawmill, Intsia bijuga M.R. Monsalud, Univ. of the Philippines, Mar 1969 Philippines Daedalea elegans, FPRI10 CBS 266.60 Mahogany wood IMI, Mar 1960 UK FPRL 9, IMI 061312 CBS 579.83 Decaying jute cutting H. Esterbauer, Oct 1983 CBS 109645 Litter López, Aug 2001 Colombia NRRL A-23867 P.A. Lemke ATCC 38548 Elm twig M.G. Paice Delmar Table 1 Strains of Schizophyllum commune used in this study Page 3 of 6 Page 3 of 6 Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Table 2 Total biomass and production of schizophyllan (dry weights) by strains of Schizophyllum communea Strain Total biomass (g/l) Schizophyllan (g/l) ATCC 20165 6.4 ± 0.5 10.1 ± 0.7 ATCC 26262 8.7 ± 0.1 8.3 ± 0.5 ATCC 26890 12.4 ± 0.4 5.9 ± 0.5 ATCC 28095 9.4 ± 0.5 2.2 ± 0.2 ATCC 44200 13.2 ± 0.2 0.6 ± 0.1 CBS 199.27 10.7 ± 0.5 2.4 ± 0.1 CBS 249.69 11.5 ± 0.4 3.4 ± 0.1 CBS 266.60 4.7 ± 0.8 12.7 ± 0.1 CBS 579.83 12.3 ± 0.1 2.1 ± 0.2 CBS 109645 13.2 ± 0.4 2.1 ± 0.1 NRRL A-23867 10 ± 0.1 2.3 ± 0.2 a Cultures grown for 8 days on 1% (w/v) distiller’s dried grains with solubles (DDGS) in malt extract basal medium. Table 2 Total biomass and production of schizophyllan (dry weights) by strains of Schizophyllum communea of 4.6 × 106 and 5.2 × 106, respectively. The values are consistent with literature values of 6–12 × 106 (Rau 1999). The interfacial tension of 0.3% (w/v) aqueous solutions of schizophyllan from S. Conclusions a Cultures grown for 8 days on 1% (w/v) distiller’s dried grains with solubles (DDGS) in malt extract basal medium. Ten of 11 diverse strains of S. commune produced schizophyllan from agricultural biomass. Two of these strains, ATCC 20165 and CBS 266.60, produced yields comparable to those from a commercial production strain. The physical properties of schizophyllan solutions from these strains also were comparable to those of commercial schizophyllan, suggesting that they would be equivalent in biomaterial applications. Properties of schizophyllan solutions from S. commune strains ATCC 20165 and CBS 266.60 Schizophyllan from S. commune strains ATCC 20165 and CBS 266.60 was structurally analyzed by Hete- ronuclear Single Quantum Coherence-NMR (Figure 2). The spectra were essentially identical to those of both a commercial schizophyllan standard and schizophyllan from commercial strain ATCC 38548. Spectra showed two anomeric sugar signals at 4.55 ppm and 4.20 ppm, due to the β-1,3-linked glucose and β-1,6-linked glucose, respectively. These signals correlated to overlapping 13C signals at 103.4 ppm, consistent with β-linked glucosyl residues. Other carbohydrate signals were apparent in the 2.7 – 4.0 ppm region for 1H, and 55 – 85 ppm for 13C nuclei. Characteristic methylene – CH2 signals were apparent at 3.40 and 3.60 ppm, coupled to a single 13C signal at 61 ppm. These were assigned to the C-6 position of the backbone glucose residues carrying a 1,6-glucose branch. These data are consistent with the isolated polysaccharides being schizophyllan. Production of schizophyllan by diverse strains of Schizophyllum commune commune strains ATCC 20165 and CBS 266.60 were 52 ± 1.9 and 55 ± 2.8 dy/cm, respectively (standard deviations shown). By compa- rison, schizophyllan from commercial strain ATCC 38548 showed similar values of 58 ± 3.5 dy/cm, while pure water exhibits an interfacial tension of 72 dy/cm (Dunlap et al. 2011). Thus, schizophyllan solutions from strains ATCC 20165 and CBS 266.60 showed relatively low surface activity, similar to commercial preparations (Rau 1999). Strains and culture conditions Strains and culture conditions Schizophyllum commune strains used in this study were obtained from the American Type Culture Collection, Manassas, VA (ATCC strains), the ARS Culture Col- lection at the National Center for Agricultural Uti- lization Research, Peoria IL (NRRL strain) and the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS strains). Malt extract (ME) basal medium contained 2% (w/v) malt extract and 0.1% (w/v) peptone. Strains were grown on ME solid medium containing 2% (w/v) glucose and 2.5% (w/v) agar at 28°C for 7–10 days. A 7 mm × 7 mm square of mycelium was used to inoculate 250 mL of ME basal medium containing 2% (w/v) glucose in a 500 ml fluted Erlenmeyer flask with three 10 mm glass beads. This preinoculum culture was incubated at 240 rpm for 4–5 days at 30°C. Experimental cul- tures containing 1% (w/v) of DDGS in 150 mL of ME basal medium (without glucose) in 500 mL flasks were inoculated with 1.5 mL of preinoculum (1% v/v) and incubated at 240 rpm for 8 days at 30°C. DDGS was obtained from the National Corn-to-Ethanol Re- search Center, Edswardville, IL. All experiments were carried out in triplicate and standard deviations are shown. The solution viscosity properties of 0.3% (w/v) aque- ous solutions of schizophyllan from strains ATCC 20165 and CBS 266.60 were compared with those of com- mercial strain ATCC 38548 (Figure 3). These strains exhibited nearly identical viscosity and shear thinning properties, characteristic of the pseudoplastic flow be- havior of schizophyllan solutions (Rau 1999). HPSEC was used to characterize the molecular weight of schizophyllan. Schizophyllan produced by S. com- mune strains ATCC 20165 and CBS 266.60 exhibited heterodisperse peaks with maximum molecular weights Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Page 4 of 6 Page 4 of 6 Figure 2 HSQC 2D-NMR spectra of schizophyllans from strains CBS 266.6 (top) and ATCC 20165 (lower). Figure 2 HSQC 2D-NMR spectra of schizophyllans from strains CBS 266.6 (top) and ATCC 20165 (lower). Figure 2 HSQC 2D-NMR spectra of schizophyllans from strains CBS 266.6 (top) and ATCC 20165 (lower). air-dried overnight to reduce the amount of ethanol, and then lyophilized. Isolation of schizophyllan Whole culture suspensions were homogenized for 20 sec (Power Gen 700, Fisher Scientific) and centrifuged at 6,166 × g for 1 h at 4°C. The supernatants were collected and the insoluble pellets (containing mycelia and re- sidual DDGS) were resuspended in 100 mL of deionized water, homogenized, and centrifuged as before. The pel- lets were dried under vacuum for 48 h at 60°C. The su- pernatants were combined and one volume of 95% ethanol was added. After 1 h at 4°C, precipitates were collected by centrifugation at 6,166 × g for 1 h at 4°C, Authors’ contributions NS, TL, and NP participated in the conception and design of the study, interpreted experimental results, and helped draft the manuscript. NS produced and characterized schizophyllan from diverse strains of Schizophyllum commune. NP conducted the NMR studies of schizophyllan from selected strains and controls. All authors read and approved the final manuscript. NMR analysis Solution NMR spectra were recorded on a Bruker AMX 500 spectrometer at normal probe temperature with standard instrument settings. Deuterated dime- thylsulfoxide (d6-DMSO) was used as the solvent. All chemical shifts were referenced to tetramethylsilane at 0 ppm. Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Page 5 of 6 Figure 3 Solution viscosity properties of 0.3% (w/v) aqueous solutions of schizophyllan produced by Schizophyllum commune on DDGS in ME basal medium. Figure 3 Solution viscosity properties of 0.3% (w/v) aqueous solutions of schizophyllan produced by Schizophyllum commune on DDGS in ME basal medium. Competing interests The authors declare that they have no competing interests. Abbreviations DDGS: Distiller’s dried grains with solubles; d6-DMSO: Deuterated dimethylsulfoxide; HPSEC: High performance size exclusion chromatography; ME: Malt extract basal medium; NMR: Nuclear magnetic resonance; USDA: United States Department of Agriculture. Schizophyllan molecular weights were determined by size exclusion chromatography as previously described, using a Shodex SB-806 M high performance size exclu- sion chromatography (HPSEC) column (Showa Denko, Tokyo, Japan) and eluted with 0.05 M sodium nitrate at a flow rate of 0.5 ml/min (Leathers et al. 2010). The column was calibrated with a set of eight pullulan mo- lecular weight standards ranging from 5.8 × 103 Da to 1.66 × 106 Da (Showa Denko, Tokyo, Japan). Separations were monitored using a Shodex OR-1 optical rotation detector (Showa Denko). Solution viscosity properties Expert technical assistance was provided by Erika Hertenstein, Melinda S. Nunnally, Suzanne Unser, and Andrew J. Thomas. The authors sincerely thank Christopher Dunlap for surface tension measurements. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2010-65504-20377 from the USDA National Institute of Food and Agriculture. Mention of any trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. Solution viscosity was measured using a TA Instruments (New Castle, DE) ARES LS-1 controlled strain rheome- ter with a 25 mm titanium parallel plate. All tests were performed at 25°C using a peltier plate. Steady rate sweeps were used to determine the viscosity of samples from 0.01-100 s-1. The Cross model was used to deter- mine the zero shear viscosity, which was 460 Pa.s. Received: 30 May 2013 Accepted: 19 September 2013 Published: 22 September 2013 References Surface activity was determined using the pendant drop method (Dunlap et al. 2011). Samples were analyzed using the FTA 4000 surface tension instrument (First Ten Angstorms Inc., USA). Measurements were made using 22 gauge blunt needles with 7 μL drops. The re- ported values are the average of triplicate cultures. Dunlap CA, Schisler DA, Price NP, Vaughn SF (2011) Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight. J Microbiol 49:603–609 Dunlap CA, Schisler DA, Price NP, Vaughn SF (2011) Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight. J Microbiol 49:603–609 Gao L, Zhou S (2008) Optimization of extraction technology of schizophyllan produced by solid fermentation. Shipin Gongye Keji 29:214–216 Kumari M, Survase SA, Singhal RS (2008) Production of schizophyllan using Schizophyllum commune NRCM. Biores Technol 99:1036–1043 Page 6 of 6 Page 6 of 6 doi:10.1186/2193-1801-2-476 Cite this article as: Sutivisedsak et al.: Production of schizophyllan from distiller’s dried grains with solubles by diverse strains of Schizophyllum commune. SpringerPlus 2013 2:476. Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Leathers TD, Nunnally MS, Price NP (2006) Co-production of schizophyllan and arabinoxylan from corn fiber. Biotechnol Lett 28:623–626 Leathers TD, Nunnally MS, Cote GL (2010) Optimization of process conditions for enzymatic modification of alternan using dextranase from Chaetomium erraticum. Carbohydr Pol 81:732–736 Li W, Zhou P, Yu L (2011) Statistical optimization of the medium composition by response surface methodology to enhance schizophyllan production by Schizophyllum commune. Zeitschrift Naturforschung C J Biosci 66:173–181 Prokop A, Rapp P, Wagner F (1992) Production of extracellular β-1,3/β-1,6-glucan by mono- and dikaryons of Schizophyllum commune. Exp Mycol 16:197–206 Rau U (1999) Production of schizophyllan. In: Bucke C (ed) Methods in biotechnology, vol 10, carbohydrate biotechnology protocols. Humana Press, Totowa, pp 43–55 Rau U (2002) Schizophyllan. In: Vandamme EJ, De Baets S, Steinbuchel A (ed) Biopolymers, vol 6, polysaccharides II: polysaccharides from eukaryotes. Wiley-VCH, Weinheim, pp 61–91 Rau U, Gura E, Olszewski E, Wagner F (1992a) Enhanced glucan formation of filamentous fungi by effective mixing, oxygen limitation and fed-batch processing. J Ind Microbiol 9:19–26 Rau U, Haarstrick A, Wagner F (1992b) Suitability of schizophyllan solutions for polymerflooding of petroleum reservoirs with high temperature and stability. Chemie Ingenieur Technik 64:576–577 Rosentrater KA, Ileleji K, Johnston DB (2012) Manufacturing of fuel ethanol and distillers grains - current and evolving processes. In: Liu K, Rosentrater KA (ed) Distillers Grains: Production, Properties, and Utilization. CRC Press, Boca Raton, pp 73–102 Rosentrater KA, Ileleji K, Johnston DB (2012) Manufacturing of fuel ethanol and distillers grains - current and evolving processes. In: Liu K, Rosentrater KA (ed) Distillers Grains: Production, Properties, and Utilization. CRC Press, Boca Raton, pp 73–102 Sanroman MA, Nunez MJ (1993) Production of extracellular polysaccharides. Anales de la Asociacion Quimica Argentina 81:15–23 Sanroman MA, Nunez MJ (1993) Production of extracellular polysaccharides. Anales de la Asociacion Quimica Argentina 81:15–23 g Schulz D, Rau U, Wagner F (1992) Characteristics of films prepared from native and modified branched β-1,3-D-glucans. Carbohydr Pol 18:295–299 Shu C, Hsu H (2011) Production of schizophyllan glucan by Schizophyllum commune ATCC 38548 from detoxificated hydrolysate of rice hull. J Taiwan Inst Chem Eng 42:387–393 Schulz D, Rau U, Wagner F (1992) Characteristics of films prepared from native and modified branched β-1,3-D-glucans. Carbohydr Pol 18:295–299 Schulz D, Rau U, Wagner F (1992) Characteristics of films prepared from native and modified branched β-1,3-D-glucans. Sutivisedsak et al. SpringerPlus 2013, 2:476 http://www.springerplus.com/content/2/1/476 Carbohydr Pol 18:295–299 Shu C, Hsu H (2011) Production of schizophyllan glucan by Schizophyllum commune ATCC 38548 from detoxificated hydrolysate of rice hull. J Taiwan Inst Chem Eng 42:387–393 Shu C, Hsu H (2011) Production of schizophyllan glucan by Schizophyllum commune ATCC 38548 from detoxificated hydrolysate of rice hull. J Taiwan Inst Chem Eng 42:387–393 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of Schizophyllum commune: cellulase and xylanase production and formation of the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of Schizophyllum commune: cellulase and xylanase production and formation of the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of Schizophyllum commune: cellulase and xylanase production and formation of the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of Schizophyllum commune: cellulase and xylanase production and formation of the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178 Sutivisedsak N, Leathers TD, Nunnally MS, Price NPJ, Biresaw G (2013) Utilization of agricultural biomass in the production of the biopolymer schizophyllan. J Ind Microbiol Biotechnol 40:105–112 Sutivisedsak N, Leathers TD, Nunnally MS, Price NPJ, Biresaw G (2013) Utilization of agricultural biomass in the production of the biopolymer schizophyllan. J Ind Microbiol Biotechnol 40:105–112 doi:10.1186/2193-1801-2-476 Cite this article as: Sutivisedsak et al.: Production of schizophyllan from distiller’s dried grains with solubles by diverse strains of Schizophyllum commune. SpringerPlus 2013 2:476. doi:10.1186/2193-1801-2-476 Cite this article as: Sutivisedsak et al.: Production of schizophyllan from distiller’s dried grains with solubles by diverse strains of Schizophyllum commune. SpringerPlus 2013 2:476. 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A chiral phosphazane reagent strategy for the determination of enantiomeric excess of amines
Chemical science
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aYusuf Hamied Department of Chemistry, Cambridge University, Lenseld Road, Cambridge, CB2 1EW, UK. E-mail: dsw1000@cam.ac.uk bThe Faraday Institution Quad One, Harwell Science and Innovation Campus, Didcot, UK † Electronic supplementary information (ESI) available: synthetic procedures and analytical data, NMR and X-ray data, DFT calculations. CCDC 2105705–2105719 and 2157891–2157896. For ESI and crystallographic data in CIF or other electronic format see https://doi.org/10.1039/d2sc01692c A chiral phosphazane reagent strategy for the determination of enantiomeric excess of amines† Cite this: Chem. Sci., 2022, 13, 5398 Andrew J. Peel,*a Alexandros Terzopoulos,a Rajesh B. Jethwa, a Dipanjana Choudhury,a Hao-Che Niu,a Andrew D. Bond, a Jonathan Slaughter ab and Dominic S. Wright *a All publication charges for this article have been paid for by the Royal Society of Chemistry Received 23rd March 2022 Accepted 12th April 2022 DOI: 10.1039/d2sc01692c rsc.li/chemical-science Methods for measuring enantiomeric excess (ee) of organic molecules by NMR spectroscopy provide rapid analysis using a standard technique that is readily available. Commonly this is accomplished by chiral derivatisation of the detector molecule (producing a chiral derivatisation agent, CDA), which is reacted with the mixture of enantiomers under investigation. However, these CDAs have almost exclusively been based on carbon frameworks, which are generally costly and/or difficult to prepare. In this work, a methodology based on the readily prepared inorganic cyclodiphosph(III)azane CDA ClP(m- NtBu)2POBorn (Born ¼ endo-(1S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl) is shown to be highly effective in the measurement of ee’s of chiral amines, involving in situ reaction of the chiral amines (R*NH2) with the P–Cl bond of the CDA followed by quaternization of the phosphorus framework with methyl iodide. This results in sharp 31P NMR signals with distinct chemical shift differences between the diastereomers that are formed, which can be used to obtain the ee directly by integration. Spectroscopic, X-ray structural and DFT studies suggest that the NMR chemical shift differences between diastereomers is steric in origin, with the sharpness of these signals resulting from conformational locking of the bornyl group relative to the P2N2 ring induced by the presence of the P(V)-bonded amino group (R*NH). This study showcases cheap inorganic phosphazane CDAs as simple alternatives to organic variants for the rapid determination of ee. Received 23rd March 2022 Accepted 12th April 2022 DOI: 10.1039/d2sc01692c rsc.li/chemical-science © 2022 The Author(s). Published by the Royal Society of Chemistry Introduction The reaction of [ClPNtBu]2 16 with the chiral, naturally occurring alcohol ()-menthol has been reported previously,23c however, the bicyclic alcohol endo-()-borneol was selected as a chiral auxiliary in the current study on the basis that it should exhibit less conformational freedom than menthol (and is nonetheless inexpensive). Furthermore, it has been reported that the formation of borneol derivatives, where O–H is replaced by O–X (X s H) reduces the number of accessible conformers that can arise due to rotation about the O–X bond, and this was anticipated to be of benet in developing a CDA.29 The proto- type CDA was prepared by reacting [ClPNtBu]2 with endo- ()-borneol in a 1 : 1 ratio in THF in the presence of Et3N (Scheme 3). Aer trituration with Et2O and extraction into toluene, the crude product was recrystallised from n-pentane to give ClP(m-NtBu)2POBorn (Born ¼ bornyl, endo-(1S)-1,7,7- trimethylbicyclo[2.2.1]heptan-2-yl) (1) in 50% yield (from two fractions). 1H NMR spectroscopy in C6D6 showed signals belonging to bornyl and tBu groups in a 1 : 2 ratio (see ESI Fig. S1†). Meanwhile, 31P{1H} NMR spectroscopy revealed two doublets, at d 190.5 ppm and 140.0 ppm, which could be attributed to the –Cl and –OR substituted P atoms of a P2N2 ring, respectively. The observed inequivalence of the 1H NMR signals of the tBu groups within the P2N2 ring unit can be attributed to the effect of spatial orientation of the chiral axil- lary, though evidently the inuence is small, with a separation of d 0.02 ppm between these signals. Furthermore, this sepa- ration appears to be solvent dependent as only one singlet is Molecules based on phosphorus–nitrogen skeletons are of interest in this regard because of the similarity between the C–C and P–N bond dissociation energies (346 kJ mol1 vs. Introduction Scheme 1 The reaction of an achiral pyridyl aluminate with a chiral alcohol, producing diastereoisomeric dimers. The result is the asym- metrisation of the reporter R2-groups of the pyridyl rings (marked in magenta, green, brown and purple), which can be detected by 1H NMR spectroscopy. R1, R2 ¼ alkyl. Scheme 2 The concept of forming diastereomeric cyclodiphosph(III) azanes, where R* is a chiral auxiliary and R0*H is a chiral analyte. 2, in which one of the P atoms is bonded to a chiral auxiliary (R*) while the other has a reactive P–Cl bond. The highly reac- tive nature of this bond means that reaction with an acidic H- atom of a chiral analyte (R0*H) leads to a pair of diastereo- mers. An important feature of cyclodiphosphazanes of this type is the general thermodynamic preference for the cis isomer in the solution and solid states,28 so that the chiral groups R* and R0* should be in close proximity. This, it was thought, should induce the maximum effect on the chemical shis of the R and S isomers of R0*. Scheme 1 The reaction of an achiral pyridyl aluminate with a chiral alcohol, producing diastereoisomeric dimers. The result is the asym- metrisation of the reporter R2-groups of the pyridyl rings (marked in magenta, green, brown and purple), which can be detected by 1H NMR spectroscopy. R1, R2 ¼ alkyl. Scheme 1 The reaction of an achiral pyridyl aluminate with a chiral alcohol, producing diastereoisomeric dimers. The result is the asym- metrisation of the reporter R2-groups of the pyridyl rings (marked in magenta, green, brown and purple), which can be detected by 1H NMR spectroscopy. R1, R2 ¼ alkyl. Scheme 1 The reaction of an achiral pyridyl aluminate with a chiral alcohol, producing diastereoisomeric dimers. The result is the asym- metrisation of the reporter R2-groups of the pyridyl rings (marked in magenta, green, brown and purple), which can be detected by 1H NMR spectroscopy. R1, R2 ¼ alkyl. discrimination relies on the in situ formation of diastereomeric dimers, rather than monomeric diastereomers (Scheme 1).12 This, together with the substantial ionic character of organo- metallic CDAs of this type, leads to hydrolytic sensitivity and oxygen intolerance, which are major drawbacks to bench-top applications. Introduction convenient determination of enantiomeric excess (ee) using NMR spectroscopy.5 Current methods involve the use of enantiomerically-pure compounds as chiral auxiliaries.6 A number are based on non-covalent interactions, including chiral solvating agents (CSAs),7 chiral lanthanide shi reagents,8 ion-pairing agents5c and liquid crystals.9 However, chiral derivatisation agents (CDAs)10 that form a covalent bond with the chiral analyte are perhaps the most commonly used species for determining ee's. Their popularity stems from the robustness of the covalent link between the auxiliary and ana- lyte, greater freedom in solvent selection and their added potential for use in assignment of absolute conguration.11 The creation of chiral molecules plays a major role in numerous chemical processes, from natural product synthesis to the design of new materials. Crucial to the search for efficient methods of producing enantiopure substances is the ability to detect and measure chiral composition.1 Many methods are established for the determination of the chiral purity of a sample, including optical rotation and circular dichroism,2 gas chromatography (GC) or liquid chromatography (HPLC)3 with a chiral stationary phase. Methods based on NMR spec- troscopy have been studied extensively and remain at the fore- front of this area due to the simplicity and availability of this technique and the potential for fast turnaround.4 The reaction of an organic CDA with a chiral analyte typically creates a new C–X bond that has a relatively high bond disso- ciation energy and low-to-moderate bond polarity, e.g. C–N or C–O. The kinetic stability of these bonds is essential to prevent interchange of chiral components during analysis, and this has led to the dominance of organic chemistry in chiral derivatisa- tion. So far, however, the potential of readily prepared inorganic CDAs has rarely been explored. Success in this endeavour hinges on selection of inorganic frameworks that can compete with the stability of classical carbon-based systems. A recent example is pyridyl aluminate reagents which can be used for the chiral discrimination of alcohols, but their mechanism of In the last few decades, considerable effort has gone into developing new methodologies and reagents for the rapid and © 2022 The Author(s). Published by the Royal Society of Chemistry 5398 | Chem. Sci., 2022, 13, 5398–5412 5398 Edge Article Edge Article Chemical Science Scheme 2 The concept of forming diastereomeric cyclodiphosph(III) azanes, where R* is a chiral auxiliary and R0*H is a chiral analyte. Results and discussion Scheme 3 Synthesis of 1. Introduction 290 kJ mol1, respectively) and their low polarity (DcP–N ¼ 0.85).13 A particularly well-developed subset of P–N compounds are cyclodiphosph(III) azanes [XP(NR)]2 (X ¼ halogen, -OR, -N(H)R, -NR2, -R; R ¼ organic group).14 Many different examples are accessible from the inex- pensive precursor [ClPNtBu]2, which has excellent stability and is easy to functionalise.15,16 Cyclodiphosphazane molecules have been investigated extensively as building blocks to inorganic macrocycles,17–20 as ligands in supramolecular coordination complexes21 and in MOFs.22 Furthermore, they have found appli- cations in homogeneous catalysis,23 anti-tumour metallodrug development24 and anion recognition/transport.25,26 A role for P–N compounds in the chiral realm is emerging with a recent report on chiral amino-phosphonium salts which exhibit highly enantiose- lective recognition of chiral molecules, including carboxylic acids, amines and alcohols, through non-covalent interactions.27 However, in this case the ready detection of chirality and the measurement of ee's was not possible. Inspired by this, and our earlier work on aluminates, we decided to explore the potential of cyclodiphosphazanes to build covalent chiral reagents that can be used to probe enantiomeric excess and absolute conguration using NMR spectroscopy. Edge Article Fig. 1 The molecular structure of 1 (ellipsoids at 30% probability) with H atoms omitted for clarity. One representative molecule from the asymmetric unit is depicted. The other molecule has essentially identical conformation, aside from rotation of one of the tBu groups. Selected bond lengths (˚A) and angles (): P1–N1 1.682(3), P1–N2 1.695(3), P2–N1 1.732(3), P2–N2 1.729(3), P1–Cl1 2.1619(3), P2–O1 1.612(2), P1–N1–P2 97.93(13), P1–N2–P2 97.52(13), N1–P1–N2 82.77(13), N1–P2–N2 80.36(12), N1–P1–Cl1 104.05(10), N2–P1–Cl1 104.14(10), N1–P2–O1 106.83(12), N2–P2–O1 106.80(12). Reactions of 1 with the amines (R)-1-phenylethylamine and (S)-1-phenylethylamine (both of which are benchmark chiral amine reagents in the literature) were undertaken to explore its potential as a CDA. In situ-prepared 1 was reacted in a 1 : 1 ratio with (R)- or (S)-1-phenylethylamine in the presence of excess Et3N (Scheme 4). Recrystallisation of the solid residues from toluene gave crystals of (R)-PhCH(Me)NHP(m-NtBu)2POBorn (4- R) or (S)-PhCH(Me)NHP(m-NtBu)2POBorn (4-S). The solid-state structures of 4-R and 4-S are signicantly different. In 4-R there are three independent molecules in the asymmetric unit (see ESI Fig. S55 and S56†). Two of these (one of which is shown in Fig. 2a) are similar, but distinct from the conformation of the third (Fig. 2b). The differences arise largely due to pivoting around the P–O and C–O bonds at the bornyl group (see ESI†). None of these arrangements allow for a good ‘t’ of the groups on the chiral amine within the pocket offered by the endo face of the bornyl ring and their conformations are probably dictated by packing effects, suggesting relatively small energy differences between them. Fig. 1 The molecular structure of 1 (ellipsoids at 30% probability) with H atoms omitted for clarity. One representative molecule from the asymmetric unit is depicted. The other molecule has essentially identical conformation, aside from rotation of one of the tBu groups. Selected bond lengths (˚A) and angles (): P1–N1 1.682(3), P1–N2 1.695(3), P2–N1 1.732(3), P2–N2 1.729(3), P1–Cl1 2.1619(3), P2–O1 1.612(2), P1–N1–P2 97.93(13), P1–N2–P2 97.52(13), N1–P1–N2 82.77(13), N1–P2–N2 80.36(12), N1–P1–Cl1 104.05(10), N2–P1–Cl1 104.14(10), N1–P2–O1 106.83(12), N2–P2–O1 106.80(12). In contrast to 4-R, the solid-state structure of 4-S reveals a single unique molecule (in space group P212121) and the structural effect of inverting the stereochemistry at the a-carbon Fig. 2 Two of the independent molecules in the asymmetric unit of 4- R, showing different conformations at the bornyl group (see ESI Fig. S55† for ellipsoid representations and selected metric parameters). Establishing the methodology Based on the mechanism of chiral discrimination we had observed previously using pyridyl aluminate reagents, involving the formation of diastereomeric dimers (Scheme 1), we reasoned that the more rigid P2N2 ring units of cyclo- diphosph(III)azanes could form the basis of a new type of inor- ganic CDA. Our prototype CDA was of type A, shown in Scheme Scheme 3 Synthesis of 1. Scheme 3 Synthesis of 1. Chem. Sci., 2022, 13, 5398–5412 | 5399 Chem. Sci., 2022, 13, 5398–5412 | 5399 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Edge Article Selected bond lengths (˚A) and angles (): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2 1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9), N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3– P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1– P2–N2 104.35(9). Edge Article Chemical Science Chemical Science Edge Article Edge Article Fig. 3 Molecular structure of 4-S (ellipsoids at 30% probability) with H atoms on C omitted for clarity. Selected bond lengths (˚A) and angles (): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2 1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9), N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3– P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1– P2–N2 104.35(9). Fig. 3 Molecular structure of 4-S (ellipsoids at 30% probability) with H atoms on C omitted for clarity. Selected bond lengths (˚A) and angles (): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2 1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9), N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3– P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1– P2–N2 104.35(9). Fig. 4 (a) Expansions of the 1H NMR spectra of 4-R and 4-S in CDCl3; (b) numbering scheme for bornyl ring hydrogens (blue ¼ exo, green ¼ endo) and (c) an expansion of the 31P{1H} NMR spectra of 4-R and 4-S. of the amine is unexpectedly large. While in 4-R the phenyl group projects away from the bornyl group, in 4-S it faces it, tting inside the pocket on the endo face (Fig. 3). Some steric repulsion between the Ph- and tBu-groups is evident (also in 4- R), which involves ‘pushing down’ on the tBu group [the sums of the bond angles at N1 and N2 in 4-S are 359 and 347, respectively]. The structural features seen in 4-R and 4-S would be expected to translate into measurable differences in the NMR spectra of these diastereomers if their solid-state structures were retained in solution. However, at a glance this does not appear to be the case. A comparison of the 1H NMR spectra of 4-R and 4-S in CDCl3 reveals few differences, with the notable exceptions being the methyl resonances of the bornyl group and of the two hydrogens of the bornyl framework in the region d 2.3–2.1 ppm (see Fig. 4a and b). Regarding the chiral amine substituents, the resonance associated with the CH group, centred around d 4.9 ppm, is broadened signicantly in both compounds, while the N–H resonances, centred around d 3.3 ppm, remain as sharp multiplets. © 2022 The Author(s). Published by the Royal Society of Chemistry Edge Article observed in more polar CDCl3. X-ray diffraction revealed that 1 crystallises in the Sohncke space group P21 (consistent with its enantiopurity), with two molecules in the asymmetric unit (Fig. 1). These molecules have essentially identical confor- mations, except for rotation of one tBu group. The P2N2 rings in 1 remain intact, as also observed in the only other solid- state structure of an alkoxy(chloro)phosphazane, Hyp2N2P2(- Cl)OCH(CF3)2 (Hyp ¼ (Me3Si)3Si).30 The P2N2 ring can be described as kite-shaped, which is a result of the different P–N bond lengths and angles at phosphorus associated with the OBorn (P–Nav ¼ 1.689 ˚A; N–P–Nav ¼ 80.3) and Cl groups (P– Nav ¼ 1.732 ˚A; N–P–Nav ¼ 82.9) that arise due to differences in electronegativity. These groups are arranged cis to one another in 1, as is seen in most other cyclodiphosphazanes in the solid state.14 NMR spectroscopy on batches of the crude product showed the only signicant impurities to be starting material, the disubstituted product [tBuNPOBorn]2 (2), and phosphinic acid- type hydrolysis product H(O)P(m-tBuN)2POBorn (3) (see ESI for full characterisation and Fig. S53 and S54† for molecular structures).31,32 Signicantly, for preparative purposes, puri- cation of crude 1 was not necessary. Scheme 4 Synthesis of 4-R and 4-S. Fig. 2 Two of the independent molecules in the asymmetric unit of 4- R, showing different conformations at the bornyl group (see ESI Fig. S55† for ellipsoid representations and selected metric parameters). Fig. 2 Two of the independent molecules in the asymmetric unit of 4- R, showing different conformations at the bornyl group (see ESI Fig. S55† for ellipsoid representations and selected metric parameters). Scheme 4 Synthesis of 4-R and 4-S. © 2022 The Author(s). Published by the Royal Society of Chemistry 5400 | Chem. Sci., 2022, 13, 5398–5412 5400 | Chem. Sci., 2022, 13, 5398–5412 Fig. 3 Molecular structure of 4-S (ellipsoids at 30% probability) with H atoms on C omitted for clarity. Selected bond lengths (˚A) and angles (): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2 1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9), N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3– P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1– P2–N2 104.35(9). Edge Article Fig. 3 Molecular structure of 4-S (ellipsoids at 30% probability) with H atoms on C omitted for clarity. Edge Article Meanwhile, 31P{1H} NMR spectroscopy shows very broad peaks at d 142 and 99 ppm in CDCl3 (Fig. 4c) that suggest that dynamic processes are occurring, such as exo–endo inter- conversion of the substituents (resulting from rotation of the P–O or P–N bonds; the resultant conformers denoted Oexo/Oendo and Nexo/Nendo, respectively) and/or cis–trans isomerism.23 Cooling of 4-R in CD2Cl2 (down to 233 K) only resulted in sharpening of the 31P NMR resonances, and the fact that the chemical shis remained in the region expected for the cis isomer (ESI Fig. S34b†) suggest that endo-exo interconversion is more likely to be responsible for the signal broadening at room temperature.33 Fig. 4 (a) Expansions of the 1H NMR spectra of 4-R and 4-S in CDCl3; (b) numbering scheme for bornyl ring hydrogens (blue ¼ exo, green ¼ endo) and (c) an expansion of the 31P{1H} NMR spectra of 4-R and 4-S. predicted.28b Hence, whilst we believe Oexo–Nendo–Oexo–Nexo interconversion to be the dominant uxional process, cis–trans isomerism cannot be ruled out. In contrast, calculations based on the experimentally observed cis Oexo–Nendo isomer show that rotation of the chiral Ph(Me)CH group in 4-S about the C–N bond, switching the C–H bond from facing the P2N2 ring unit (as in the observed solid-state structure) to away from the P2N2 ring unit, is thermodynamically unfavourable (with an activa- tion energy of around 45.6 kJ mol1) (see ESI Table S7 and Fig. S81†). DFT calculations on 4-S in chloroform indicate that its various conformers possess energies in very close proximity (see ESI Table S6 and Fig. S74–S80†); the three most stable are shown in Fig. 5. Though NMR spectroscopy hints at the pref- erence for cis isomers in solution, and the calculations reveal a small energy difference between the Oexo–Nendo and Oexo–Nexo forms of these, energetically accessible trans isomers are also While these results clearly show measurable differences in the 1H NMR spectra of the diastereomers formed from the © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5401 Chemical Science Edge Article Edge Article Fig. 5 Optimised structures for the three most stable conformers of 4-S and their relative energies compared to the cis Oexo–Nendo conformer (experimentally observed in the solid-state structure). Calculations are at the B3LYP/TZVP level and included an applied polarised continuum model (PCM) using the dielectric constant for chloroform to model the solution structure. Edge Article Analogous isomers of 4-R are depicted in ESI Fig. S82 and S83.† Fig. 5 Optimised structures for the three most stable conformers of Fig. 5 Optimised structures for the three most stable conformers of 4-S and their relative energies compared to the cis Oexo–Nendo conformer (experimentally observed in the solid-state structure). Calculations are at the B3LYP/TZVP level and included an applied polarised continuum model (PCM) using the dielectric constant for chloroform to model the solution structure. Analogous isomers of 4-R are depicted in ESI Fig. S82 and S83.† Fig. 5 Optimised structures for the three most stable conformers of 4-S and their relative energies compared to the cis Oexo–Nendo conformer (experimentally observed in the solid-state structure). Calculations are at the B3LYP/TZVP level and included an applied polarised continuum model (PCM) using the dielectric constant for chloroform to model the solution structure. Analogous isomers of 4-R are depicted in ESI Fig. S82 and S83.† reactions of 1, the small chemical shiseparations encountered in a mixture would lead to overlap of signals and make it impossible to measure enatiomeric excess. Similarly, the oper- ation of uxional processes at ambient temperature eliminates 31P NMR spectroscopy as a viable spectroscopic alternative in these P(III) dimers. Attempts to obtain sharp 31P NMR signals by formation of an internally coordinated lithiate (with Li envis- aged to bridge the N and O atoms) were unsuccessful. However previous reports on the selective methylation of phosphorus atoms in cyclodiphosphazanes using methyl iodide hinted at another option.21b,c Quaternisation has been observed to provide advantages for this type of P/N compound such as improved solubility in polar solvents and even air/water stability. Therefore we reasoned that quaternisation might also suppress the uxional processes observed above and provide a further means of increasing the difference in the chemical shis between the P atoms in diasteromeric pairs. 4-R and 4-S were therefore combined with excess methyl iodide in n- hexane which led to the formation of the salts [{(R)-PhCH(Me) NH}P(Me)(m-NtBu)2POBorn]I (5-R) and [{(S)-PhCH(Me)NH} P(Me)(m-NtBu)2POBorn]I (5-S) (Scheme 5, Fig. 6). Fig. 6 (a) The molecular structure of 5-R (ellipsoids at 30% probability) and with H atoms on C omitted for clarity. Edge Article Selected bond lengths (˚A) and angles (): P1–N1 1.640(3), N1–P2 1.739(3), P2–N2 1.741(3), N2–P1 1.655(3), P1–N3 1.602(3), P2–O1 1.629(2), P1–C27 1.782(4); P1–N1–P2 97.11(16), N1–P2–N2 80.34(14), P2–N2–P1 96.48(15), N1–P1–N2 85.90(15), N1–P1–N3 116.25(17), N2–P1–N3 116.89(15), N1–P2–O1 103.91(14), N2–P2–O1 100.75(13), N1–P1–C27 116.07(17), N2–P1– C27 116.09(17), N3–P1–C27 105.41(18). (b) The molecular structure of 5-S (ellipsoids at 30% probability) and with H atoms on C omitted for clarity. Selected bond lengths (˚A) and angles (): P1–N1 1.646(3), N1–P2 1.755(2), P2–N2 1.731(3), N2–P1 1.640(3), P1–N3 1.600(3), P2–O1 1.627(2), P1–C27 1.781(3); P1–N2–P2 96.37(13), N1–P2–N2 80.06(12), P2–N2–P1 97.54(14), N2–P1–N2 86.03(13), N1–P1–N3 115.37(14), N2–P1–N3 117.40(14), N1–P2–O1 103.46(12), N2–P2–O1 100.54(11), N1–P1–C27 117.55(16), N2–P1–C27 114.83(16), N3–P1–C27 105.45(15). Following recrystallisation from THF, X-ray diffraction reveals that the amino/alkoxide substituents of 5-R and 5-S remain in a cis relationship with respect to each other and conrms that selective reaction of the more electron-rich amine-substituted P atom has occurred in both cases, leaving the O-substituted P and N atom of the chiral amino groups untouched. The orientation of the substituents of the amino groups in 5-R is identical to one of the molecules in 4-R (specically the one depicted in Fig. 2b), and the conformation of 5-S is identical to 4-S. Not unexpectedly, the exocyclic P–N bond lengths associated with the P(V) centre in 5-R (1.602(3) ˚A) and 5-S (1.600(3) ˚A) are all shortened with respect to the cor- responding P(III) centre in 4-R (1.667(5) ˚A [average]) and 4-S (1.671(2) ˚A). While the asymmetry in these cycles does not allow for a direct comparison, it is evident that a shortening of the endocyclic PV–N bonds also occurs to within the range 1.640– 1.655 ˚A (compared with 1.716–1.747 ˚A for 4-R and 4-S). Osten- sibly, due to bond shortening there is an expansion of the Nchiral–P–Nring angles around the newly generated PV centre (range 103.7–108.1 for 4-R/4-S; 115.3–117.4 for 5-R/5-S). Nonetheless, the effect on the exocyclic N/O distance between the amino-N and alkoxide-O atoms is marginal (being ca. 4 ˚A in every case). Both 5-R and 5-S show near linear interactions between the N–H hydrogen atoms and the nearby iodide ions in their lattices, at distances comparable to those reported in the of 5-S is identical to 4-S. © 2022 The Author(s). Published by the Royal Society of Chemistry Edge Article Not unexpectedly, the exocyclic P–N bond lengths associated with the P(V) centre in 5-R (1.602(3) ˚A) and 5-S (1.600(3) ˚A) are all shortened with respect to the cor- responding P(III) centre in 4-R (1.667(5) ˚A [average]) and 4-S (1.671(2) ˚A). While the asymmetry in these cycles does not allow for a direct comparison, it is evident that a shortening of the endocyclic PV–N bonds also occurs to within the range 1.640– 1.655 ˚A (compared with 1.716–1.747 ˚A for 4-R and 4-S). Osten- sibly, due to bond shortening there is an expansion of the Nchiral–P–Nring angles around the newly generated PV centre (range 103.7–108.1 for 4-R/4-S; 115.3–117.4 for 5-R/5-S). Nonetheless, the effect on the exocyclic N/O distance between the amino-N and alkoxide-O atoms is marginal (being ca. 4 ˚A in every case). Both 5-R and 5-S show near linear interactions between the N–H hydrogen atoms and the nearby iodide ions in their lattices, at distances comparable to those reported in the Scheme 5 Selective reaction of 4-R and 4-S with methyl iodide. Scheme 5 Selective reaction of 4-R and 4-S with methyl iodide. © 2022 The Author(s). Published by the Royal Society of Chemistry 5402 | Chem. Sci., 2022, 13, 5398–5412 Edge Article Chemical Science literature for N–H/I contacts (2.65 ˚A for 5-R, 2.68 ˚A for 5-S based on calculated positions; literature value 2.69 ˚A).34 The most important conclusion from this structural analysis is that despite clear changes in bonding within the P2N2 ring, inter- actions between the exocyclic substituents are not signicantly affected by methylation. NMR spectroscopy reveals much more pronounced differ- ences between parent molecules 4-R/4-S and iodide salts 5-R/5-S. In CDCl3, the most noticeable change is the shiof the N–H resonance, from ca. d 3.2 ppm to d 8.4 ppm, which is associated with incorporation of the P(V) centre (Fig. 7a).24a The increased electron withdrawing character of this P atom is also evident from deshielding of the Ph- and Me-group hydrogens. Mean- while, the CH resonances of the Ph(Me)CH groups, formerly broad singlets centred at d 4.94 ppm in 4-R/4-S, transform into clearly dened multiplets, at d 4.65 and 4.68 ppm for 5-R and 5-S, respectively. This was the rst indication that the dynamic processes evident in solutions of 4-R and 4-S are suppressed by methylation in 5-R and 5-S. Edge Article This was reasoned to arise from enhanced N(p) / P–N(s*) donation upon oxidation of the PN(III) centre in 4-R/4-S to PN(V) increasing the barrier to P–N bond rotation. Possibly as result of this, the separation of the inequi- valent 1H NMR resonances of the tBu groups is amplied approximately twofold moving from 4-R/4-S to 5-R/5-S, as the phenyl ring is more rigidly located with respect to each tBu group. The bornyl ring hydrogens still appear at slightly different posi- tions in the new diastereomers but the differences between 5-R and 5-S are no larger than between 4-R and 4-S (Fig. 7b). On the other hand, the bornyl Me-peaks show a more distinctive pattern, whereby Me* (Fig. 7c) appears more deshielded in 5-S (d 0.96 ppm) than in 5-R (d 0.92 ppm), consistent with Me* being posi- tioned further from the shielding Ph-ring. 31P{1H} NMR spectroscopy provides more concrete evidence of the change in the dynamics by showing the disappearance of the broad resonances for 4-R/4-S (ca. d 140 and 100 ppm), which are replaced by sharp doublets, at d 122.7 and 38.8 ppm for 5-R, and d 123.0 and 38.8 ppm for 5-S (Fig. 8a). The signicance of this is that the d 0.3 ppm separation in chemical shiof the O- bound P atom is sufficient to avoid overlap (see Fig. 8b), in turn allowing for accurate integration of the 31P NMR peaks. This provided the necessary proof of principle for the use of cyclo- diphosphazanes as CDAs since integration can be used directly to determine the ee. This is described in more detail later in this section. In the meantime, it is worth noting that the physical characteristics of diastereomers 5-R and 5-S are substantially different. For example, the decomposition temperature of 5-S is some 20 C higher than 5-R. More importantly, from the reac- tion of the roughly 50 : 50 mixture of (S)- and (R)-1-phenyleth- ylamine with 1 followed by in situ quaternisation with methyl iodide, 5-S is selectively crystallised, as veried by X-ray crys- tallography and analysis of the bulk crystalline material by 31P NMR spectroscopy (Fig. 8c and d). The ratio of 5-S and 5-R was found to be 93 : 7. Edge Article Condi- tions: (A) Et3N (2 eq.), THF, 4 h at RT; (B) methyl iodide (3 eq.), THF, 3 h at RT. The reaction was stopped after stages (A) and (B) by removal of the volatiles. Below are shown expansions of the 31P NMR spectra for scalemic mixtures of 1-phenylethylamine. The determined %ee's are given on the right of the spectra, and their predicted ee's based on the ratios of the enantiomers added are shown in parentheses. The ee's predicted for the pure enantiomers are those stated by the supplier (Acros Organics). Edge Article Fig. 9 Method for the in situ derivatisation and analysis of a non- enantiopure amine (in this case 1-phenylethylamine) using 1. Condi- tions: (A) Et3N (2 eq.), THF, 4 h at RT; (B) methyl iodide (3 eq.), THF, 3 h at RT. The reaction was stopped after stages (A) and (B) by removal of the volatiles. Below are shown expansions of the 31P NMR spectra for scalemic mixtures of 1-phenylethylamine. The determined %ee's are given on the right of the spectra, and their predicted ee's based on the ratios of the enantiomers added are shown in parentheses. The ee's predicted for the pure enantiomers are those stated by the supplier (Acros Organics). Fig. 8 Expanded regions of (a) 31P{1H} NMR spectra of 5-R and 5-S; (b) 31P NMR spectra of 5-R and 5-S; (c) an aliquot from a reaction mixture containing roughly equal portions of (R)- and (S)-1-phenylethylamine and (d) the product of this reaction following recrystallization. compositions, with the main error in the measurement of the ee being due to the sensitivity of 31P NMR as well as the presence of trace amounts of either isomer in the commercially-supplied samples of the R- and S-isomers. Having established the ability to distinguish 5-R and 5-S in their isolated form using 31P NMR spectroscopy, we developed an in situ methodology that can be used to measure enantio- meric excess in mixtures of commercial (R)/(S)-1-phenylethyl- amine. Predetermined mixtures of these amines on the 50 mmol scale (yielding sufficient product for NMR spectroscopy) were treated in situ with 1 and then methyl iodide (Fig. 9, top). In all cases, some hydrolysis of 1 was unavoidable (producing 3), however, residual 1 in the reaction mixture indicated that sufficient CDA was available to consume all of the amine added. The results of 31P NMR spectroscopy (Fig. Edge Article 9, bottom) show that the in situ method works well across a range of chiral Edge Article The tendency of these iodide salts to crys- tallise presents a promising avenue for further exploration, since the presence of a heavy atom allows reliable assignment of absolute structure—essential in assessing a reaction's stereoselectivity. Fig. 7 Expansions of the 1H NMR spectra of 4-R, 4-S, 5-R and 5-S, showing (a) the signals of selected bornyl ring hydrogens and (b) tBu groups/Me groups as indicated in (c). Chem. Sci., 2022, 13, 5398–5412 | 5403 © 2022 The Author(s). Published by the Royal Society of Chemistry Fig. 8 Expanded regions of (a) 31P{1H} NMR spectra of 5-R and 5-S; (b) 31P NMR spectra of 5-R and 5-S; (c) an aliquot from a reaction mixture containing roughly equal portions of (R)- and (S)-1-phenylethylamine and (d) the product of this reaction following recrystallization. Chemical Science Having established the ability to distinguish 5-R and 5-S in their isolated form using 31P NMR spectroscopy, we developed compositions, with the main error in the measurement of the ee being due to the sensitivity of 31P NMR as well as the presence of trace amounts of either isomer in the commercially-supplied samples of the R- and S-isomers. Fig. 8 Expanded regions of (a) 31P{1H} NMR spectra of 5-R and 5-S; (b) 31P NMR spectra of 5-R and 5-S; (c) an aliquot from a reaction mixture containing roughly equal portions of (R)- and (S)-1-phenylethylamine and (d) the product of this reaction following recrystallization. Fig. 9 Method for the in situ derivatisation and analysis of a non- enantiopure amine (in this case 1-phenylethylamine) using 1. Condi- tions: (A) Et3N (2 eq.), THF, 4 h at RT; (B) methyl iodide (3 eq.), THF, 3 h at RT. The reaction was stopped after stages (A) and (B) by removal of the volatiles. Below are shown expansions of the 31P NMR spectra for scalemic mixtures of 1-phenylethylamine. The determined %ee's are given on the right of the spectra, and their predicted ee's based on the ratios of the enantiomers added are shown in parentheses. The ee's predicted for the pure enantiomers are those stated by the supplier (Acros Organics). Chemical Science Edge Article Chemical Science Edge Article Chemical Science Chemical Science Edge Article Fig. 9 Method for the in situ derivatisation and analysis of a non- enantiopure amine (in this case 1-phenylethylamine) using 1. Edge Article Chemical Science Chemical Science Table 1 A comparison of the distances between the hydrogen atoms of 6-R/6-S indicated in Fig. 10, based upon the calculated positions in their crystal structures, normalised to average neutron C–H distances Table 1 A comparison of the distances between the hydrogen atoms of 6-R/6-S indicated in Fig. 10, based upon the calculated positions in their crystal structures, normalised to average neutron C–H distances offering a slightly more sterically hindered chiral centre. Though the intermediate phosphazane analogous to 4-R/4-S could not be isolated in pure form from the reactions of R- and (S)-1-phenylpropylamine with 1, in situ reactions of them with methyl iodide proceeded smoothly to give diffraction-quality crystals of [{(R)-PhCH(Et)NH}P(Me)(m-NtBu)2POBorn]I (6-R) and [{(S)-PhCH(Et)NH}P(Me)(m-NtBu)2POBorn]I (6-S). X-ray crystallography demonstrated that 6-R/6-S retain effectively identical conformations to the related salts 5-R/5-S (see ESI Fig. S57 and S58†), with the aromatic group sitting above opposite tBu groups within the pair of diastereomers and hence the C–H group pointing towards the P2N2 ring units. Bearing in mind this similarity it is not surprising that many spectro- scopic features found in 5-R/5-S are also seen in the NMR spectra of 6-R/6-S in CDCl3. Most importantly, two well- separated low-eld multiplets are observed in their 31P NMR spectra, at d 122.6 ppm for 6-R and d 123.1 ppm for 6-S, showing that assessment of the ee will be possible here also. Ph-Hortho/H contact Distance 6-R (˚A) 6-S (˚A) H6 3.91 2.59 H8 2.88 2.11 H9 2.10 2.32 solution, in which the phenyl group and endo face of the bornyl group are next to each other. This is also consistent with the crystallographically determined H/H distances involved Several features of the 1H NMR spectra of diastereomers 6-R and 6-S provide greater insight into their solution-state struc- tures (than was possible for 5-R and 5-S). The rst of these is the emergence of well-dened multiplets for the diastereotopic CH2-hydrogens of the Et group within the chiral Ph(Et)CH- substituent. In the parent amine (R)/(S)-Ph(Et)CHNH2, only one multiplet (a doublet of quintets, at d 1.69 ppm) is seen. However, incorporation into 6-R/6-S results in resolution into two signals, at d 2.30 and 2.09 ppm, suggesting conformational stability. NOESY experiments in CDCl3 reveal weak NOE's for 6- R/6-S between the phenyl group ortho protons and bornyl ring protons (see ESI Fig. S9e†). Solution studies and detection limit Although we had established the use of 1 as a CDA, there were two important questions which we wanted to address, namely, (i) the relationship between solid-state and solution structure, and (ii) the detection limit of our methodology. The rst of these was explored (somewhat fortuitously) using the related amine 1-phenylpropylamine in which the methyl group of 1-phenylethylamine is replaced by an ethyl group, © 2022 The Author(s). Published by the Royal Society of Chemistry 5404 | Chem. Sci., 2022, 13, 5398–5412 Edge Article A cross peak to H6endo is revealed in each case and while for 6-R overlap of H8endo and H9exo precludes rm conclusions regarding the spatial relationship to the amino group, H8endo and H9exo are well-separated for 6-S and there is a clear correlation of H8endo with the phenyl group ortho protons. Taken together with the visible NOEs between H9endo and the ortho-phenyl protons in both 6-R and 6-S, we conclude that their solid-state structures are preserved in Fig. 10 Diagram showing the hydrogen atoms for which NOEs are observed in the cations of 6-R and 6-S. The red-dotted line indicates a likely NOE Fig. 11 The structure of a representative molecule from the asym- metric unit of 8-R (ellipsoids at 30% probability). H atoms on C are omitted for clarity. Selected bond lengths (˚A) and angles (): P1B–N1B 1.635(6), N1B–P2B 1.748(5), P2B–N2B 1.721(6), N2B–P1B 1.642(5), P1B–N3B 1.609(6), P2B–O1B 1.613(5), P1B–C28B 1.780(7); P1B–N1B– P2B 96.5(3), N1B–P2B–N2B 80.2(3), P2B–N2B–P1B 97.3(3), N2B– P1B–N1B 85.9(3), N1B–P1B–N3B 116.3(3), N2B–P1B–N3B 117.0(3), N1B–P1B–C28B 116.2(3), N2B–P1B–C28B 115.7(3), N3B–P1B–C28B 105.5(3), N1B–P2B–O1B 104.3(2), N2B–P2B–O1B 100.4(3). (b) The structure of a representative molecule from the asymmetric unit of 8-S (ellipsoids at 30% probability). H atoms on C are omitted for clarity. Selected bond lengths (˚A) and angles (): P1B–N1B 1.639(6), N1B–P2B 1.764(6), P2B–N2B 1.733(6), N2B–P1B 1.637(6), P1B–N3B 1.608(6), P2B–O1B 1.614(6), P1B–C28B 1.781(8); P1B–N1B–P2B 95.9(3), N1B– P2B–N2B 80.0(3), P2B–N2B–P1B 97.2(3), N1B–P1B–N3B 115.3(4), N2B–P1B–N3B 117.0(4), N1B–P1B–C28B 116.9(4), N2B–P1B–C28B 115.4(4), N3B–P1B–C28B 105.4(4), N1B–P2B–O1B 104.2(3), N2B– P2B–O1B 100.3(4). Fig. 10 Diagram showing the hydrogen atoms for which NOEs are observed in the cations of 6-R and 6-S. The red-dotted line indicates a likely NOE. Fig. 10 Diagram showing the hydrogen atoms for which NOEs are observed in the cations of 6-R and 6-S. The red-dotted line indicates a likely NOE. Fig. 10 Diagram showing the hydrogen atoms for which NOEs are observed in the cations of 6-R and 6-S. The red-dotted line indicates a likely NOE. © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5405 Chemical Science Edge Article (Fig. 10 and Table 1). This conclusion is important because it shows that there are no signicant uxional processes occur- ring which would alter the spatial arrangement between the OBorn and chiral substituents. explicitly by the crystal structures of the racemic compounds 7- R/S and 8-R/S, which show disorder indicative of a solid solution of the two diastereomers. If this situation should be retained in solution, we would expect the NMR spectra of the diastereomers of 8-R and 8-S to show much smaller differences compared to the previous examples 5 and 6. Ultimately, the utility of our protocol will be limited by the peak separation of the 31P NMR resonances of R- and S- analytes and therefore the ability to integrate accurately. In order to explore this, we next considered 2-phenylpropylamine, PhCH(Me)CH2NH2, in which the chiral centre is separated by a CH2 group from the amine functionality (anticipating a reduction in peak separation). The previous protocol was used, in which 1 was prepared without purication and subse- quently reacted with 2-phenylpropylamine in the presence of a Brønsted base (NEt3) to give the cyclodiphosph(III)azane intermediates (R)-PhCH(Me)CH2NHP(m-tBuN)2POBorn (7-R) and (S)-PhCH(Me)CH2NHP(m-tBuN)2POBorn (7-S) with the pure enantiomers and (R/S)-PhCH(Me)CH2NHP(m-tBuN)2POBorn (7- R/S) with racemic 2-phenylpropylamine. Further treatment of crude intermediates 7-R, 7-S, 7-R/S with methyl iodide and recrystallization from toluene afforded excellent crystals of [{(R)- PhCH(Me)CH2NH}(Me)P(m-tBuN)2POBorn]I (8-R), [{(S)- PhCH(Me)CH2NH}(Me)P(m-tBuN)2POBorn]I (8-S) and [{(R/S)- PhCH(Me)CH2NH}(Me)P(m-tBuN)2POBorn]I (8-R/S). The solid- state structures of 7-R, 7-S, 7-R/S, 8-R, 8-S and 8-R/S (ESI Fig. S60–S64†) were obtained. The structures of 8-R and 8-S (one of the crystallographically independent molecules) are shown in Fig. 11. The 1H NMR spectra of 8-R and 8-S are indeed very similar (see ESI Fig. S13a, S14a and S15a†). Importantly, however, very sharp 31P{1H} NMR signals are still observed (at d 124.3 and 45.7 ppm for 8-R and d 124.2 and 45.7 ppm for 8-S; Fig. 12). This can be compared to 7-R and 7-S which show two broad peaks at d 138 and 103 ppm for both. 31P{1H} NMR spectroscopy gave sufficient line separation for integration of the PN peaks at ca. d 46 ppm for the two diastereomers in racemic 8-R/S, with the best peak resolution obtained by inverse-gated decoupling (with recycle delays $30 s, 30 pulse) and this was additionally required in order to obtain reliable integration values. Fig. Chemical Science 13 shows the determination of the ee's of mixtures of the R- and S-isomers of 2-phenylpropylamine using our in situ protocol. It is clear from these data that the measured and predicted values are reasonably close to the values determined Fig. 13 Determination of the ee's for mixture of 2-phenylpropylamine using 1 The conditions are the same as those given in the caption to Fig. 9. The determined %ee's are shown on the right, and their pre- dicted ee's based on the ratios of the enantiomers added are given in parentheses. The ee's of the pure enantiomers and racemic amine were determined independently by chiral HPLC (since these were not stated by the supplier, Sigma). The amines were converted into amides by reaction with acetic anhydride for this purpose. There is an esti- mated error of up to 2% in the determined ee's due to poor separation on the column. The other predicted values (1 : 3 and 3 : 1) were calculated based on the ee's of the ‘pure’ enantiomers determined from HPLC (so an error of up to ca 2% applies to these values too). Notably, the crystal structures of the diastereomers are iso- structural for both 7-R/7-S and 8-R/8-S, showing that the molecular conformation (in the solid state) is effectively invariant for the two diastereomers in each case. The R or S conguration at the chiral centre of the amine is accommo- dated with no change in conformation for the remainder of the molecule, and in particular no change to the orientation of the bornyl group. The molecular similarity is demonstrated Fig. 12 Expansions of the 31P{1H} NMR spectra (CDCl3) of 8-R and 8-S. Fig. 13 Determination of the ee's for mixture of 2-phenylpropylamine using 1 The conditions are the same as those given in the caption to Fig. 9. The determined %ee's are shown on the right, and their pre- dicted ee's based on the ratios of the enantiomers added are given in parentheses. The ee's of the pure enantiomers and racemic amine were determined independently by chiral HPLC (since these were not stated by the supplier, Sigma). The amines were converted into amides by reaction with acetic anhydride for this purpose. There is an esti- mated error of up to 2% in the determined ee's due to poor separation on the column. Substrate scope This is particularly noticeable in the case of 13 in which the conformational exibility present in the n-butyl group results in the smallest PO and PN peak separation between the diastereomers. (ii) The poorest peak separations are seen in 8, 12, 13 and 17 in which there is apparently the greatest degree of conforma- tional exibility in the aliphatic groups of the primary amines. This is particularly noticeable in the case of 13 in which the conformational exibility present in the n-butyl group results in the smallest PO and PN peak separation between the diastereomers. (iii) Perhaps unsurprisingly bearing in mind the mild conditions and reagents involved, there appears to be good functional group tolerance across the spectrum of primary amines investigated, especially in the case of amino acid esters which bear greater chemical functionality (15–17). (iv) There is a noticeably strong correlation between the PO chemical shiand the absolute conguration for the eleven a- amines explored (i.e. 5, 6, 9–17). With the exception of 13 (where dS z dR), the PO chemical shiof the S-isomer is always higher than for the R-isomer (dS > dR). It can be noted that the b-amine 2-phenylpropylamine (8) does not t the trend. In contrast, the PN resonances do not exhibit any such clear correlation; 5, 8, 9, 11, 12, 13 have higher PN chemical shis for the R-isomer (dR > dS) whereas the opposite holds for 6, 14, 15, 16, 17 (dS > dR), and only for 10 dS z dR. (iv) There is a noticeably strong correlation between the PO chemical shiand the absolute conguration for the eleven a- amines explored (i.e. 5, 6, 9–17). With the exception of 13 (where dS z dR), the PO chemical shiof the S-isomer is always higher than for the R-isomer (dS > dR). It can be noted that the b-amine 2-phenylpropylamine (8) does not t the trend. In contrast, the PN resonances do not exhibit any such clear correlation; 5, 8, 9, 11, 12, 13 have higher PN chemical shis for the R-isomer (dR > dS) whereas the opposite holds for 6, 14, 15, 16, 17 (dS > dR), and only for 10 dS z dR. Our reagent 1 can therefore be used to determine the abso- lute conguration of chiral a-amines, with the PO chemical shi being a reliable indicator. Edge Article discussed 5, 6 and 8) are summarised in Table 2 which also indicates whether 31P or 31P{1H} NMR provide sufficient sepa- ration of the resonances for measurement of the ee. by chiral HPLC (bearing in mind the estimated ca 2% error in these values due to poor separation of the acetyl amide on the column). However, the extent to which the overall molecular shape is affected by changing the chiral centre in the amine from R to S probably makes our method less suitable for amines in which the chiral substituent is remote from the amine functionality. Products 9–11 were also prepared using our in situ method outlined in Fig. 8 (see Scheme 7) to conrm its applicability over a larger number of substrates. The resulting 31P NMR spectra starting from racemic and 2 : 1 R : S scalemic mixtures of 9–11 are well resolved and appended in the ESI (Fig. S37–S39, S52†). The assignment thereof is supported by the spectra of the cor- responding enantiopure compounds prepared individually. Substrate scope The most important condition that must be met to apply our methodology for measuring ee is adequate line separation of the 31P NMR signals. In order to establish the scope of the method, a greatly expanded range of commercially available chiral a-primary amines incorporating a variety of functional groups and steric characteristics was explored to establish if there are sufficiently large chemical shiseparations of the 31P NMR resonances (Scheme 6). The eighteen new compounds (9– 17, -R and -S) were prepared starting from enantiopure amines and fully characterised, including the single-crystal X-ray structures of 10-R, 10-S, 12-R, 12-S, 13-R and 13-S. Full details of the spectroscopic analyses and characterisation of 9–17 are included in the ESI. Of particular interest are the expansions of 31P{1H} NMR spectra showing the peak separation between products with the pairs of enantiomeric substrates (ESI Fig. S43–S51†). The results (including those for previously The major conclusion that can be drawn from the data shown in Table 2 is that 1 can be used to discriminate between all of the chiral amines studied, although the small values of Dd for both PO and PN in the cases of 8, 12 and 13 would lead to less accurate determination of ee. In these cases the use of an inverse gated 31P{1H} decoupling routine is, however, found to provide sufficient peak resolution for these substrates. While the inherent difficulties in obtaining accurate ee's using the integration method limit the sensitivity of the method for 31P NMR spectroscopy, the sensitivity is clearly more than adequate to provide a quick assessment of chiral purity on a convenient scale and in a straightforward manner (using the PO or PN signals). There are four other conclusions that we can draw from these data: (i) the presence of aromatic substituents on the primary amine is not required to promote signicant chiral inequiva- lence; rather, the origin of the separation of the PO and PN resonances of the R and S diastereomers is probably steric/ spatial in origin. S h 6 S th i f 9 17 R/S Chi l t i th i (ii) The poorest peak separations are seen in 8, 12, 13 and 17 in which there is apparently the greatest degree of conforma- tional exibility in the aliphatic groups of the primary amines. Chemical Science The other predicted values (1 : 3 and 3 : 1) were calculated based on the ee's of the ‘pure’ enantiomers determined from HPLC (so an error of up to ca 2% applies to these values too). Fig. 12 Expansions of the 31P{1H} NMR spectra (CDCl3) of 8-R and 8-S. 5406 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Chemical Science Substrate scope This is important because, uniquely, this can be done at the same time as the determination of the ee in a single 31P NMR experiment of a scalemic mixture. Scheme 6 Synthesis of 9–17-R/S. Chiral centre in the amino groups marked *. © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5407 Chemical Science Edge Article Edge Article Edge Article Table 2 A summary of the 31P NMR data, separation of low-field (PO) and high-field (PN) signals, and an indication of the suitability of 31P NMR for measuring ee (with and without decoupling) Table 2 A summary of the 31P NMR data, separation of low-field (PO) and high-field (PN) signals, and an indication of the suitability of 31P NMR for measuring ee (with and without decoupling) Substrate (compound number) PO (d ppm) PN (d ppm) DPO DPN Suitability for ee measurement 31P 31P{1H} 123.0 (S) 38.8 (R/S) 0.33 0.03 Good Good 122.7 (R) dR > dS 123.1 (S) 38.9 (S) 0.47 0.04 Good Good 122.6 (R) 38.8 (R) 124.3 (R/S) 45.7 (R/S) 0.09 0.09 Poor Medium dR > dS dR > dS 122.9 (S) 39.2 (R/S) 0.34 0.07 Good Good 122.6 (R) dR > dS 122.5 (S) 41.2 (R/S) 0.22 0 Poor Good 122.2 (R) dR z dS 123.7 (S) 41.9 (S) 0.16 0.10 Poor Good 123.5 (R) 42.0 (R) 122.4 (R/S) 41.2 (R/S) 0.01 0.04 Poor Medium dS > dR dR > dS 122.2 (R/S) 40.7 (R/S) 0 0.07 Poor Good dS z dR dR > dS 121.8 (S) 39.2 (R/S) 0.52 0.02 Good Good 121.3 (R) dS > dR Substrate (compound number) 5408 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry © 2022 The Author(s). Published by the Royal Society of Chemistry Table 2 (Contd.) Edge Article Chemical Science Edge Article Chemical Science Chemical Science Substrate (compound number) PO (d ppm) PN (d ppm) DPO DPN Suitability for ee measurement 31P 31P{1H} 124.1 (S) 44.2 (S) 0.39 0.28 Good Good 123.7 (R) 43.9 (R) 123.4 (S) 42.3 (S) 0.30 0.67 Good Good 123.1 (R) 41.6 (R) 123.9 (R/S) 43.5 (S) 0.02 0.16 Poor Good dS > dR 43.4 (R) A clue to the probable feature inuencing the PO chemical shis is provided by the numerous snapshots of the molecular conformation established within the crystal structures. Substrate scope Since several of the structures show more than one independent molecule and/or disorder of the bornyl group, more than 30 such snapshots are available (see ESI Section 4†). The orienta- tion of the bornyl group with respect to the P2N2 ring is described by the P/P–O–CBorn (s1) and P–O–CBorn–C(Me) (s2) torsion angles, as shown in Fig. 14a. A scatterplot of s1 vs. s2 (ESI, Fig. S73†) shows an overall broad spread of points (indi- cating that the bornyl conformation is not inherently “locked”), but with a prominent cluster comprising 14 points centred at s1 z 157, s2 z 120, which corresponds to the bornyl confor- mation shown in Fig. 2b and 6a. The cluster includes at least one molecule from all characterised compounds showing the Oexo conformation, which indicates that it is likely to represent (at least a local) energy minimum for the orientation of the bornyl group. Although the snapshots are taken from the crystal structures, while the PO resonances are measured in solution, any conformation observed in the solid state must be intrinsi- cally accessible in solution, while a conformation not observed in the solid state is not proven to be. Considering the quater- nised compounds, both the R- and S- diastereomers are seen within the cluster for 8, 12 and 13, and little difference is seen between dS and dR in the 31P NMR. For 5, 6 and 10, however, the R-diastereomer is seen within the cluster but the S-diasteromer is not, and there is a clear distinction between dS and dR in the 31P NMR. Hence, we conclude that discrimination between dS and dR for the PO resonance arises when the S-substrate blocks access to the low-energy bornyl conformation. The sharpness of the signals results from conformational locking of the bornyl group, while the difference between dS and dR indicates the extent of the difference between the two locked conformations. For amines where both the R- and the S-substrate permit access to the same bornyl conformation, the discrimination will be reduced. The observed conformational differences are interpreted to be steric in origin. The ‘Me-side’ of the bornyl group appears to have a large steric inuence, with the most sterically demanding group of the amine (R2 in Fig. 14b) being diagonally opposite to Scheme 7 In situ formation of 9–11-R/S. Chiral centre in the amines marked *. © 2022 The Author(s). Conclusions 14 (a) Defining the P/P–O–CBorn (s1) and P–O–CBorn–C(Me) (s2) torsion angles; (b) diagrammatic representation of the solid-state structures of R- and S-isomers (in which the O atoms are all sp2 hybridised, consistent with the X-ray structures which show C–O–P angles close to 120), showing the orientation of the lone pair in p and sp2 orbitals on the O atom. The blue circles are used to highlight the relative steric sizes of the groups. The steric interactions between the two ends of the molecule are mediated through the t-butyl groups, which show rotational variation. the Me-side in the S-isomer, but adjacent in the R-isomer. The distance between bornyl and amine is too large to have a direct effect, but the steric interaction is mediated by the tert-butyl group, which changes its orientation in the R- and S- diaste- reomers. The effect is clearest in 5 and 6, where the tert-butyl groups show an eclipsed conformation in the R- but a staggered conformation in the S-diastereomer. Substrate scope Published by the Royal Society of Chemistry © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5409 Edge Article Chemical Science Fig. 14 (a) Defining the P/P–O–CBorn (s1) and P–O–CBorn–C(Me) (s2 Conclusions In summary, we have developed a simple inorganic chiral derivatisation agent based on a cyclodiphosphazane, which can be used to detect chirality and measure enantiomeric excess in chiral amines and employs a cheap chiral auxiliary. A two-step process, involving the substitution of the P–Cl group with the amine, followed by selective quaternisation of one of the P centres gives diastereomeric iodide salts of the type [(R*NH)(Me)P(m-NtBu)2POBorn]I, where R* is a chiral group and OBorn is the chiral ancillary. 31P NMR spectroscopy proves to be particularly useful for measuring the ratio of diastereomers produced from scalemic mixtures, with the small but useable separations in chemical shis of diastereomeric pairs. The origin of the effect is the steric inuence of the chiral amine on the orientation of the bornyl group. Though the method does not give highly accurate measures of enantiomeric excess, it is nonetheless sensitive enough to provide a rapid assay of chiral composition. Screening a range of commercial chiral amines indicates that measurement of ee is possible by integration of the PO and/or PN 31P/31P{1H} NMR resonances. The method is most suited to a-chiral amines in which greatest separation of the PO and PN resonances occurs, but can also be applied to b- chiral amines. Using 31P NMR spectroscopy allows detection of chiral derivatives in relatively small amounts (suitable for NMR assays) without any signicant interference of side products with the signals of interest. Finally, the relative values of the PO chemical shis are diagnostic of the absolute conguration of a-alkyl-amines, so that our CDA performs all three tasks; detection of chirality, measurement of ee and determination of absolute conguration; in one simple experiment. This contrasts with previously developed organic CDAs (like Mosher's acid) where two separate experiments are required to determine ee's and absolute conguration.11a Clearly, our CDA design concept is general, and better selectivity and more accurate measurement of ee's is likely to be possible using other chiral auxiliaries and/or P2N2 ring substituents. Work on rening these systems and on extending the scope to chiral alcohols and carboxylic acids is underway. Fig. Conflicts of interest There are no conicts to declare. Conflicting interests DFT (B3LYP/TZVP) geometry optimisations of 4-R, 4-S and 5-R in CHCl3 reproduce their solid-state structures closely (Fig. S74, S82 and S84, ESI†), with calculated CBorn–O–P/P dihedrals being very similar to those found experimentally. A survey of the frontier orbitals of 5-R and 5-S does not indicate any donation of the O-lone pairs into the s* orbital of the ring P–N bonds, and NBO analysis shows that the charges on the principal atoms of both are almost identical (ESI, Table S8†). Given the above, we conclude that the most likely origin of the difference in the chemical shiarising from a change in orientation of the bornyl group is the relative orientation of the O- and P-lone pairs.35 The authors declare no conicting interests. References 1 D. Leung, S. O. Kang and E. V. Anslyn, Chem. Soc. Rev., 2012, 41, 448–479. 2 (a) L. A. Joyce, M. S. Maynor, J. M. Dragna, G. M. da Cruz, V. M. Lynch, J. W. Canary and E. V. Anslyn, J. Am. Chem. Soc., 2011, 133, 13746–13752; (b) S. Nieto, V. M. Lynch, E. V. Anslyn, H. Kim and J. Chin, J. Am. Chem. Soc., 2008, 130, 9232–9233. 22 M. M. Siddiqui, S. M. Mobin, I. Senkovska, S. Kaskel and M. S. Balakrishna, Chem. Commun., 2014, 50, 12273–12276. 23 (a) K. V. Axenov, V. Kotov, M. Klinga, M. Leskel¨a and T. Repo, Eur. J. Inorg. Chem., 2004, 695–706; (b) S. Mohanty and M. S. Balakrishna, J. Chem. Sci., 2010, 122, 137–142; (c) R. Rama Suresh and K. C. Kumara Swamy, Tetrahedron Lett., 2009, 50, 6004–6007; (d) T. Roth, H. Wadepohl, D. S. Wright and L. H. Gade, Chem. - Eur. J., 2013, 19, 13823–13837. 3 E. Badaloni, W. Cabri, A. Ciogli, R. Deias, F. Gasparrini, F. Giorgi, A. Vigevani and C. Villani, Anal. Chem., 2007, 79, 6013–6019. 4 T. J. Wenzel, Discrimination of Chiral Compounds Using NMR Spectroscopy, Wiley-VCH, Weinheim, 2007. 24 (a) D. 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J., 2021, 27, 289–297. 6 D. Parker, Chem. Rev., 1991, 91, 1441–1457. 27 P. Rajasekar, C. Data availability Synthetic procedures for all new compounds, their character- isation and NMR data can be found in the ESI.† Crystallo- graphic data for 1, 2, 3, 4-R, 4-S, 5-R, 5-S, 6-R, 6-S, 7-R, 7-S, 7-R/S, 8-R, 8-S, 8-R/S, 10-R, 10-S, 12-R, 12-S, 13-R, 13-S have been deposited at the CCDC (deposition numbers 2105705, 2105711, 2105707, 2105709, 2105718, 2105714, 2105706, 2105708, 2105717, 2105710, 2105715, 2105712, 2105716, 2105713, O- and P-lone pairs.35 5410 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article 2105719, 2157896, 2157893, 2157895, 2157894, 2157891, 2157892, respectively) and can be obtained on request. Full coordinates of DFT optimised structures are also available as a separate data le in the ESI.† 9 P. Lesot, C. Aroulanda, H. Zimmermann and Z. Luz, Chem. Soc. Rev., 2015, 44, 2330–2375. 10 J. A. Dale, D. L. Dull and H. S. Mosher, J. Org. Chem., 1969, 34, 2543–2549. 11 (a) T. R. Hoyle, C. S. Jeffrey and F. Shao, Nat. Protoc., 2007, 2, 2451–2458; (b) D. A. Allen, A. E. Tomaso Jr., O. P. Priest, D. F. Hindson and J. L. Hurlburt, J. Chem. Educ., 2008, 85, 698–700. Acknowledgements A. J. Peel thanks the Leverhulme Trust (grant RPG-2017-146) for funding. R. B. Jethwa would like to thank the U.K. EPSRC and the Royal Dutch Shell plc. for an I-Case (EP/R511870/1). J. Slaughter thanks the Faraday Institution (grant numbers FIRG001 and FIRG024) for funding. We thank Duncan Howe for assistance with NMR spectroscopy. Calculations were carried out on the in-house Odyssey HPC cluster and the authors are grateful for the calculation time used therein. We also thank Dr Robert Phipps and Robert Pearce-Higgins for running chiral HPLC on one of the amines. 17 A. J. Plajer, R. Garc´ıa-Rodr´ıguez, C. G. M. Benson, P. D. Matthews, A. D. Bond, S. Singh, L. H. Gade and D. S. Wright, Angew. Chem., Int. Ed., 2017, 56, 9087–9090. 18 H.-C. Niu, A. J. Plajer, R. Garc´ıa-Rodr´ıguez, S. Singh and D. S. Wright, Chem. - Eur. J., 2018, 24, 3073–3082. 19 A. J. Plajer, J. Zhu, P. Proehm, A. D. Bond, U. F. Keyser and D. S. Wright, J. Am. Chem. Soc., 2019, 141, 8807–8815. 20 A. J. Plajer, S. Lee, A. D. Bond, J. M. Goodman and D. S. Wright, Dalton Trans., 2020, 49, 3403–3407. 21 For examples see(a) D. Suresh, M. S. Balakrishna and J. T. Mague, Dalton Trans., 2008, 3272–3274; (b) C. Ganesamoorthy, J. T. Mague and M. S. Balakrishna, Eur. J. Inorg. Chem., 2008, 596–605; (c) P. Chandrasekaran, J. T. Mague and M. S. Balakrishna, Dalton Trans., 2009, 5478–5486; (d) M. M. Siddiqi, J. T. Mague and M. S. Balakrishna, Inorg. Chem., 2015, 54, 1200–1202. Author contributions A. J. P. and A. T. undertook experimental work, which was supervised by A. J. P. and J. S.; R. B. J. performed DFT calcula- tions; A. J. P., A. T. and D. C. undertook NMR studies; H.-C. N. aided in experimental work; A. D. B. carried out X-ray crystal- lography and analysis of the solid-state structures; D. S. W. and A. J. P. conceived the original idea. All authors were involved in the writing of the paper. 12 R. Garc´ıa-Rodr´ıguez, S. Hanf, A. D. Bond and D. S. Wright, Chem. Commun., 2017, 53, 1225–1228. 13 Y.-R. Luo, Comprehensive Handbook of Chemical Bond Energies, CRC Press: Boca Raton, 1st edn, 2007. 14 M. S. Balakrishna, Dalton Trans., 2016, 45, 12252–12282. 15 O. J. Scherer and P. Klusmann, Angew. Chem., Int. Ed., 1969, 8, 752–753. 16 A. Bashall, E. L. Doyle, C. Tubb, S. J. Kidd, M. McPartlin, A. D. Woods and D. S. Wright, Chem. Commun., 2001, 2542–2543. References Jose, M. Sarkar and R. Boomishankar, Angew. Chem., Int. Ed., 2020, 60, 4023–4027. 7 G. Storch, M. Siebert, F. Rominger and O. Trapp, Chem. Commun., 2015, 51, 15665–15668. 28 (a) I. Silaghi-Dumitrescu and I. Haiduc, Phosphorus Sulfur Relat. Elem., 1994, 91, 21; (b) L. Stahl, Coord. Chem. Rev., 2000, 210, 203. 8 M. D. McCreary, D. W. Lewis, D. l. Wernick and G. M. Whitesides, J. Am. Chem. Soc., 1974, 96, 1038–1054. Chem. Sci., 2022, 13, 5398–5412 | 5411 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article 29 F. J. Devlin and P. J. Stephens, J. Org. Chem., 2005, 70, 2980– 2993. 33 A. Tarrasoli, M. L. Thompson, R. C. Haltiwanger, T. G. Hill and A. D. Norman, Inorg. Chem., 1988, 27, 3382–3386. 33 A. Tarrasoli, M. L. Thompson, R. C. Haltiwanger, T. G. Hill and A. D. Norman, Inorg. Chem., 1988, 27, 3382–3386. 34 T. Steiner, Acta Crystallogr., Sect. B: Struct. Sci., 1998, 54, 456– 463. 34 T. Steiner, Acta Crystallogr., Sect. B: Struct. Sci., 1998, 54, 456– 463. 30 M. Kurpat, R. Kuzora, M. Lehman, A. Shultz, A. Villinger and R. Wustrak, J. Organomet. Chem., 2010, 695, 1006–1011. 35 Few 31P NMR studies have identied lone pair orientation as an inuence on chemical shi; a notable example is that of diphosphanes—vide J. P. Albrand and C. Ta¨ıeb, Phosphorus Chemistry: Proceedings of the 1981 International Conference, ACS Symp. Ser. 171, ed. L. D. Quin and J. G. Verkade, American Chemical Society, Washington, DC, 1981. 35 Few 31P NMR studies have identied lone pair orientation as an inuence on chemical shi; a notable example is that of diphosphanes—vide J. P. Albrand and C. Ta¨ıeb, Phosphorus Chemistry: Proceedings of the 1981 International Conference, ACS Symp. Ser. 171, ed. L. D. Quin and J. G. Verkade, American Chemical Society, Washington, DC, 1981. 35 Few 31P NMR studies have identied lone pair orientation as an inuence on chemical shi; a notable example is that of diphosphanes—vide J. P. Albrand and C. Ta¨ıeb, Phosphorus Chemistry: Proceedings of the 1981 International Conference, ACS Symp. Ser. 171, ed. L. D. Quin and J. G. Verkade, American Chemical Society, Washington, DC, 1981. 31 E. L. Doyle, F. Garc´ıa, S. M. Humphrey, R. A. Kowenicki, L. R´ıera, A. D. Woods and D. S. Wright, Dalton Trans., 2004, 807–812. 5412 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry 34 T. Steiner, Acta Crystallogr., Sect. B: Struct. Sci., 1998, 54, 456– 463. 33 A. Tarrasoli, M. L. Thompson, R. C. Haltiwanger, T. G. Hill and A. D. Norman, Inorg. Chem., 1988, 27, 3382–3386. 34 T. Steiner, Acta Crystallogr., Sect. B: Struct. Sci., 1998, 54, 456– 463. 35 Few 31P NMR studies have identied lone pair orientation as an inuence on chemical shi; a notable example is that of diphosphanes—vide J. P. Albrand and C. Ta¨ıeb, Phosphorus Chemistry: Proceedings of the 1981 International Conference, ACS Symp. Ser. 171, ed. L. D. Quin and J. G. Verkade, American Chemical Society, Washington, DC, 1981. References 32 This was identied by the presence of a doublet of doublets in the 31P NMR spectrum (in CDCl3) at, d ¼ 7.0 ppm, with 1JP-H ¼ 583 Hz and 2JP-P ¼ 10 Hz. 5412 | Chem. Sci., 2022, 13, 5398–5412
https://openalex.org/W2127431419
https://discovery.ucl.ac.uk/id/eprint/1378611/1/1378611.pdf
English
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A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data
Bioinformatics
2,012
cc-by
8,480
ABSTRACT Motivation: The Illumina Infinium 450 k DNA Methylation Beadchip is a prime candidate technology for Epigenome-Wide Association Studies (EWAS). However, a difficulty associated with these beadarrays is that probes come in two different designs, characterized by widely differ- ent DNA methylation distributions and dynamic range, which may bias downstream analyses. A key statistical issue is therefore how best to adjust for the two different probe designs. Results: Here we propose a novel model-based intra-array normal- ization strategy for 450 k data, called BMIQ (Beta MIxture Quantile dilation), to adjust the beta-values of type2 design probes into a stat- istical distribution characteristic of type1 probes. The strategy involves application of a three-state beta-mixture model to assign probes to methylation states, subsequent transformation of probabilities into quantiles and finally a methylation-dependent dilation transformation to preserve the monotonicity and continuity of the data. We validate our method on cell-line data, fresh frozen and paraffin-embedded tumour tissue samples and demonstrate that BMIQ compares favour- ably with two competing methods. Specifically, we show that BMIQ improves the robustness of the normalization procedure, reduces the technical variation and bias of type2 probe values and successfully eliminates the type1 enrichment bias caused by the lower dynamic range of type2 probes. BMIQ will be useful as a preprocessing step for any study using the Illumina Infinium 450 k platform. Motivation: The Illumina Infinium 450 k DNA Methylation Beadchip is a prime candidate technology for Epigenome-Wide Association Studies (EWAS). However, a difficulty associated with these beadarrays is that probes come in two different designs, characterized by widely differ- ent DNA methylation distributions and dynamic range, which may bias downstream analyses. A key statistical issue is therefore how best to adjust for the two different probe designs. Results: Here we propose a novel model-based intra-array normal- ization strategy for 450 k data, called BMIQ (Beta MIxture Quantile dilation), to adjust the beta-values of type2 design probes into a stat- istical distribution characteristic of type1 probes. The strategy involves application of a three-state beta-mixture model to assign probes to methylation states, subsequent transformation of probabilities into quantiles and finally a methylation-dependent dilation transformation to preserve the monotonicity and continuity of the data. We validate our method on cell-line data, fresh frozen and paraffin-embedded tumour tissue samples and demonstrate that BMIQ compares favour- ably with two competing methods. *To whom correspondence should be addressed ABSTRACT Specifically, we show that BMIQ improves the robustness of the normalization procedure, reduces the technical variation and bias of type2 probe values and successfully eliminates the type1 enrichment bias caused by the lower dynamic range of type2 probes. BMIQ will be useful as a preprocessing step for any study using the Illumina Infinium 450 k platform. A key statistical issue with the Illumina 450k beadchip is that probes come in two different designs, which causes the methyla- tion values derived from these two designs to exhibit widely dif- ferent distributions (Dedeurwaerder et al., 2011). Indeed, type2 probes are typically characterized by a much lower dynamic range compared with type1 probes, even after adjustment for dif- ferences in biological characteristics such as CpG density (Dedeurwaerder et al., 2011). Comparison with bisulphite pyrose- quencing data further showed that type2 probe values are biased and generally less reproducible (Dedeurwaerder et al., 2011). To correct for this bias, a peak-based correction (PBC) method was proposed (Dedeurwaerder et al., 2011) which normalises type2 design probes so as to render them comparable with type1 probes. Making the statistical distributions of type1 and type2 probes comparable is important for several reasons. Not doing so may introduce an enrichment bias towards type1 probes when ranking probes in supervised analyses, as the dynamic range of type1 probes is significantly higher. Moreover, methods that seek to determine differentially methylated regions (Jaffe et al., 2012) also assume that probes within these regions are comparable and thus one would want to avoid any sources of technical variation within them. Finally, one would wish to apply unsupervised di- mensional reduction algorithms (Houseman et al., 2008; Koestler et al., 2010) and classification algorithms (Zhuang et al., 2012) to one single dataset, and not separately to two different assays. Availability: BMIQ is freely available from http://code.google. com/p/bmiq/. Contact: a.teschendorff@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online Supplementary information: Supplementary data are available at Bioinformatics online Received on June 27, 2012; revised on October 9, 2012; accepted on November 16, 2012 Received on June 27, 2012; revised on October 9, 2012; accepted on November 16, 2012  The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data Andrew E. Teschendorff1,*, Francesco Marabita2, Matthias Lechner3, Thomas Bartlett1, Jesper Tegner2, David Gomez-Cabrero2 and Stephan Beck3 A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data Andrew E. Teschendorff1,*, Francesco Marabita2, Matthias Lechner3, Thomas Bartlett1, Jesper Tegner2, David Gomez-Cabrero2 and Stephan Beck3 p g , p 1Statistical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK, 2Department of Medicine, Unit of Computational Medicine, Centre for Molecular Medicine, Karolinska Institute, Solna 171 76, Stockholm, Sweden and 3Medical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK Associate Editor: Olga Troyanskaya (Baylin and Ohm, 2006; Feinberg et al., 2006; Jones and Baylin, 2007). One particular epigenetic mark of interest is DNA methy- lation. Indeed, DNA methylation markers have been proposed as early detection, diagnostic and prognostic markers in a wide range of different diseases (Rakyan et al., 2011). Underpinning this increased interest in epigenomics are significant advances in bea- darray technology, which now allow routine measurement of DNA methylation at over thousands of CpG dinucleotides (Bibikova et al., 2009, 2011; Sandoval et al., 2011). Among these, the Illumina Infinium 450 k Human Methylation Beadchip offers both scalability and coverage (4480 000 probes) and is thus suitable for Epigenome-Wide Association Studies (EWAS) (Dedeurwaerder et al., 2011; Rakyan et al., 2011; Sandoval et al., 2011). Gene expression Advance Access publication November 21, 2012 Advance Access publication November 21, 2012 A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data Andrew E. Teschendorff1,*, Francesco Marabita2, Matthias Lechner3, Thomas Bartlett1, Jesper Tegner2, David Gomez-Cabrero2 and Stephan Beck3 1Statistical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK, 2Department of Medicine, Unit of Computational Medicine, Centre for Molecular Medicine, Karolinska Institute, Solna 171 76, Stockholm, Sweden and 3Medical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK Associate Editor: Olga Troyanskaya BIOINFORMATICS ORIGINAL PAPER BIOINFORMATICS ORIGINAL PAPER ORIGINAL PAPER Vol. 29 no. 2 2013, pages 189–196 doi:10.1093/bioinformatics/bts680 1 INTRODUCTION In the past few years, the field of epigenomics has risen to prom- inence (Feinberg, 2010; Petronis, 2010). Epigenomics not only offers an improved understanding of fundamental biological processes such as cellular differentiation and early embryogenesis, but is also widely recognized to be key in understanding the pathogenesis of complex genetic diseases like cancer Although the PBC method was validated in one dataset (Dedeurwaerder et al., 2011) and has now been implemented in a pipeline for 450 k data (Wang et al., 2012), two recent studies have exposed potential problems with PBC, specially when  The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. A.E.Teschendorff et al. (HNCs), of which 18 were HPVþ and 14 HPV, as well as five fresh frozen HNCs (FF), of which 2 were HPVþ and 3 HPV. The data are available from GEO under accession number GSE38271. (HNCs), of which 18 were HPVþ and 14 HPV, as well as five fresh frozen HNCs (FF), of which 2 were HPVþ and 3 HPV. The data are available from GEO under accession number GSE38271. applied to tissue samples (Maksimovic et al., 2012; Touleimat and Tost, 2012). In fact, as noted in these studies, PBC breaks down when the methylation density distribution does not exhibit well-defined peaks/modes. Hence, both studies proposed subset quantile normalization methods (SQN and SWAN) to correct for the type2 bias and which avoid the pitfalls of PBC (Maksimovic et al., 2012; Touleimat and Tost, 2012). In this work, we show that PBC often leads to discontinuities (‘holes’) in the type2 density distribution. To address this problem, we here propose a novel mixture model-based normalization algo- rithm, called Beta MIxture Quantile dilation (BMIQ). We sub- ject BMIQ to a rigorous evaluation using numerous independent datasets and using a number of different evaluation criteria to assess its robustness and performance. Specifically, we assess BMIQ in terms of reducing (i) the technical variance, (ii) the type2 bias, (iii) and the above-mentioned type1 enrichment bias. We further benchmark BMIQ against PBC and SWAN. 1 INTRODUCTION For assessing technical variance and to allow a comprehensive comparison of BMIQ to PBC/SWAN across many datasets, we use in addition to replicates, a novel evaluation framework based on using adjacent type1–type2 probe pairs within probe clusters, a framework which we show leads to consistent and robust con- clusions across 10 independent datasets. We demonstrate that, overall, BMIQ compares favourably to PBC and SWAN. Dataset 5: (GBM) This 450 k dataset consists of 81 glioblastoma multiformes (GBMs) (Turcan et al., 2012), 49 of which were categorized as CpG island methylator positive (CIMPþ) and 32 as CIMP. Datasets 6–10: TCGA, LIV, LC, BLDC, HCC These 450k samples are all from the TCGA: Specifically, Dataset6 (TCGA) consists of 10 samples as provided in the Bioconductor data package TCGAmethylation 450 k, Dataset7 (LIV) consists of nine normal liver tissue samples from Batch203 in the TCGA data portal, Dataset8 (LC) consists of 22 lung cancer samples from Batch196, Dataset9 (BLDC) consists of 12 bladder cancer samples from Batch86 and Dataset10 (HCC) consists of 10 hepatocellular carcinoma samples from Batch153. 2.2 BMIQ: Beta MIxture Quantile dilation normalization strategy The normalization of type2 probe values into type1 must satisfy the following criteria. (i) It must allow for the different biological character- istics of type1 and type2 probes, i.e type1 probes are significantly more likely to map to CpG islands than type2 probes, and hence the relative proportion of methylated and unmethylated probes will vary between the two designs. In the case of the type2 probes, this means that these pro- portions must be invariant under the normalization transformation. (ii) The transformation of the type2 probe values should reduce the bias, which amounts to matching of the density distributions of the two design types, specially at the unmethylated and methylated extremes. (iii) The transformation must be monotonic, that is, the relative ranking of beta values of the type2 probes must be invariant under the transform- ation. Next, we propose a normalization strategy for the type2 probes satisfying the above properties and which is based on three steps: 2.1 Biological data: DNA methylation Illumina Infinium 450k DNAm assay The DNA methylation data considered in this work were all generated using Illumina’s Infinium Human Methylation 450 k beadchip. Full details of this tech- nology are described in Bibikova et al. (2011) and Sandoval et al. (2011). Briefly, the methylation value of each probe follows an approximate - valued distribution, with  constrained to lie between 0 (unmethylated locus) and 1 (methylated). This follows from the definition of  as the ratio of methylated to combined intensity values, i.e  Fitting of a three-state (unmethylated-U, hemimethylated-H, fully methylated-M) beta mixture model to the type1 and type2 probes separately. For sake of convenience we refer to intermediate allelic methylation as hemimethylation even though hemimethylation is most often used in the context of strand-specific methylation. Let aI U, bI U   , aI H, bI H   , aI M, bI M     denote the parameters of the three beta distributions for the type1 probes, and similarly let aII U, bII U   , aII H, bII H   , aII M, bII M     describe the estimated parameters of the three beta components for the type2 probes. State membership of individual probes is determined by the maximum probability criterion.  ¼ M U þ M þ e ð1Þ ð1Þ where U and M are the unmethylated and methylated intensity values of the probe (averaged over bead replicates) and e ¼ 100 is a small correc- tion term to regularize probes of low total signal intensity (i.e. probes with U þ M  0 after background subtraction). Throughout we used non–background-corrected DNAm data. Of the 485 577 probes, 72% are of a type2 design in which the U and M measurements are obtained in different colour channels, while the rest (28%) of the probes are of the old type1 design in which both U and M measurements are obtained in the same colour channel. Importantly, type1 and type2 probes differ sig- nificantly in terms of CpG density, with CpGs mapping to CpGs islands overrepresented among type1 probes (Bibikova et al., 2011; Sandoval et al., 2011). where U and M are the unmethylated and methylated intensity values of the probe (averaged over bead replicates) and e ¼ 100 is a small correc- tion term to regularize probes of low total signal intensity (i.e. 2.1 Biological data: DNA methylation probes with U þ M  0 after background subtraction). Throughout we used non–background-corrected DNAm data. Of the 485 577 probes, 72% are of a type2 design in which the U and M measurements are obtained in different colour channels, while the rest (28%) of the probes are of the old type1 design in which both U and M measurements are obtained in the same colour channel. Importantly, type1 and type2 probes differ sig- nificantly in terms of CpG density, with CpGs mapping to CpGs islands overrepresented among type1 probes (Bibikova et al., 2011; Sandoval et al., 2011).  For those type2 probes assigned to the U-state, transform their probabilities of belonging to the U-state to quantiles using the in- verse of the cumulative beta distribution with beta parameters aI U, bI U   estimated from the type1 U component. Let II U denote the normalized values of the type2 U-probes.  For those type2 probes assigned to the M-state, transform their probabilities of belonging to the M-state to quantiles using the in- verse of the cumulative beta distribution with beta parameters aI M, bI M   estimated from the type1 M component. Let II M denote the normalized values of the type2 M-probes. Datasets 1 and 2: (BT) and (CL) This is a subset of the 450 k dataset considered in Dedeurwaerder et al. (2011). We used the data from eight fresh frozen (FF) breast tumours and eight normal breast tissue specimens [hereafter refered to as (BT)], as well as the three replicates from the HCT116 WT cell-line [hereafer refered to as (CL)]. For these cell-lines, matched bisulphite pyrosequencing (BPS) data were available for nine type2 probes.  For the type2 probes assigned to the H-state, we perform a dilation (scale) transformation to ‘fit’ the data into the ‘gap’ with endpoints defined by max II U   and min II M  . We next describe each of the above steps in detail. We first model each beta value  as, We next describe each of the above steps in detail. 2.1 Biological data: DNA methylation S1) to then obtain type2-specific thresholds using a simple correction reflecting the difference in the modes between the type1 and type2 distributions. Specifically, if tðIÞ U is the lower threshold (i.e. type1  values less than tðIÞ U are called unmethylated) and UMðIÞ and UMðIIÞ are the estimated modes of the unmethylated type1 and type2 components, the intial prior estimate for tðIIÞ U would be tðIIÞ U ¼ tðIÞ U þ ðUMðIIÞ  UMðIÞÞ. Similarly, the threshold for calling probes fully methylated or just hemi-methylated would be tðIIÞ M ¼ tðIÞ M þ ðMMðIIÞ  MMðIÞÞ where MM denotes the mode of the methylated state. We note that resulting thresholds would normally fall within the ranges 0.2–0.3 and 0.60–0.8, respectively. Having thus identified reasonable initial estimates for the weights fðIIÞ U , ðIIÞ H , ðIIÞ M g, the algorithm will then automatically determine the unmethylated, hemi- methylated and methylated fractions for each sample individually. (ii) A second important observation is the overall robustness of BMIQ to the goodness of the type2 EM-fit. This is important, since we consis- tenly observe that the methylated type2 distribution is not well described by a beta function (Supplementary Fig. S1). In this regard we have also verified that using a beta mixture model with more than three states does not improve the overall type2 fit. Fortunately however, as explained above, the goodness of fit problem associated with the H and M-probes can be easily circumvented by modelling only the right tail of the methylated component as the corresponding tail of a beta distribution. In this case, the left tail is modelled together with the H- probes using the observed empirical distribution. Hence, the probe values that are not well described by a beta distribution are not normalized using estimated beta parameters, which means that their normalization is in- sensitive to the goodness of fit. mt s ¼ at s at s þ bt s ð3Þ ð3Þ Further, let UII, HII, MII denote the set of type2 probes assigned to unmethylated, hemimethylated or fully methylated states (using the max- imum probability criterion), and let UL II (UR II) denote the set of UII probes with -values smaller (larger) than mII U. Similarly, let ML II (MR II) denote the set of MII probes with -values smaller (larger) than mII M. 2.1 Biological data: DNA methylation We first model each beta value  as, Datasets 3 and 4: (FFPE) and (FF) This 450k dataset consists of 32 formalin-fixed paraffin-embedded (FFPE) head and neck cancers Datasets 3 and 4: (FFPE) and (FF) This 450k dataset consists of 32 formalin-fixed paraffin-embedded (FFPE) head and neck cancers ð2Þ pðtÞ ¼ t UBðjat U, bt UÞ þ t HBðjat H, bt HÞ þ t MBðjat M, bt MÞ 190 Adjusting design bias in 450 k studies when performing the conformal transformation. This is because we observed that it is the left tail end of the methylated type2 distribution that is generally not well described by a beta-distribution (Supplementary Fig. S1), presumably as a result of the dye bias, which is specific to the type2 distribution. where B denotes the beta probability density function and t denotes the design type t ¼ ðI, IIÞ. We infer the parameters ð, a, bÞ using an Expectation Maximization (EM) algorithm as described in (Ji et al., 2005). The estimated parameters we denote again by ðt s, at s, bt sÞ where t labels the design and s one of the three states (U, H, M). The resulting means of the estimated beta-distributions are denoted by mt s where There are a number of other important points to note about BMIQ: (i) First, it is important to choose reasonable initial weight parameters fðIIÞ U , ðIIÞ H , ðIIÞ M g in the EM-algorithm. As these fractions can vary signifi- cantly from study to study, or even sample to sample depending on the nature of the samples assayed, it is important to choose reasonable initial values on a per-sample basis. Not doing so may result in mild disconti- nuities in the type2 density distribution. To obtain estimates for these prior weight parameters, we first note that their estimation only requires estimates for the two thresholds used for calling the three states, since the weights for a given sample are determined given a choice of thresholds. Moreover, although the thresholds will show little inter-sample variabil- ity, the weights may not, reflecting the biological differences in the number of probes that are unmethylated, hemimethylated or fully methy- lated. In BMIQ, the estimation of the initial thresholds proceeds in an automatic fashion on a per-sample basis: in detail, we use the estimated thresholds from the type1 distribution (which always gives an excellent fit, Supplementary Fig. HM ¼ maxH þ minM We want the new normalized maximum and minimum values of H-probes to satisfy We want the new normalized maximum and minimum values of H-probes to satisfy nmaxH ¼ minfII Mg  HM nminH ¼ maxfII Ug þ UH so that ðÞ H ¼ nmaxH  nminH. The normalized -values for the H-probes is then given by the conformal (shiftþ dilation) transformation 2.1 Biological data: DNA methylation This subdivision into values which fall left (L) or right (R) of the mean are necessary since the state membership probabilities estimated from the EM algorithm are two tailed. Next, for the UL II probes we estimate their type2 tail probabil- ities of belonging to the U-state, i.e p ¼ PðUjUL IIÞ ¼ FðUL IIjaII U, bII UÞ where F denotes the cumulative distribution beta function. We then transform these probabilities back to quantiles (i.e -values), but using the type1 parameters, i.e q ¼ F1ðpjaI U, bI UÞ ð4Þ ð4Þ and finally set the normalized -value, UL II ¼ q. An identical transform- ation (using 1  F instead of F) is performed for the UR II probes. Next, we perform the analogous operation for the ML II and MR II probes. This therefore yields normalized type2 values for all type2 U and M probes. Finally, it remains to normalize the type2 H probes. Since the type2 H probe value distribution is sandwiched between the U and M probe dis- tributions, we can use an empirical approach to normalize these values, thus also bypassing the difficulty that type2 H probe values are not well described by a beta distribution (Supplementary Fig. S1). Specifically, we first identify the minima and maxima of the type2 H-probes, maxH ¼ maxfII Hg and minH ¼ minfII Hg, and let ðÞ H ¼ maxH  minH. We also find the minimum of the M-probes, i.e minM ¼ minfII Mg and the maximum of the U-probes, i.e. maxU ¼ maxfII Ug. We point out that in fact all of these extrema represent robust values, because they do not represent extrema on the bounded (0,1) support, i.e. the values maxU, minH, maxH and minM are not close to 0 or 1. Next, we define distances College London on June 18, 2013 UH ¼ minH  maxU HM ¼ maxH þ minM UH ¼ minH  maxU HM ¼ maxH þ minM 3.1 Improved robustness of BMIQ II H ¼ nminH þ dfðII H  minHÞ II H ¼ nminH þ dfðII H  minHÞ ð5Þ ð5Þ To validate BMIQ, we first applied it to data where the PBC method has been shown to work reasonably well. Thus, we applied it to a fresh frozen breast tumour sample from Dataset1 (Dedeurwaerder et al., 2011) (Fig. 1A). We can see that for this particular sample, the methylated type1 peak is well defined and as a result both PBC and BMIQ appear to do well in generating smooth density distributions for the type2 probes, which at the methylation extremes are also reasonably well matched to the type1 density distribution. Next, we applied both PBC and BMIQ to the FFPE tumour samples from where df ¼ ðÞ H =ðÞ H is the dilation factor. It is important to observe that the conformal transformation involves a non-uniform rescaling of the H probe beta values since it depends on the beta-value of the probe. This is absolutely key in order to avoid gaps or holes from emerging in the normalized distribution. This algorithm is flexible in that the dilation can be performed also including the ML II (and/or the URÞIIÞ probes, which means that the match- ing of the density distributions is only done on the respective tails (i.e. the MR II and UL II probes). We point that in practice we find that the optimal performance is attained by including the ML II probes with the H-probes 191 A.E.Teschendorff et al. Dataset3, for which the type1 methylated peak was not well defined (Fig. 1B). In these samples, PBC generated a type2 dens- ity distribution that exhibited relatively sharp changes (‘holes’) (Fig. 1B), suggestive of a non-optimal adjustment and indicating that in such cases PBC breaks down. This is not suprising since PBC relies heavily on the ability to detect clear unmethylated and methylated modes in the type1 density distribution in order to then adjust the type2 distribution accordingly. Importantly, BMIQ does not use the type1 modes to adjust the type2 data, and hence BMIQ normalization of the type2 probes generated a much smoother density distribution, suggestive of an improved normalization framework (Fig. 1B). Moreover, the tail ends of the BMIQ type2 distribution better matched those of the type1 distribution without affecting the fractions of unmethylated, hemimethylated and fully methylated probes, which are pre- served by the BMIQ transformation. Fig. 1. 3.3 BMIQ reduces bias of type2 methylation values Fig. 1. (A) Density distributions of beta-values for the type1 probes, type2 probes (unnormalized) and normalized type2 probes for a breast tumour sample from Dataset1. (B) Density distributions of beta-values for the type1 probes, type2 probes (unnormalized) and normalized type2 probes for a head and neck tumour sample from Dataset3. Left panels are for PBC, right panels for BMIQ Using replicates to evaluate normalization methods assesses the method in terms of reducing technical variability but does not evaluate whether the actual values of the replicates are closer to the true estimate. This requires comparison with a gold-standard, which is provided by matched BPS data (Dedeurwaerder et al., 2011). Hence, we compared the methods in terms of the devi- ations from BPS methylation values for the nine type2 probes in Dedeurwaerder et al. (2011) for which matched 450k BPS data were available. Similar to PBC, we observed that BMIQ signifi- cantly reduced the bias of type2 values (Fig. 3), although there was no improvement over PBC itself, presumably owing to the ations from BPS methylation values for the nine type2 probes in Dedeurwaerder et al. (2011) for which matched 450k BPS data were available. Similar to PBC, we observed that BMIQ signifi- cantly reduced the bias of type2 values (Fig. 3), although there was no improvement over PBC itself, presumably owing to the Fig. 2. (A) Boxplots comparing the standard deviation (y-axis) of type1 (1) and type2 (2) probes over the three HCT116 WT replicates from (Dedeurwaerder et al., 2011), for the case of no design normalization (NONE), PBC and BMIQ. (B) As (A) but now comparing the Manhattan distances over type2 probes only for each pair of replicates Fig. 3. Barplot comparing the maximum and mean absolute deviation of 450 k values from their corresponding bisulphite pyrosquencing values over the nine type2 probes considered in Dedeurwaerder et al., (2011). We compare these deviation measures for the case of no design normal- ization (NONE), PBC normalization and BMIQ Fig. 2. (A) Boxplots comparing the standard deviation (y-axis) of type1 (1) and type2 (2) probes over the three HCT116 WT replicates from (Dedeurwaerder et al., 2011), for the case of no design normalization (NONE), PBC and BMIQ. (B) As (A) but now comparing the Manhattan distances over type2 probes only for each pair of replicates Fig. 3. 3.1 Improved robustness of BMIQ (A) Density distributions of beta-values for the type1 probes, type2 probes (unnormalized) and normalized type2 probes for a breast tumour sample from Dataset1. (B) Density distributions of beta-values for the type1 probes, type2 probes (unnormalized) and normalized type2 probes for a head and neck tumour sample from Dataset3. Left panels are for PBC, right panels for BMIQ 3.2 BMIQ reduces technical variation To further test BMIQ we applied it to Dataset2 (CL) consisting of three replicates of a given cell-line, to investigate if reprodu- cibility is improved. First, we computed for each of the probes its standard deviation across the three replicates and for each of the three scenarios: no normalization, PBC and BMIQ. As seen, BMIQ performed similarly to PBC and led to a significant re- duction in inter-replicate variability (Fig. 2A). To check this fur- ther, we compared the normalization methods in terms of the Euclidean distance between the three possible pairs of replicates across the type2 probes (Fig. 2B). Using this measure, BMIQ not only led to a significant improvement, but was also marginally better than PBC (Fig. 2B). 3.5 Reduced technical variability within probe clusters 3.5 Reduced technical variability within probe clusters To further assess BMIQ, we devised an evaluation framework which exploits the well known spatial correlation of DNA methylation at scales 5500bp (Eckhardt et al., 2006). Approximately 27% of the 450k probes fall into 12 501 probe clusters, defined as contiguous regions containing at least seven probes with no two adjacent probes separated by4300 bp (Jaffe et al., 2012). Within these probe clusters, we posited that pairs of adjacent probes, one from each design and within 200 bp of each other, should have similar methylation values. Among the 12 501 probe clusters we identified on the order of 30 000 of such adjacent type1–type2 probe pairs. Thus, to evaluate the normal- ization algorithms, we asked which one minimizes the absolute difference in methylation between such closely adjacent type1– type2 pairs. We considered a total of 10 independent datasets, seven of which had idat files, thus allowing also for a direct comparison with SWAN (Maksimovic et al., 2012). For each dataset, we computed the mean of the absolute deviations over probe pairs and samples. Comparison of these average deviations revealed that BMIQ consistently reduced the technical variation, while also outperforming PBC and SWAN (Table 1). In fact, in 9 of 10 datasets, BMIQ was substantially better as assessed using a paired Wilcoxon rank sum test over all probe pairs and samples (Table 1). Example methylation profiles within these probe clusters confirmed that BMIQ successfully reduces the technical variability, while PBC can break down either overcorrecting or suppressing the type2 data values, leading to substantial dif- ferences in methylation between neighbouring probes, even at scales of5100bp (Fig. 5 and Supplementary Table S1). Fig. 4. OR of relative enrichment of type1 probes over type2 probes among the top 100, top 250, top 500 and top 1000 ranked probes, where probes were ranked according to the absolute difference in methylation (beta-values). ORs are shown for the case of no design normalization (NONE), PBC and BMIQ normalization. Supervised analysis and ranking was performed only on probes mapping to CpG islands and within 200bp upstream of transcription start site to correct for biological differences between type1 and type2 probes. The line OR¼ 1 represents the ideal scenario of no relative enrichment of type1 versus type2 probes. The 95% confidence envelope around OR¼ 1 is shown to assess significant deviations from OR¼ 1. 3.3 BMIQ reduces bias of type2 methylation values Barplot comparing the maximum and mean absolute deviation of 450 k values from their corresponding bisulphite pyrosquencing values over the nine type2 probes considered in Dedeurwaerder et al., (2011). We compare these deviation measures for the case of no design normal- ization (NONE), PBC normalization and BMIQ Fig. 3. Barplot comparing the maximum and mean absolute deviation of 450 k values from their corresponding bisulphite pyrosquencing values over the nine type2 probes considered in Dedeurwaerder et al., (2011). We compare these deviation measures for the case of no design normal- ization (NONE), PBC normalization and BMIQ Fig. 2. (A) Boxplots comparing the standard deviation (y-axis) of type1 (1) and type2 (2) probes over the three HCT116 WT replicates from (Dedeurwaerder et al., 2011), for the case of no design normalization (NONE), PBC and BMIQ. (B) As (A) but now comparing the Manhattan distances over type2 probes only for each pair of replicates 192 Adjusting design bias in 450 k studies fact that in these specific samples the methylated type1 peak was well defined, a scenario in which PBC works well. fact that in these specific samples the methylated type1 peak was well defined, a scenario in which PBC works well. probes. In contrast, BMIQ eliminated the type1 enrichment bias in all three datasets (the resulting OR was always close to 1) without overcorrecting the data and avoiding the type2 enrichment bias seen for PBC. 3.4 BMIQ eliminates the type1 enrichment bias To further test BMIQ, we considered the supervised context, in which a ranked list of probes correlating with a phenotype of interest is derived. Given the higher dynamic range of type1 probes, one expects that this would favour type1 probes and that therefore there would be a relative over enrichment of type1 over type2 probes in a top ranked list of probes. However, one key difficulty when assessing whether there is a bias towards type1 probes is that type1 and type2 probes differ significantly in terms of their biological characteristics, in par- ticular in terms of CpG density. Hence, in order to avoid con- founding by CpG density, we only selected probes that mapped to CpG islands and to 200 bp upstream of the TSS, thus allowing a sensible comparison between type1 and type2 probes. We con- sidered three different datasets and derived for each a ranked list of probes associated with a phenotype of interest: breast cancer versus normal breast [Dataset1 (BT)], HPVþ versus HPVHNC [Dataset3 (FFPE)], and CIMPþ versus CIMP (GBM) (Dataset5). The ranking was performed using the magnitude of differential methylation. Although this ranking does not take the within-phenotype variability into account, it remains a popular method (Dedeurwaerder et al., 2011; Du et al., 2010), and for our purposes, using the absolute difference in beta-values allows us to better interpret the performance of the different normalization methods. To assess any potential bias towards type1 probes, we computed for a given number of top ranked probes the odds ratio (OR) of relative enrichment of type1 over type2 probes. Across all three datasets, we indeed observed a bias towards type1 probes, although the severity of this bias varied substan- tially from study to study (Fig. 4). Using PBC, in one dataset this bias was eliminated; however, in the other two datasets, PBC overcorrected the data leading to a bias favouring type2 3.5 Reduced technical variability within probe clusters (A) Eight breast cancers versus eight normal breast (Dataset1), (B) 18 HPVþ HNCs versus 14 HPV HNCs (Dataset3), (C) 49 CIMPþ GBMs versus 32 CIMP GBMs (Dataset5) Fig. 4. OR of relative enrichment of type1 probes over type2 probes among the top 100, top 250, top 500 and top 1000 ranked probes, where probes were ranked according to the absolute difference in methylation (beta-values). ORs are shown for the case of no design normalization (NONE), PBC and BMIQ normalization. Supervised analysis and ranking was performed only on probes mapping to CpG islands and within 200bp upstream of transcription start site to correct for biological differences between type1 and type2 probes. The line OR¼ 1 represents the ideal scenario of no relative enrichment of type1 versus type2 probes. The 95% confidence envelope around OR¼ 1 is shown to assess significant deviations from OR¼ 1. (A) Eight breast cancers versus eight normal breast (Dataset1), (B) 18 HPVþ HNCs versus 14 HPV HNCs (Dataset3), (C) 49 CIMPþ GBMs versus 32 CIMP GBMs (Dataset5) 193 A.E.Teschendorff et al. 3.6 BMIQ robustly identifies features associated with HPV status of sensitivity and positive predictive value (PPV) between the different normalization methods. To perform this analysis, we used Dataset4(FF) consisting of 2 HPVþ and 3 HPV fresh frozen head and neck cancers to derive features associated with HPV status. As test set we used Dataset3(FFPE) consisting of 18 HPVþ and 14 HPV head and neck cancers (FFPE tissue). Using limma (Smyth, 2005) and an FDR threshold of 0.35, we observed that BMIQ identified substantially more differentially methylated features than PBC or SWAN (Table 2). Importantly, this was not at the expense of a smaller PPV, and so, overall, BMIQ identified substantially more true positives (Table 2). Finally, it must be verified that the reduction in technical vari- ance obtained with BMIQ is not at the expense of a reduced biological signal. Since it is difficult to establish what constitutes a true positive, we used a training test set strategy, to identify features in a training set and calling them true positives if vali- dated in a test set. This strategy thus allows for a comparison Table 1. For each dataset we compare the absolute deviation in methy- lation between adjacent type1–type2 probe pairs (probes within 200 bp of each other), averaged over probe pairs and samples, for four different normalisation methods 4 DISCUSSION Dataset NONE (%) PBC (%) SWAN (%) BMIQ (%) P BT 7.8 6.3 NA 6.2 51010 CL 8.6 18.4 NA 7.2 51010 FFPE 9.2 8.0 8.5 7.8 51010 FF 8.5 8.1 7.6 7.3 51010 GBM 9.2 7.6 NA 7.5 51010 TCGA 9.4 7.8 8.3 7.4 51010 LIV 10.0 6.3 7.4 6.4 1 LC 10.3 7.0 7.7 6.7 51010 BLDC 11.0 8.0 7.9 7.6 51010 HCC 12.0 8.5 8.7 8.1 51010 NONE refers to the case of no adjustment for probe design type. The last column give the paired Wilcoxon rank sum test P-value (treating each probe-pair deviation in each sample as a separate value), assessing the statistical significance that the absolute deviation for BMIQ is smaller than the next best competing method. NA indicates non-available owing to lack of access to idat files needed for processing by SWAN. In bold-face we show the smallest deviation across methods. Dataset NONE (%) PBC (%) SWAN (%) BMIQ (%) P BT 7.8 6.3 NA 6.2 51010 CL 8.6 18.4 NA 7.2 51010 FFPE 9.2 8.0 8.5 7.8 51010 FF 8.5 8.1 7.6 7.3 51010 GBM 9.2 7.6 NA 7.5 51010 TCGA 9.4 7.8 8.3 7.4 51010 LIV 10.0 6.3 7.4 6.4 1 LC 10.3 7.0 7.7 6.7 51010 BLDC 11.0 8.0 7.9 7.6 51010 10 In this work we have presented a novel mixture-model-based algorithm (BMIQ) for correcting the bias associated with type2 probe values in 450k studies. Confirming the observations Table 2. Table listing the number of differentially methylated probes (nDMPs) associated with HPV status in Dataset4 (FF), and the corres- ponding estimates for the positive predictive value (PPV) and number of true positives (nTPs) estimated using Dataset3 (FFPE) as test set Metric NONE PBC SWAN BMIQ nDMP 51 70 41 252 PPV 0.25 0.18 0.19 0.20 nTP 13 13 8 51 DMPs were defined at an FDR threshold of 0.35, and those with the same sign of limma t-statistic in the two sets and with a corresponding P-value50.01 in the test set were deemed true positives. NONE refers to the case of no adjustment for probe design type. The last column give the paired Wilcoxon rank sum test P-value (treating each probe-pair deviation in each sample as a separate value), assessing the statistical significance that the absolute deviation for BMIQ is smaller than the next best competing method. 4 DISCUSSION NA indicates non-available owing to lack of access to idat files needed for processing by SWAN. In bold-face we show the smallest deviation across methods. DMPs were defined at an FDR threshold of 0.35, and those with the same sign of limma t-statistic in the two sets and with a corresponding P-value50.01 in the test set were deemed true positives. Fig. 5. Examples of methylation profiles, from Dataset4(FF), of three probe clusters on chromosomes 19, 1 and 15, respectively. The design type of each probe is indicated with 1 and 2. The non design normalized data (NONE), PBC, SWAN and BMIQ corrected data are superimposed. Observe how across the three loci, BMIQ generally corrects the data in a way which is more consistent with the neighbouring type1 values. In the left panel PBC overcorrects the data, in the right panel there is suppression, while in the middle panel PBC both overcorrects and suppresses beta values. The data values can be found in Supplementary Table S1 Fig. 5. Examples of methylation profiles, from Dataset4(FF), of three probe clusters on chromosomes 19, 1 and 15, respectively. The design type of each probe is indicated with 1 and 2. The non design normalized data (NONE), PBC, SWAN and BMIQ corrected data are superimposed. Observe how across the three loci, BMIQ generally corrects the data in a way which is more consistent with the neighbouring type1 values. In the left panel PBC overcorrects the data, in the right panel there is suppression, while in the middle panel PBC both overcorrects and suppresses beta values. The data values can be found in Supplementary Table S1 194 Adjusting design bias in 450 k studies made in Touleimat and Tost (2012) and Maksimovic et al. (2012), we have seen that PBC can break down in samples with ill-defined type1 methylation peaks, causing sharp, almost discontinuous changes (which we call ‘holes’) in the density dis- tributions (Fig. 1B), which motivated our quest to find a more robust algorithm. We have shown that BMIQ improves the robustness and can successfully normalize the type2 distribution, avoiding the appearance of such ‘holes’ (Fig. 1B). Moreover, BMIQ successfully matches the tail-ends of the type1 and type2 distributions, while faithfully preserving the proportions of unmethylated and methylated probes within each of the two designs. 4 DISCUSSION In summary, using a number of different evaluation criteria and numerous datasets, we have seen that BMIQ compares fa- vourably with both PBC and SWAN. Although we did not com- pare BMIQ to SQN (Touleimat and Tost, 2012), this latter method is very similar to SWAN, as they both rely on a probe subset quantile normalization. Like SQN/SWAN, BMIQ uses quantiles to normalize the type2 probe values into a distribution that is comparable with that of type1 probes. However, unlike SQN and SWAN, BMIQ is based on an explicit beta-mixture modeling framework, and uses state-membership probabilities under this beta mixture model to reassign the quantiles of the type2 probes according to the type1 distribution. Thus, BMIQ is assumption-free, as it does not require a separate normalization to be performed on selected subsets of probes that are matched for biological characteristics (e.g. CpG density), as done in SQN and SWAN. In fact, under the BMIQ frame- work, all the biological differences (including CpG density) between the type1 and type2 probes are captured by the esti- mated fractions of unmethylated, hemimethylated and methy- lated probes, which will be different between the two assays. Thus, BMIQ does not depend on a priori and somewhat arbitrary choices of which biological characteristics to use when matching the type1 and type2 distributions. For instance, in SQN the normalization is performed on probe subsets defined by specific CpG characteristics (e.g. shelves, shores, CpG is- lands); however, multiple different definitions for say CpG is- lands exist (Gardiner-Garden and Frommer, 1987; Takai and Jones, 2002; Wu et al., 2010; Zhao et al., 2009). Similarly, in SWAN the number of CpGs in the probe body, even if they differ by one, is used to define probe normalization categories, and thus it is unclear whether these probe categories represent an optimal way of dividing the probes up. Therefore, we see the beta-mixture model framework of BMIQ as an important con- ceptual advantage over SQN/SWAN, since, as demonstrated here, it successfully normalises type2 probe values, faithfully pre- serving the numerous and complex biological differences that exist between the two designs without ever needing to define probe subsets. Nevertheless, it will be interesting to conduct a comprehensive and detailed comparison of BMIQ, SQN and SWAN on matched 450k BPS data on a sufficiently large number of loci and samples. 4 DISCUSSION To further test BMIQ, we used data on technical replicates (to show that it reduces technical variability) and matched BPS data (to show that it reduces the bias of type2 probe values). Using these criteria, we have seen that BMIQ leads to significant im- provements, similar to the improvements noted for PBC (Figs 2 and 3). In relation to these evaluation criteria, it is worth point- ing out that BMIQ was compared with PBC on samples with well-defined type1 methylation peaks, i.e. on data that were used to develop PBC itself. Hence, it is likely that an evaluation of technical reproducibility (using replicates) and type2 value bias (using matched BPS data) on data where the methylated type1 peaks are less well-defined would favour BMIQ over PBC. However, we did not have access to technical replicates or matched BPS data in the other specific datasets considered here. Therefore, in order to further assess BMIQ, we devised a supervised framework across three independent datasets to ob- jectively compare the algorithms in their ability to reduce the expected enrichment bias of type1 probes. First, we showed that if no design normalization is performed then there is indeed an enrichment bias towards type1 probes, even when ad- justed for CpG density (Fig. 4). We also showed that in two datasets, PBC overcorrected the type2 data, leading to an over- inflated dynamic range, thus favouring type2 probes and causing an ‘overshooting’ of the enrichment scores, reflected by a signifi- cant underenrichment of type1 probes (Fig. 4B and C). In con- trast, BMIQ successfully avoided any type1/type2 enrichment bias in all three datasets, indicative of an improved normaliza- tion of type2 values (Fig. 4). We should point out that the over- correction of type2 values and the associated overinflated dynamic range caused by PBC is consistent with the presence of ‘holes’ in the hemimethylated region of the type2 density dis- tribution. Thus, with PBC there is an artificial expulsion of data points from the hemimethylated region to the unmethylated and/ or methylated extremes. In a further assessment of BMIQ, we conducted a detailed spatial analysis of DNA methylation at the level of probe clusters across 10 independent datasets. 4 DISCUSSION By care- fully analysing adjacent type1–type2 probe pairs, we observed that PBC can often overcorrect or suppress the data (in some cases inducing abnormally large 30% changes in methylation), in contrast to BMIQ, which normalized type2 values in a way that rendered them more consistent with the values of neighbouring type1 probes (Fig. 5 and Table 1). Interestingly, BMIQ also appeared to outperform SWAN (Table 1), which is part of the popular and widely used minfi package (Hansen and Aryee, 2012). Of note, the reduction in technical variance achieved by BMIQ was not at the expense of a lower biological signal (Table 2). at University College London on June 18, 2013 http://bioinformatics.oxfordjournals.org/ Downloaded from Conflict of Interest: none declared. REFERENCES Koestler,D.C. et al. (2010) Semi-supervised recursively partitioned mixture models for identifying cancer subtypes. Bioinformatics, 26, 2578–2585. Baylin,S.B. and Ohm,J.E. (2006) Epigenetic gene silencing in cancer—a mechanism for early oncogenic pathway addiction? Nat. Rev. Cancer, 6, 107–116. Maksimovic,J. et al. (2012) Swan: Subset quantile Within-Array Normalization for illumina infinium humanmethylation450 beadchips. Genome Biol., 13, R44. r early oncogenic pathway addiction? Nat. Rev. Cancer, 6, 107–116 lumina infinium humanmethylation450 beadchips. Genome Biol., 13 Bibikova,M. et al. (2011) High density DNA methylation array with single CpG site resolution. Genomics, 98, 288–295. Petronis,A. (2010) Epigenetics as a unifying principle in the aetiology of complex traits and diseases. Nature, 465, 721–727. Bibikova,M. et al. (2009) Genome-wide DNA methylation profiling using Infinium assay. Epigenomics, 1, 177–200. Rakyan,V.K. et al. (2011) Epigenome-wide association studies for common human diseases. Nat. Rev. Genet., 12, 529–541. Dedeurwaerder,S. et al. (2011) Evaluation of the infinium methylation 450k tech- nology. Epigenomics, 3, 771–784. Sandoval,J. et al. (2011) Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome. Epigenetics, 6, 692–702. Sandoval,J. et al. (2011) Validation of a DNA methylation m CpG sites in the human genome. Epigenetics, 6, 692–702 Du,P. et al. (2010) Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics, 11, 587. Smyth,G.K. (2005) Limma: linear models for microarray data. In: Gentleman,R. et al. (eds) Bioinformatics and Computational Biology Solutions using R and Bioconductor. Springer, New York, pp. 397–420. methylation levels by microarray analysis. BMC Bioinformatics, 11, Eckhardt,F. et al. (2006) DNA methylation profiling of human chromosomes 6, 20 and 22. Nat. Genet., 38, 1378–1385. Takai,D. and Jones,P.A. (2002) Comprehensive analysis of cpg islands in human chromosomes 21 and 22. Proc. Natl Acad. Sci. USA, 99, 3740–3745. Feinberg,A.P. (2010) Epigenomics reveals a functional genome anatomy and a new approach to common disease. Nat. Biotechnol., 28, 1049–1052. Touleimat,N. and Tost,J. (2012) Complete pipeline for infinium() human methy- lation 450k beadchip data processing using subset quantile normalization for accurate dna methylation estimation. Epigenomics, 4, 325–341. Feinberg,A.P. et al. (2006) The epigenetic progenitor origin of human cancer. Nat. Rev. Genet., 7, 21–33. lation 450k beadchip data processing using subset quantile normalization for accurate dna methylation estimation. Epigenomics, 4, 325–341. Gardiner-Garden,M. and Frommer,M. (1987) CpG islands in vertebrate genomes. J. Mol. Biol., 196, 261–282. Turcan,S. et al. (2012) IDH1 mutation is sufficient to establish the glioma hyper- methylator phenotype. Nature, 483, 479–483. Hansen,K.D. 5 CONCLUSIONS We have presented a mixture model assumption-free normaliza- tion algorithm, BMIQ, which will be useful for correcting the bias associated with the type2 assay in DNA methylation studies using the Illumina Infinium 450k platform. Funding: A.E.T. is supported by a Heller Research Fellowship. M.L. was supported by a Wellcome Trust Research Training Fellowship (093855). S.B. was supported by a Royal Society Wolfson Research Merit Award (WM100023) and grants from the Wellcome Trust (084071) and EU-FP7 BLUEPRINT (282510). We also thank FP7 SYNERGY-COPD (F.B., D.G., J.T.), BILS (D.G.) and Stockholm County (J.T.). Conflict of Interest: none declared. 195 A.E.Teschendorff et al. REFERENCES and Aryee,M. (2012) Minfi: Analyze Illumina’s 450k Methylation Arrays. R package version 1.2.0. Wang,D. et al. (2012) IMA: an R package for high-throughput analysis of illumi- na’s 450k infinium methylation data. Bioinformatics, 28, 729–730. Houseman,E.A. et al. (2008) Model-based clustering of dna methylation array data: a recursive-partitioning algorithm for high-dimensional data arising as a mixture of beta distributions. BMC Bioinformatics, 9, 365. Wu,H. et al. (2010) Redefining CpG islands using hidden Markov models. Biostatistics, 11, 499–514. of beta distributions. BMC Bioinformatics, 9, 365 Zhao,Q. et al. (2009) Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line. Proc. Natl Acad. Sci. USA, 106, 1886–1891. Jaffe,A.E. et al. (2012) Bump hunting to identify differentially methylated regions in epigenetic epidemiology studies. Int. J. Epidemiol., 41, 200–209. Ji,Y. et al. (2005) Applications of beta-mixture models in bioinformatics. Bioinformatics, 21, 2118–2122. Zhuang,J. et al. (2012) A comparison of feature selection and classification methods in DNA methylation studies using the illumina 27k platform. BMC Bioinformatics, 24, 59. Jones,P.A. and Baylin,S.B. (2007) The epigenomics of cancer. Cell, 128, 683–692. 196
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Aqueous proton transfer across single-layer graphene
Nature communications
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ARTICLE Received 5 Nov 2014 | Accepted 6 Feb 2015 | Published 17 Mar 2015 & 2015 Macmillan Publishers Limited. All rights reserved. Aqueous proton transfer across single-layer graphene B r n s charge density, in the duration of the jumps or in the rates of the jumps when single-layer graphene is present. These findings indicate that the acid–base chemistry at the fused silica/water interface occurs in an unimpeded fashion in the presence of single-layer graphene. Importance of macroscopic defects ruled out. Scanning electron microscopy (SEM, Methods) images of graphene single layers deposited on fused silica windows show a low density of macro- scopic pinholes and that the graphene is free of cracks or folds (Fig. 1c). Two-dimensional (2D) diffusion from those locations to the location of the laser beam is considered by calculating, for a given proton diffusion coefficient D, the mean-square displace- ment, hDr2i, according to hDr2i ¼ z  t  D, where t is time and z is the number of neighbouring sites to which the proton can hop23 (six in for the case of the hexagonal graphene lattice). In the literature, reported theoretically and experimentally determined proton surface diffusion coefficients range between 1.01  10  7 and 9.00  10  5 cm2 s  1 (refs 24–39). While the bulk diffusion coefficient for a proton in water is accepted to range between 8  10  5 and 9  10  5 cm2 s  1, there are disagreements in the literature about whether the surface proton diffusion coefficient is similar to the bulk coefficient or slower than the bulk coefficient on hydrophobic and hydrophilic surfaces for a variety of different systems24,28,38. Reactivity is expected to substantially slow down the 2D diffusion of the proton (approximately magnitude  20 reduction)30,40,41 when it moves across an amphoteric oxide whose protonation effectively terminates the diffusion path. Reactive proton diffusion coefficients reported for Nafion42,43 are similarly low. Indeed, our own reactive force field calculations containing partially hydroxylated quartz surfaces show the proton diffusion is quickly terminated by protonation of the surface SiO  groups (Supplementary Note 4 and Supplementary Fig. 6). This result indicates that proton diffusion is significantly slower in the presence of surface anionic species due to proton trapping at these sites. Aqueous proton transfer across single-layer graphene ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 B rick-and-mortar networks of stacked graphene oxide nanosheets can act as effective membranes1–8, while single-layer graphene exhibits dramatically lower permeabilities towards gases4,9. In fact, graphene is thought to be unfit even for proton transfer, which is associated with computed gas-phase energy barriers exceeding 1.4 eV (ref. 10) unless dopants or nanoscale openings are externally introduced6,7,10,11, or an external potential bias is applied12. To determine whether graphene is indeed impermeable to protons, we place well-characterized single-layer graphene13 on top of a fused silica substrate and cycle, at room temperature and constant ionic strength, the bulk pH of an aqueous solution above the graphene layer between basic and acidic. We test for proton exchange through graphene by probing the underlying silica surface with an interfacial potential-dependent version of second harmonic generation (SHG)14,15 using 120 fs input pulses at energies well below the graphene damage threshold13. With a detection limit of 10  5 to 10  6 V (ref. 16), the method is sensitive enough to follow protonation or deprotonation of as little as 1% of the available silanol groups present in the area probed by SHG. The interfacial potential vanishes at the point of zero charge (PZC of fused silica B2.5) (ref. 17), and the SHG signal intensity is small14,18,19. Increasing the pH at constant ionic strength shifts the relevant interfacial acid–base equilibria SiOH2þ þ OH "SiOH þ H2O and SiOH þ OH "SiO  þ H2O (pKa B4.5 and B8.5)14,18,20 to the right, and the resulting interfacial potential polarizes the interfacial water molecules such that the SHG signal intensity increases14,18. Intuitively, the close proximity of the graphene layer and the charged fused silica surface, combined with the sensitivity of the method, make our approach akin to an Å-scale voltmeter for detecting even rare occurrences of proton exchange. We find no significant difference between the SHG versus time traces recorded in the presence and absence of graphene. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defect sites in the graphene layer. Computer simulations reveal low energy processes for water-mediated proton transfer across hydroxyl-terminated atomic defect sites that participate in a Grotthuss-type relay, while defects terminated by pyrylium-like ether bridges shut down proton exchange. Aqueous proton transfer across single-layer graphene In our experiments, the continuous proton supply from the aqueous bulk is expected to form a propagating reaction front: our calculations show a drastically increased proton diffusion coefficient of 4.944  10  5 cm2 s  1, or just half of that of bulk water, once protons arriving through any opening within the graphene sheet interact with the hydroxylated portion of the surface that is located behind the reaction front. To conservatively assess an upper bound limit for our estimations, we calculated the proton mean-square displacement using a D-value of 1  10  6 cm2 s  1. The probability of placing our laser beam within the propagating reaction front emanating from a given macroscopic pinhole was then estimated to be 4% and 21% for 1 and 10 s SHG jump times, respectively (Supplementary Note 5, Supplementary Figs. 7–10 and Supplementary Table 3). Given that the pH jumps were repeated on at least 18 different days with eight different graphene samples and delays in changes of the SHG response were not observed with statistical significance; so we conclude that the diffusion of protons from the few macroscopic pinholes that are present in our samples, or, alternatively, from the sample edge, to the area probed by the laser cannot explain our observations of proton transfer through graphene. Aqueous proton transfer across single-layer graphene Jennifer L. Achtyl1, Raymond R. Unocic2, Lijun Xu3, Yu Cai3, Muralikrishna Raju5, Weiwei Zhang6, Robert L. Sacci7, Ivan V. Vlassiouk7, Pasquale F. Fulvio8,9, Panchapakesan Ganesh2, David J. Wesolowski9, Sheng Dai9, Adri C.T. van Duin6, Matthew Neurock3,4 & Franz M. Geiger1 Jennifer L. Achtyl1, Raymond R. Unocic2, Lijun Xu3, Yu Cai3, Muralikrishna Raju5, Weiwei Zhang6, Robert L. Sacci7, Ivan V. Vlassiouk7, Pasquale F. Fulvio8,9, Panchapakesan Ganesh2, David J. Wesolowski9, Sheng Dai9, Adri C.T. van Duin6, Matthew Neurock3,4 & Franz M. Geiger1 Proton transfer across single-layer graphene proceeds with large computed energy barriers and is therefore thought to be unfavourable at room temperature unless nanoscale holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer graphene supported on fused silica to cycles of high and low pH, and show that protons transfer reversibly from the aqueous phase through the graphene to the other side where they undergo acid–base chemistry with the silica hydroxyl groups. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defects. Computer simulations reveal low energy barriers of 0.61–0.75 eV for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a Grotthuss-type relay, while pyrylium-like ether terminations shut down proton exchange. Unfavourable energy barriers to helium and hydrogen transfer indicate the process is selective for aqueous protons. 1 Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60201, USA. 2 Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. 3 Departments of Chemical Engineering and Chemistry, University of Virginia, 102 Engineers’ Way, Charlottesville, Virginia 22904-4741, USA. 4 Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota 55455, USA. 5 Department of Physics, Pennsylvania State University, University Park, Pennsylvania 16802, USA. 6 Department of Mechanical and Nuclear Engineering, Pennsylvania State University, University Park, Pennsylvania 16801, USA. 7 Measurement Science and Systems Engineering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37931, USA. 8 Department of Chemistry, University of Puerto Rico, Rı´o Piedras Campus; San Juan, Puerto Rico 00931, USA. 9 Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. Correspondence and requests for materials should be addressed to F.M.G. (email: geigerf@chem.northwestern.edu). 1 NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications URE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 (b) p-in/all-out polarized SHG response recorded as a function of time from the fused silica/water interface during pH jumps from 7 to 3 to 10 and subsequent pH cycling between 3 and 10 at a bulk aqueous flow of 0.9 ml/s and 1 mM NaCl concentration in the absence (crimson, bottom) and presence (black, top, offset for clarity) of single-layer graphene placed between the fused silica substrate and the flowing bulk aqueous phase. Five-point boxcar indicated by dark lines. (c) Composite of 25 SEM images of single-layer graphene on a fused silica substrate, showing seven macroscopic pinholes, marked by white circles. (d) High-resolution aberration-corrected ADF STEM images of defect-free single-layer graphene on a transmission electron microscopy grid and (e) of a rarely imaged atomic defect. Scale, 1 nm. We find that the main restriction for aqueous proton transfer through pristine, defect-free graphene is the energy required to push the proton through the center of an aromatic ring in the hydrophobic graphene layer as shown in Supplementary Fig. 11. While the protons readily migrate in the solution phase above and below the graphene surface via proton shuttling, they are unable to pass through the hydrophobic graphene layer. The energy costs to desolvate the proton from the aqueous layer and drive it through the center of an intact aromatic ring within the graphene layer are quite high and result in an activation barrier, that is, over 3.8 eV. point defects , , we find, albeit rarely, atomic defects (Fig. 1e). Unless hydrocarbons or heavy metal atoms46 are present in graphene, defect formation due to electron beam-induced etching (as opposed to ion bombardment or oxidative etching)47 of pristine chemical vapour deposition (CVD) graphene at the energies employed here is unlikely. Rather, the rare defects we observe on occasion are more likely to originate from the synthesis process or cosmic rays, as the STEM experiments are carried out below the knock-on damage threshold for graphene48, and the femtosecond laser pulses are attenuated below the onset of processes other than SHG13. Given a lower limit to the estimated defect-to-defect distance of B0.1 mm (ref. 49) (while difficult to determine accurately from Raman spectroscopy, the actual distance is likely to be longer), we assess the probability of placing our laser beam within the propagating reaction front emanating from a given atomic defect to be 100%. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 Mirror /2 plate Lens Lens Lens Fundamental (ω) 600 nm Mirror Graphene film Ring of water Teflon flow cell pH 7 10 10 10 10 10 10 10 3 3 3 3 3 3 3 3 2 Normalized lSHG 1 0 0 1,200 2,400 3,600 4,800 6,000 7,200 8,400 Time (s) Outlet Inlet Solution #2 PMT 40 × 30 μm2 Solution #1 Waste Fused silica hemisphere Fused silica window SHG (2ω) pH conductivity Filter Figure 1 | Experimental approach. (a) Experimental setup using a waveplate (l/2) to prepare 600 nm light plane-polarized parallel to the plane of incidence (p-in) while a photomultiplier tube (PMT) detects the SHG photons at l ¼ 300 nm. (b) p-in/all-out polarized SHG response recorded as a function of time from the fused silica/water interface during pH jumps from 7 to 3 to 10 and subsequent pH cycling between 3 and 10 at a bulk aqueous flow of 0.9 ml/s and 1 mM NaCl concentration in the absence (crimson, bottom) and presence (black, top, offset for clarity) of single-layer graphene placed between the fused silica substrate and the flowing bulk aqueous phase. Five-point boxcar indicated by dark lines. (c) Composite of 25 SEM images of single-layer graphene on a fused silica substrate, showing seven macroscopic pinholes, marked by white circles. (d) High-resolution aberration-corrected ADF STEM images of defect-free single-layer graphene on a transmission electron microscopy grid and (e) of a rarely imaged atomic defect. Scale, 1 nm Mirror /2 plate Lens Lens Lens Fundamental (ω) 600 nm Mirror Graphene film Ring of water Teflon flow cell Outlet Inlet Solution #2 PMT Solution #1 Waste Fused silica hemisphere Fused silica window SHG (2ω) pH conductivity Filter 40 × 30 μm2 pH 7 10 10 10 10 10 10 10 3 3 3 3 3 3 3 3 2 Normalized lSHG 1 0 0 1,200 2,400 3,600 4,800 6,000 7,200 8,400 Time (s) Figure 1 | Experimental approach. (a) Experimental setup using a waveplate (l/2) to prepare 600 nm light plane-polarized parallel to the plane of incidence (p-in) while a photomultiplier tube (PMT) detects the SHG photons at l ¼ 300 nm. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 The basal planes of pristine graphene can, and do, contain rare atomic-scale defect sites comprised of carbon atom vacancies, as shown in Fig. 1e. Our calculations indicate that while the activation barrier for proton transfer through a single-vacancy site is over 1.9 eV lower than that for transfer through the pristine graphene surface, it is still nearly 2.0 eV due to the small size of the vacancy and the hydrophobicity of the surface. The formation of di- and tri-vacancy sites increases the diameter of the opening in the graphene layer and reduces the barrier further to B1.5 eV, but this barrier is still too high to permit aqueous proton transfer at room temperature. Results l l Silanol protonation and deprotonation unimpeded by graphene. Using a dual-pump flow system (Fig. 1a) at a flow rate of at B0.9 ml s  1, we varied the bulk solution pH between 3 and 10 while maintaining constant 1 mM ionic strength (see Methods). As shown in Fig. 1b, we find no significant difference between the SHG versus time traces recorded in the presence and absence of graphene, and no statistically significant differences in the kinetic rates and jump durations (Supplementary Note 1, Supplementary Figs 1–2 and Supplementary Tables 1–2). The SHG responses to pH changes are consistent with the acid–base equilibria of the fused silica/water interface14,15,19,21, yielding effective pKa,eff values of 3.5(1) and 8.3(2), which fall within the reported literature values (Supplementary Note 2 and Supplementary Fig. 3)22. This finding indicates that the SHG experiments do not track merely ion adsorption to the graphene/water interface but acid–base chemistry at the fused silica surface underneath it, for which proton transfer across the membrane is a necessary condition. As expected from refs 1–5, porous graphene multilayers do not inhibit proton transfer either (Supplementary Note 3 and Supplementary Figs 4–5). On the basis of these results we conclude that the fused silica/water interface does not behave differently in terms of relative surface Imaging rare atomic defects. Scanning transmission electron microscopy (STEM) was then used to search for atomic defects using annular dark field (ADF) STEM imaging at 60 kV (see Methods). The majority of the images show perfect six-fold symmetry in the position of the carbon atoms and vast areas that lack grain boundaries and atomic, or vacancy, defects (Fig. 1d). Nevertheless, similar to prior reports of atomic-scale vacancy NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 2 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 ARTICLE & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Discussion T l d Snapshots (c,d) and energetics (e,f) from the nudged elastic band calculations for proton transfer through the oxygen- and OH- terminated defect sites marking (region I) release of proton from H3O þ to oxygen and OH groups, respectively; (region II) relay of proton between oxygen and OH groups, respectively; (region III) release of proton from oxygen and OH groups to H3O þ, respectively. Denotations of spheres: grey ¼ carbon; red ¼ oxygen; white ¼ hydrogen atoms. Additional ReaxFF simulations show that a proton transfer channel, consisting o water molecules that transfer the protons through Grotthuss-type reactions, thins and finally vanishes when the pairs of OH groups terminating the defect site are successively replaced with oxygen atoms (Fig. 3a–d). These transfer paths are selective to aqueous protons as helium and H2 transfer requires barriers exceeding 1.9 eV (Supplementary Note 7 and Supplemen- tary Fig. 14). Table 1 gives the comparison of activation barriers for proton transfer through graphene in water calculated by ReaxFF and DFT. The barriers given by DFT for the pristine and single-vacancy case are high (3.9 and 42.0 eV, respectively) and insurmountable during molecular dynamics (MD) simulations at 300 K. ReaxFF overpredicts the barriers for proton transfer in the pristine and single vacancy case. Yet, the barriers for the relevant quad-vacancy cases given by ReaxFF are in good agreement with DFT. Note that ReaxFF was not specifically trained against any of these barriers. three oxygen atoms in epoxide-like arrangements reminiscent of pyrylium cations (different from the crown ethers recently reported by Guo et al.)52, or with six hydroxide groups. Proton transfer through the pyrylium-terminated quad-vacancy site requires 1.7 eV (Fig. 2a), attributed to the protophobicity of pyrylium cations and their in-plane localization, which leaves a 3.4 Å gap between water and the graphene substrate that prevents proton transfer. The hydroxyl-terminated site (Fig. 2b), however, provides hydrogen-bonding networks (Fig. 2d) that interconnect the graphene surface to the water layers above and below it. DFT and ReaxFF simulations indicate that these hydrogen-bonding networks serve as conduits that facilitate proton transfer from the solution phase to the surface through the center of the defect site and into the solution on the opposite side of the membrane via a Grotthuss mechanism53 involving proton shuttling. Discussion T l d Snapshots (c,d) and energetics (e,f) from the nudged elastic band calculations for proton transfer through the oxygen- and OH- terminated defect sites marking (region I) release of proton from H3O þ to oxygen and OH groups, respectively; (region II) relay of proton between oxygen and OH groups, respectively; (region III) release of proton from oxygen and OH groups to H3O þ, respectively. Denotations of spheres: grey ¼ carbon; red ¼ oxygen; white ¼ hydrogen atoms. Figure 3 | Reactive force field calculations. Proton channel formation fro ReaxFF calculations of water-mediated proton transfer through atomic defects terminated in six OH groups (a), four OH groups and one oxyge atom (b), two OH groups and two oxygen atoms (c), and three oxygen atoms (d). Denotations of spheres: grey ¼ carbon; red ¼ oxygen; white ¼ hydrogen atoms. Figure 3 | Reactive force field calculations. Proton channel formation from ReaxFF calculations of water-mediated proton transfer through atomic defects terminated in six OH groups (a), four OH groups and one oxygen atom (b), two OH groups and two oxygen atoms (c), and three oxygen atoms (d). Denotations of spheres: grey ¼ carbon; red ¼ oxygen; white ¼ hydrogen atoms. Figure 3 | Reactive force field calculations. Proton channel formation from ReaxFF calculations of water-mediated proton transfer through atomic defects terminated in six OH groups (a), four OH groups and one oxygen atom (b), two OH groups and two oxygen atoms (c), and three oxygen atoms (d). Denotations of spheres: grey ¼ carbon; red ¼ oxygen; white ¼ hydrogen atoms. 2.0 1.5 1.0 0.5 0.0 0 2 4 6 8 10 12 Reaction coordinate (Å) (I) (III) (II) Energy (eV) 0 2 4 6 8 Reaction coordinate (Å) 0.75 (I) (III) (II) 0.50 0.25 Energy (eV) 0.00 bottom three defect hydroxyl groups on the other side, and finally release into the aqueous phase. While solution-phase proton shuttling occurs with activation barriers o0.2 eV, the barrier for transferring the proton through the defect sites in graphene via the proton relay mechanism is just 0.68 eV (DFT, Fig. 2b, well reproduced by ReaxFF (0.61 eV)), indicating proton transfer will occur at room temperature. Reaction coordinate (Å) Reaction coordinate (Å) Figure 2 | Density functional theory calculations. Side and top views of Figure 2 | Density functional theory calculations. Side and top views of oxygen- (a) and OH- (b) terminated defect models used in the DFT calculations. Discussion T l d To elucidate the mechanisms for proton transfer, we discuss findings from density functional theory (DFT) calculations (Fig. 2) and ReaxFF reactive force field molecular dynamics (Fig. 3)50,51 simulations (Methods). DFT simulations track the detailed changes in the electronic structure and quantify the corresponding activation barriers as protons transfer from the water layer above the surface through the graphene interface and exit into solution on the opposite side of the surface. The ReaxFF simulations provide a larger scale representation of the interfaces and explicitly include dynamics. All of the defect terminations considered are energetically favourable as compared with the bare quad-vacancy system (Supplementary Note 6 and Supplementary Fig. 12). The removal of four carbon atoms in a central aromatic ring in the graphene layer leads to the formation of the quad-vacancy site as shown in Supplementary Fig. 13. This site is comprised of six coordina- tively unsaturated carbon atoms that are either terminated with 3 & 2015 Macmillan Publishers Limited. All rights reserved ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 bottom three defect hydroxyl groups on the other side, and final release into the aqueous phase. While solution-phase proto shuttling occurs with activation barriers o0.2 eV, the barrier fo transferring the proton through the defect sites in graphene v the proton relay mechanism is just 0.68 eV (DFT, Fig. 2b, we reproduced by ReaxFF (0.61 eV)), indicating proton transfer w occur at room temperature. Additional ReaxFF simulations show that a proton transf channel, consisting o water molecules that transfer the proton through Grotthuss-type reactions, thins and finally vanishes whe the pairs of OH groups terminating the defect site are successive replaced with oxygen atoms (Fig. 3a–d). These transfer paths a selective to aqueous protons as helium and H2 transfer requir barriers exceeding 1 9 eV (Supplementary Note 7 and Supplemen 2.0 1.5 1.0 0.5 0.0 0 2 4 6 8 0 2 4 6 8 10 12 Reaction coordinate (Å) Reaction coordinate (Å) 0.75 (I) (III) (II) (I) (III) (II) 0.50 0.25 Energy (eV) Energy (eV) 0.00 Figure 2 | Density functional theory calculations. Side and top views of oxygen- (a) and OH- (b) terminated defect models used in the DFT calculations. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications 2015 M ill P bli h Li it d All i ht d ARTICLE Solution pH was adjusted with minimum amounts of dilute solutions of B1 M NaOH (Sigma- Aldrich, 99.99%) and HCl (EMD ACS grade). The pH-jump experiments were carried out using a fused silica hemisphere (ISP Optics, 1’’ diameter, QU-HS-25) pressed against either a fused silica window (ISP Optics, 1’’ diameter, QI-W-25-1) or a CVD-prepared graphene film transferred onto a silica window in an experimental setup previously reported13,56. The hemisphere and the fused silica window were cleaned before experiments by first treating the surface of interest with NoChromix (Godax Laboratories) for 1 h, rinsing with Millipore water and then storing in Millipore water overnight for SHG experiments the next day. On the day of the experiment, the bare fused silica window and hemisphere were sonicated in methanol for 6 min, dried in a 110 C oven for 30 min, oxygen plasma cleaned (Harric Plasma) on high for 30 s, and then stored in Millipore water until the experiment. The graphene samples were not cleaned with this procedure, but were instead cleaned by flushing with B2 l of Millipore water before each experiment. Supplementary Note 9 describes the graphene characterization and analysis by Raman and ultraviolet–visible spectroscopy (Supplementary Fig. 16) prior and after the pH-jump experiments. barriers are comparable to recent experimentally determined activation energy barriers for proton transfer through graphene subjected to an externally applied potential12. From the SHG signal jump rates and the time required for 2D proton diffusion, we estimate that the presence of as few as a handful of atomic defects in a 1 mm2 area sample of single-layer graphene is sufficient to allow for the apparent unimpeded protonation and deprotonation of the interfacial silanol groups within 10 s (Supplementary Note 8 and Supplementary Fig. 15). Yet, we caution that given the limited accuracy with which the defect density can be determined in large (mm)-scale graphene, aqueous protons may transfer across single-layer graphene not only along the path discussed here but also along others as well. The identification of low barriers specifically for water-assisted transfer of protons through OH-terminated atomic defects in graphene, and high barriers for oxygen-terminated defects could be an important step towards the preparation of zero-crossover proton-selective membranes. Flow system and flow cell. As shown in Fig. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 Table 1 | DFT and ReaxFF-calculated activation barriers for proton transfer through different vacancy sites on graphene in water. Graphene surface (number of vacancies) Bottom layer Defect termination Activation barrier DFT (eV)* Activation barrier ReaxFF* None Water No termination 3.9 Not computed 1 Water No termination 42.0 3.54 eV 4 Water No termination 0.25 0.22 eV 4 Water 3O ether capped 1.8 1.7 eV 4 Water 6 OH hydroxyl capped 0.68 0.61 eV 4 Water þ SiO2 3 O ether capped 2.5 2.53 eV 4 Water þ SiO2 6 OH hydroxyl capped 0.7 0.75 eV DFT, density functional theory. *The energy difference that is reported is due to the initial reference (or reactant) state. Table 1 | DFT and ReaxFF-calculated activation barriers for proton transfer through different vacancy sites on graphene in water. Aberration-corrected scanning transmission electron microscopy. To confirm the single-layer nature of graphene synthesized using the CVD method13, atomic- resolution STEM imaging was performed at room temperature with an aberration- corrected Nion UltraSTEM-100 (ref. 58) equipped with a cold field-emission electron source. The microscope was operated at 60 kV, which is below the knock- on damage threshold for graphene. The CVD-prepared graphene specimens were transferred to a SiN-supported silicon microchip transmission electron microscopy grid. Before STEM imaging, the specimen was heated at 160 C in vacuum (10  5 torr) for 8 h to remove surface contamination. Following heating in vacuum, the specimen was immediately transferred to the UltraSTEM for ADF STEM imaging. The surface of the graphene still retains residual PMMA that was used in the transfer processes to the transmission electron microscopy grid as shown in Supplementary Fig. 26; however, there are large areas that are devoid of the PMMA, which made it feasible to directly image the lattice structure and confirm the single-layer nature using atomic-resolution STEM imaging. The images were filtered using a smoothing function in Digital Micrograph, and the contrast and brightness were adjusted to enhance the contrast of the graphene. Aqueous solution and substrate preparation. The aqueous solutions were pre- pared with Millipore water, prepared the day before an experiment and left open to air overnight to equilibrate with atmospheric CO2 and NaCl (Alfa Aesar, 99 þ %). The concentration of NaCl was confirmed using a conductivity meter (Fisher Traceable Conductivity and TDS meter, Fisher Scientific). ARTICLE 1a, the graphene-on-fused silica sample or the silica window were clamped face down against a Viton O-ring on the Teflon flow cell13,56 so that the surface of interest was in contact with the aqueous phase. The fused silica hemisphere was then clamped on top of the window with a Millipore water layer in between in order to minimize the change of refractive index between the phases and to avoid the use of an index-matching fluid. Throughout the duration of the experiment, it was also necessary to maintain a ring of Millipore water around the bottom of the hemisphere in order to avoid evaporation of the sandwiched water layer. All of the experiments were completed with a 0.9 ml s  1 flow using variable flow peristaltic pumps as previously reported13,56. Using the flow system depicted in Fig. 1a, the pumps were switched to pull solutions from two different reservoirs. For the experiments reported here (excluding pKa experiments, see Supplementary Note 2), the two reservoirs contained 1 mM NaCl Millipore solutions adjusted to either pH 3 or 10. At the start and end of each pH-jump experiment, a 1 mM NaCl aqueous solution adjusted to pH 7 was pumped through the system, and the SHG signal was collected until it reached a steady state. It is assumed that the steady-state conditions were reached once the SHG signal remained at a stable intensity for a minimum of 300 s. After the system reached the steady state at pH 7, the flow was switched back and forth between the pH 3 and 10 aqueous solutions, each time allowing the SHG signal to reach steady state before switching to the next pH. After several pH 3 to 10 and pH 10 to 3 jumps were completed, the pH was adjusted back to pH 7, and the SHG signal was collected until the steady state was reached one last time. None of the liquid flow effects, reported for fused silica/water interfaces subjected to high shear rates59, were observed under the creeping flow conditions used here. Supplementary Note 10 and Supplementary Table 2 assess the flow dynamics in the cell. Methods CVD h CVD graphene synthesis. We used graphene having a grain size of B100 mm (ref. 54) grown on copper foils by atmospheric pressure CVD54,55. The graphene was transferred using spin coating of poly(methyl methacrylate) (PMMA) followed by copper etching in a FeCl3 solution and PMMA removal in acetone. The transfer was made onto clean fused silica substrates (ISP Optics, 1’’ diameter, QI-W-25-1, flatness 1 wave per inch at 633 nm) to fill B1 cm2 with a single layer. Following annealing in a flow of 4% H2 in Ar for 30 min at 300 C, vibrational sum-frequency generation spectra showed no evidence for CH stretches56. Similar to the finding of water layers between graphene and mica by atomic force microscopy57, there is probably water located between the graphene samples and the fused silica substrates used here. Laser and detection system. A detailed description of our SHG setup has been described previously60–63. Briefly, we use a regeneratively amplified Ti:sapphire system (Hurricane, Spectra Physics) that operates at a kHz repetition rate to produce 120 fs pulses to pump an optical parametric amplifier (OPA-CF, Spectra Physics) tuned to produce 600 nm light. After exiting the OPA, the beam is then directed through a variable density filter to attenuate the pulse energy to either 0.3±0.05 mJ per pulse for bare silica studies or 0.15±0.05 mJ per pulse for graphene studies. The pulse energy used for the graphene films equates to a power density of 2.1(7)  104 mJ cm  2 per pulse with a 30 mm focal spot, which is well below the damage threshold of graphene as previously reported13,56. At an angle just below Scanning electron microscopy. SEM images were collected from the center of the graphene film. Graphene on an optical window was imaged using a Hitachi S-4800 scanning electron microscope (SEM) operating at 2 kV with a probing current of 10 mA and an Everhart–Thornley detector. Copper tape was used to reduce charging effects. Individual images were taken at  1,200 magnification with 1,280  960 resolution. An array of 5  5 images (529  397 mm, pixel size 176 nm) was stitched together using Adobe Illustrator. Automatic brightness and contrast adjustment on each frame was carried out using the ‘auto adjust’ feature in Preview (Apple, Inc.). No other postedit feature or change was applied. Discussion T l d This proton transfer mechanism identified here involves relaying the proton from one of the top three defect hydroxyl groups to the next hydroxyl group and the next, subsequent transfer to one of the three oxygen atoms in epoxide-like arrangements reminiscent of pyrylium cations (different from the crown ethers recently reported by Guo et al.)52, or with six hydroxide groups. Proton transfer through the pyrylium-terminated quad-vacancy site requires 1.7 eV (Fig. 2a), attributed to the protophobicity of pyrylium cations and their in-plane localization, which leaves a 3.4 Å gap between water and the graphene substrate that prevents proton transfer. The hydroxyl-terminated site (Fig. 2b), however, provides hydrogen-bonding networks (Fig. 2d) that interconnect the graphene surface to the water layers above and below it. DFT and ReaxFF simulations indicate that these hydrogen-bonding networks serve as conduits that facilitate proton transfer from the solution phase to the surface through the center of the defect site and into the solution on the opposite side of the membrane via a Grotthuss mechanism53 involving proton shuttling. This proton transfer mechanism identified here involves relaying the proton from one of the top three defect hydroxyl groups to the next hydroxyl group and the next, subsequent transfer to one of the We conclude that aqueous protons transfer through single- layer graphene via rare, OH-terminated atomic defects at room temperature. While the rarity of the atomic defect sites would make it challenging to follow proton exchange across graphene using the pH-sensitive electrodes, the close proximity of the graphene layer and the charged fused silica surface, where the experimental observation of surface protonation and deprotona- tion is made by SHG, allows for the experimental observation of proton exchange across these rare defects. The associated energy NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 4 ARTICLE Am. Chem. Soc. 130, 15444–15447 (2008). 16. Konek, C. T. et al. Interfacial acidities, charge densities, potentials, and energies of carboxylic acid-functionalized silica/water interfaces determined by second harmonic generation. J. Am. Chem. Soc. 126, 11754–11755 (2004). pp y g All of the calculations were carried out within the generalized gradient approximation using Perdew–Burke–Ernzerhof functional66 to treat exchange and correlation gradient corrections and projector augmented wave pseudopotentials67 to describe the electron–ion interactions. Plane wave basis sets with a cutoff energy of 400 eV were used to solve the Kohn–Sham equations for calculations for systems without water. Calculations for systems that include water solvation were carried out with cutoff energies for C and O of 283 eV. The surface Brillouin zone was sampled using a Monkhorst–Pack mesh of 3  3  1. All electronic energies were converged to within a tolerance of 1  10  5 eV. All of the atoms were allowed to relax in the geometry optimizations until the forces on each atom were o0.03 eV Å  1. Spin polarization was examined for all of the systems explored and applied when needed. Transition states were isolated using the nudged elastic band method44,45 together with the dimer method68. The nudged elastic band method was used to provide an initial transition state structure that was used in the subsequent dimer simulations to isolate the transition state. The reaction barrier was defined as the energy difference between the transition state and the reaction state minimum. The intrinsic barrier is defined as the energy gap between a transition state and its immediate reaction state. Given the importance of surface relaxation in atomically defected graphene layers69, all of our calculations on the single, di and quad carbon vacancy sites and the oxygen-terminated sites explicitly modeled surface relaxation (Supplementary Note 11 and Supplementary Figs 17–19). g 17. Stumm, W. & Morgan, J. J. Aquatic Chemistry, Chemical Equilibria and Rates in Natural Waters 3rd edn (John Wiley & Sons, 1996). 18. Higgins, S. R., Stack, A. G., Knauss, K. G., Eggleston, C. M. & Jordan, G. Probing molecular-scale adsorption and dissolution-growth processes using nonlinear optical and scanning probe methods suitable for hydrothermal applications. Geochem. Soc. Special Publication No. 7 111 (2002). 19. Azam, M. S., Weeraman, C. N. & Gibbs-Davis, J. M. Halide-induced cooperative acid–base behavior at a negatively charged interface. J. Phys. Chem. C 117, 8840–8850 (2013). 20. Duval, Y., Mielczarski, J. ARTICLE A., Pokrovsky, O. S., Mielczarski, E. & Ehrhardt, J. J. Evidence of the existence of three types of species at the quartz-aqueous solution interface at pH 0-10: XPS surface group quantification and surface complexation modeling. J. Phys. Chem. B 106, 2937–2945 (2002). 21. Stack, A. G., Higgins, S. R. & Eggleston, C. M. Point of zero charge of a corundum-water interface probed with optical second harmonic generation (SHG) and atomic force microscopy (AFM): new approaches to oxide surface charge. Geochim. Cosmochim. Acta 65, 3055–3063 (2001). charge. Geochim. Cosmochim. Acta 65, 3055–3063 (2001) 22. Azam, M. S., Weeraman, C. N. & Gibbs-Davis, J. M. Specific cation effects on the bimodal acid–base behavior of the silica/water interface. J. Phys. Chem. Lett. 3, 1269–1274 (2012). g The ReaxFF simulations were performed using the stand-alone ReaxFF implementation to study proton transfer through pristine graphene and graphene with di and quad vacancies. We then compared with results from long ab initio MD to validate predictions of force field in describing water/graphene systems (Supplementary Note 12 and Supplementary Fig. 20). In our simulations, we used a (6  6) periodic graphene sheet with water molecules placed in random configurations on either side of the graphene sheet. The dimensions of the simulation cell are 15.01  17.83 Å parallel to the sheet and 30 Å in the direction perpendicular to the sheet. All MD simulations have been performed in the canonical (NVT, constant number of atoms (N), constant volume (V) and constant temperature (T)) ensemble, with a time step of 0.25 fs using the Berendsen thermostat with a coupling time constant of 100 fs to control temperature of the entire system. To obtain the density plots in Fig. 3, we first divided the simulation cell into a mesh of cubic boxes with dimensions (0.30  0.30  0.30 Å). We then counted the number of times a particular atom type (for example, oxygen) was located in each of the grids through the entire length of simulation and normalized these numbers by the highest count recorded in any of the grids. We used these normalized values to obtain the resulting density plots in Fig. 3. The ReaxFF results reproduce the DFT results, described well in further detail in Supplementary Note 13 and Supplementary Figs 13 and 21–25. 23. Oura, K., Lifshits, V. G, Saranin, A. A., Zotov, A. V. & Katayama, M. Surface Science: An Introduction (Springer, 2003). ARTICLE 3, 209–213 (2011). 7. Banerjee, S. et al. Electrochemistry at the edge of a single graphene layer in a nanopore. ACS Nano 7, 834–843 (2013). 8. Wang, X. D. et al. Atomistic origins of high rate capability and capacity of N-doped graphene for lithium storage. Nano Lett. 14, 1164–1171 (2014). 9. Bunch, J. S. et al. Impereable atomic membranes from graphene sheets. Nano Lett. 8, 2458–2462 (2008). 10. Miao, M., Nardelli, M. B., Wang, Q. & Liu, Y. First principles study of the permeability of graphene to hydrogen atoms. Phys. Chem. Chem. Phys. 15, 16132–16137 (2013). Computer simulations. First-principles periodic DFT calculations were carried out to determine the lowest energy interfacial water/graphene, water/graphene/ water/silica structures and the activation barriers for proton diffusion through these interfaces using the Vienna Ab Initio Simulation Package64,65. In the DFT calculations, the reaction systems were modelled by optimizing a water phase above and below a single-graphene sheet. The simulations were carried out in a 5  5 supercell comprised of 50 carbon atoms, extended infinitively in the x and y dimensions. A 15 Å gap was inserted between the graphene layer perpendicular to the surface. The gap was subsequently filled with enough water molecules to match the overall density of water at 1.0  103 kg m  3. The initial simulations were carried out with water on both sides of the graphene layer. The lower SiO2 substrate was initially simplified by using additional water. Subsequent calculations were carried out with more realistic slabs comprised of water/graphene/water/SiO2 substrates. The reaction rates and mechanisms of proton transfer through the graphene were described in the framework of transition state theory and within the harmonic approximation, which is robust for systems of high densities. 11. Jiang, D., Cooper, V. 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The reflected fundamental and second harmonic lights are directed through a Schott filter and a monochromator to remove any contributions at the fundamental frequency before amplification with a photomultiplier tube and detection using a gated single-photon counting system. Correct power dependencies and spectral responses are verified regularly, the SHG responses are well polarized, and sample damage does not occur13,56. Given that the SHG jump rates are independent of the mean stream velocity (Supplementary Note 1), we are confident that the acid–base reactions occurring at the fused silica surface are not mass transfer limited. Ultraviolet–visible and Raman spectra indicate that the samples are resistant to acid–base cycling under the conditions employed here. total internal reflection, the p-polarized attenuated fundamental light is then directed through a fused silica hemisphere and focused at the graphene/water or silica/water interface. The reflected fundamental and second harmonic lights are directed through a Schott filter and a monochromator to remove any contributions at the fundamental frequency before amplification with a photomultiplier tube and detection using a gated single-photon counting system. Correct power dependencies and spectral responses are verified regularly, the SHG responses are well polarized, and sample damage does not occur13,56. Given that the SHG jump rates are independent of the mean stream velocity (Supplementary Note 1), we are confident that the acid–base reactions occurring at the fused silica surface are not mass transfer limited. Ultraviolet–visible and Raman spectra indicate that the samples are resistant to acid–base cycling under the conditions employed here. total internal reflection, the p-polarized attenuated fundamental light is then directed through a fused silica hemisphere and focused at the graphene/water or silica/water interface. 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DOI: http://dx.doi.org/10.14236/ewic/EVA2017.83 DOI: http://dx.doi.org/10.14236/ewic/EVA2017.83 1. INTRODUCTION work. For example, our visualisation “Sky Pacers” was partially inspired by an ancient Chinese star catalogue in the form of a poem specifying the names of stars, “The Song of the Sky Pacers”. The ancient Chinese stone carvings representing the constellations were also inspirational in our deliberations. Another line of poetry that influenced the work came from William Blake’s poem “The Tyger”. “When the serenity of the great void and the fury of the cosmos intersect, when quantum mechanics and relativity come face to face, tiptoe and waddle, skitter and scuttle, whirl and whisk, behold: The Prophecy of the Sky Pacers is now! There is no escaping our cosmic destiny.” “Glints of light, passing of shadows, the choreography of perpetual existence sets the stage for this Delphian ballet. The impenetrable flow of life’s rhythms, their Sisyphean inceptions and cessations are punctuated with eternity’s ephemeral modulations. The transmigration has begun, the beginning of the end, the end of the beginning.” “When the stars thew down their spears and watered heaven with their tears.” “When the stars thew down their spears and watered heaven with their tears.” “When the stars thew down their spears and watered heaven with their tears.” Emily Dickinson’s “The First Day's Night Had Come” creates an inspiring poetic image for another section of the piece: “She said her Strings were snapt— Her Bow—to Atoms blown— And so to mend her—gave me work Until another Morn—” Above are two descriptions of some of the works we will look examine. The essence of the presentation is to demonstrate how two seemingly opposite approaches, a humanistic, poetic approach and that of digital imaging technology can be integrated in the creation of new works of art. Robert Frost’s epigram "Pertinax," contains the following illuminating lines: Let chaos storm! Let cloud shapes swarm! I wait for form. Let chaos storm! Let cloud shapes swarm! I wait for form. Poetry Inspired, Physics Driven, Experimental Animation Harvey Goldman University of Massachusetts, Dartmouth 340 Barney’s Joy Road Dartmouth, MA 02748, USA hg_art@icloud.com 2. THE POETICS OF DIGITAL VISUALISATION. Once these poetic depictions are gathered and internalised, our process is to let them percolate while filtering the feelings they evoke through some highly sophisticated software. Both 2D and 3D animation techniques along with physics based particle systems are employed with the hope of capturing the organic nature of the poetic imagery. The ability to work with gravity, wind, air, viscosity and turbulence as creative tools has opened up new areas of exploration for us. For the last 5 years composer Jing Wang and I have been collaborating on a series of experimental electronic visualisations. The work falls under the genres of “Visual Music” or “Experimental Animation.” One of our less luminous objectives has been to bring a more organic and natural feel to our digital based creative work. The field of electronic visualisation has a strong inclination towards mathematics. Geometric forms and movements are common place. In an attempt to move past these stereotypes, we have attempted to incorporate the use of poetry as inspiration for both the form and structure of our © Goldman. Published by BCS Learning and Development Ltd. Proceedings of Proceedings of EVA London 2017, UK 420 Poetry Inspired, Physics Dri Harvey Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. Poetry Inspired, Physics Driven, Experimental Animation Harvey Goldman Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. We are currently worki “Uriel” based on a poem transcendentalist poet R this work, we are not o imagery created in the w and form/structure of the more poignant sections f “With a look that solved and stirred the devils e Gave his sentiment div Against the being of a l Poetry Inspired, Physics Driven, Experimental Animation Harvey Goldman Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. We are currently working on a new piece titled “Uriel” based on a poem of the same name by the transcendentalist poet Ralph Waldo Emerson. In this work, we are not only inspired by the poetic imagery created in the work but the “dramatic arc” and form/structure of the poem as well. One of the more poignant sections follows: “With a look that solved the sphere and stirred the devils everywhere, Gave his sentiment divine Against the being of a line.” Poetry Inspired, Physics Driven, Experimental Animation Harvey Goldman Figure 1: Still from: “Sky Pacers”. 2. THE POETICS OF DIGITAL VISUALISATION. We are currently working on a new piece titled “Uriel” based on a poem of the same name by the transcendentalist poet Ralph Waldo Emerson. In this work, we are not only inspired by the poetic imagery created in the work but the “dramatic arc” and form/structure of the poem as well. One of the more poignant sections follows: “With a look that solved the sphere and stirred the devils everywhere, Gave his sentiment divine Against the being of a line.” Figure 1: Still from: “Sky Pacers”. Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. Figure 2: Still from: “Ouroboros”. Figure 2: Still from: “Ouroboros”. 421
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https://wjso.biomedcentral.com/counter/pdf/10.1186/s12957-024-03319-3
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The management of uterine tumor resembling an ovarian sex cord tumor (UTROSCT): case series and literature review
World journal of surgical oncology
2,024
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Open Access Open Access Abstract Aims  To present a case series of 11 rare uterine tumors resembling ovarian sex cord tumors (UTROSCTs), and review the literature on this topic to offer up-to-date treatment management for UTROSCTs. Method  Eight cases from Fujian Cancer Hospital between January 2017 and May 2023 and three patients from Fujian Union Hospital between October 2012 and October 2020 were retrospectively reviewed. All cases were pathologically confirmed as UTROSCTs by two senior and experienced pathologists. Clinical behaviors, medical data, histopathologi- cal features, therapy approaches, and survival outcomes were discussed. Results  The median age at initial diagnosis was 53 years (29–70 years). 3 (27.3%) patients were under 40. Seven cases presented with abnormal vaginal bleeding, one with menstrual disorder, one with abnormal vaginal secre- tion, and two patients were accidentally found by physical examination without any symptoms. Three patients were initially misdiagnosed with endometrial cancer by MRI. Curettage was performed in all cases. Nine of them were well diagnosed by routine curettage, except for two samples, which were identified after surgery. Immunohistochemical biomarkers, such as CD99, Desmin, WT-1, CK, Vimentin, SMA, α-Inhibin, Ki67, CD56, ER, PR, and CR, tend to be positive in UTRO SCs patients. Six patients underwent hysterectomy with bilateral salpingo-oophorectomy. Two cases received a radical hysterectomy with bilateral salpingo-oophorectomy, retroperitoneal lymph node dissection, and omentum dissection. Three UTROSCTs were under observation after mass resection. The median PFS was 24 months (range 1–125 months). Conclusion  UTROSCT is a rare mesenchymal tumor with low malignant potential. Treatment modalities should be carefully considered to balance the therapy outcomes and patient needs. Surgery conservative management might be suitable for young women with fertility desires. Keywords  UTROSCT, Prognosis, Hysterectomy, Conservative management †Jie Lin and Linying Liu contributed equally to this work. Lin et al. World Journal of Surgical Oncology (2024) 22:42 https://doi.org/10.1186/s12957-024-03319-3 Lin et al. World Journal of Surgical Oncology (2024) 22:42 https://doi.org/10.1186/s12957-024-03319-3 World Journal of Surgical Oncology Lin et al. World Journal of Surgical Oncology https://doi.org/10.1186/s12957-024-03319-3 Radiological imaging characteristics d l l ld h Radiological imaging sometimes could have been deceiv- ing due to the small population size and limited knowl- edge about UTROSCT. The max SUV value in the myometrium is 4.5 under PET-CT examination after hysteroscopic curettage in case 3 (Fig. 1A) with con- servative therapy. An increasing endometrial thickness was observed in cases 4 and 10 (Fig. 1B, C). A mass with myometrial invasion of case 11 which was misdiagnosed as endometrial cancer was finally proved to be UTRO- SCT (Fig. 1D). Case 6 was also suspected as endometrial cancer by MRI examination (Fig. 1E), so as case 10. No residual tumor was noted after curettage (Fig. 1F). Other patients didn’t conduct a radiology examination. The radiological imaging findings are presented in Fig. 1. †Jie Lin and Linying Liu contributed equally to this work. g sunyang@fjzlhospital.com Full list of author information is available at the end of the article © The Author(s) 2024. 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://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 2 of 9 Lin et al. World Journal of Surgical Oncology (2024) 22:42 Pathological findings Gl d l h l Glandular epithelium, basal-like, and myoepithelial cells are the three main kinds of cells that could be found in UTROSCT patients. Under the light microscope, we could see specific common structures: myxedema, sex cord-like structure, collagen degeneration, foam-like cells, organ-like structure, and muscle layer infiltration, which are often mixed and concurrence (Fig. 2). Materials and methods Clinical data A total of 11 UTROSCT patients from two institutions from October 2012 and October 2020 were included. Intrauterine tissue and surgical specimens were reviewed and confirmed by two senior and experienced pathol- ogists. The clinical and follow-up information was obtained through medical files and telephone contact. We collected information such as menopausal status, parity, symptoms, tumor positions, tumor biomarkers, image data, immunohistochemical biomarkers, treat- ment approaches, and survival status. May 31, 2023, was the final censoring date for assessing the survival time. Results Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a sporadic and controversial disease with unclear origins, named for resembling the morphol- ogy of ovarian sex cord tumors [1]. It mainly occurs in women in their 50  s and occasionally in young women with childbearing needs [2]. UTROSCT patients always present with irregular vaginal bleeding or chronic pelvic pain. However, some can be discovered by accidents [3]. Although UTROSCT is generally considered benign or has a low-malignant potential, it could metastasize some- times. There are no established treatment protocols for UTROSCT so far. Surgical intervention is usually recom- mended with a favorable prognosis. Clinical features and treatment outcomes The age ranged from 29 to 70 (median = 53) at the initial diagnosis. Seven cases presented with vaginal bleeding, one with menstrual disorder, and one with vaginal secretion. While two showed no symptoms. No elevation of serum tumor biomarkers such as CA125, CA199, and CEA were observed in 11 cases. The mass positions were in the endometrium (4 cases) and myo- metrium (7 cases). None of the patients received fur- ther postoperative treatment. The median follow-up time was 24  months (1–125  months), and no recur- rence or metastasis was noted. Details are presented in Table 1. Although there has been modest literature on UTRO- SCT in recent years, focusing mainly on clinicopatho- logical characteristics and gene variations, relatively few advances have been made in treatment strategies due to the low incidence. Here we reported 11 UTROSCT cases with different therapy approaches together with an updated literature review to expand our knowledge of UTROSCT management. Interventions Pathology is the golden standard for UTROSCT diag- nosis. Patients with UTROSCT showed a diverse phe- notype profile, according to immunohistochemistry performed on UTROSCT patients (Fig. 3, Table 2). Bio- markers such as inhibin, CD99, CD56, WT-1, and CK tend to be positive in UTROSCTs. Most tumors had exhibited at least one of the standard sex-cord mark- ers, including CD99, WT-1, and α-Inhibin (Table 2). The positive expression rates of α-inhibin (Fig. 3A), CD99 (Fig. 3B), Vimentin (Fig. 3C) and WT-1 (Fig. 3D) were 3/10, 7/9, 5/6, 8/8, respectively. Almost all patients (9/11) expressed one or more smooth muscle markers, and the positive expression rates of desmin (Fig. 3E) and SMA All patients underwent diagnostic curettage, three of which were hysteroscopically assisted. For further steps, six patients underwent hysterectomy with bilat- eral salpingo-oophorectomy, two cases received radi- cal hysterectomy with bilateral salpingo-oophorectomy, retroperitoneal lymph node dissection, and omentum dissection, and three were under observation after tumo- rectomy due to their desires for children-bearing in the near future. All surgeries were performed by experienced gynecology surgeons. After treatment, patients were advised to have follow-up visits every three months in the first 2 years, every six months in the 3–5 years, and once a year after 5 years. Lin et al. Abbreviations: NA not available, − negative Interventions World Journal of Surgical Oncology (2024) 22:42 Page 3 of 9 Table 1  Clinical features of UTROSCT patients bb l bl Case Age Menopausal status Parity Symptoms Tumor position Tumor biomarkers Treatment approaches Recurrence CA199 U/ml CEA ng/ml CA125 U/ml 1 39 No 2–0-0–2 Vaginal bleeding Submucosa (myometrium) 13.61 2.59 9.06 Mass resection No (1 month) 2 34 No 1–0-0–1 Menstrual disorder Submucosa (myometrium)  −  NA NA Mass resection No (2 months) 3 29 No 1–0-1–1 No symptoms Submucosa (myometrium)  −  NA NA Mass resection No (54 months) 4 40 No 0–0-0–0 No symptoms Endometrium 9.39 NA 23 Hysterectomy No (24 months) 5 67 Yes 2–0-0–2 Vaginal bleeding Submucosa (myometrium) 17.72 2.5 6.64 Hysterectomy No (5 months) 6 53 No 2–0-0–2 Vaginal bleeding Endometrium 6.64 1.49 12.44 Hysterectomy No (6 months) 7 55 Yes 2–0-1–2 Vaginal bleeding Submucosa (myometrium) NA NA NA Hysterectomy No (66 months) 8 70 Yes 5–0-1–5 Vaginal bleeding Submucosa (myometrium) NA NA NA Hysterectomy No (125 months) 9 52 Yes 2–0-0–2 Vaginal bleeding Endometrium 14.64 2 8 Hysterectomy No (29 months) 10 57 Yes 3–0-0–3 Vaginal secretion Endometrium NA NA NA Radical surgery No (31 months) 11 59 Yes 2–0-0–2 Vaginal bleeding Submucosa (myometrium) - 1.54 NA Radical surgery No (13 months) Fig. 1  UTROSCT (arrow) in different images of PET-CT and MRI. A A max SUV was 4.5 in the myometrium. B–D UTROSCT is mildly hypointense compared to endometrium signals on sagittal T2 sequence. E UTROSCT is similar to uterus signal and well-delineated on dynamic contrast-enhanced sequence. F No mass observed after curettage Fig. 1  UTROSCT (arrow) in different images of PET-CT and MRI. A A max SUV was 4.5 in the myometrium. B–D UTROSCT is mildly hypointense compared to endometrium signals on sagittal T2 sequence. E UTROSCT is similar to uterus signal and well-delineated on dynamic contrast-enhanced sequence. F No mass observed after curettage PR (Fig. 3I) expressed variably among UTROSCTs. A low Ki67(Fig. 3J) index was observed among the patients (median 5%, range 2–30%). While CD56 (Fig. 3K) and (Fig. 3F) were 8/10 and 6/10, respectively. CK (Fig. 3G) as an epithelial biomarker was positive in most cases (10/11), and hormone receptors like ER (Fig. 3H) and Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 4 of 9 Page 4 of 9 Fig. 2  Morpholohistopathology of UTROSCT. Interventions UTROSCT is characterized by various architectures including A myxedema, B sex cord-like structure, C collagen degeneration, D foam-like cell, E organ-like structure. F muscle layer infiltration (H&E × 10) Fig. 2  Morpholohistopathology of UTROSCT. UTROSCT is characterized by various architectures including A myxedema, B sex cord-like structure, C collagen degeneration, D foam-like cell, E organ-like structure. F muscle layer infiltration (H&E × 10) Fig. 3  Immunohistochemical biomarkers of UTROSCT. A α-inhibin. B CD99. C Vimentin. D WT-1. E desmin. F SMA. G CK. H ER. I PR. J Ki67. K CD56. L Calretinin (IHC × 10) Fig. 3  Immunohistochemical biomarkers of UTROSCT. A α-inhibin. B CD99. C Vimentin. D WT-1. E desmin. F SMA. G CK. H ER. I PR. J Ki67. K CD56. L Calretinin (IHC×10) Fig. 3  Immunohistochemical biomarkers of UTROSCT. A α-inhibin. B CD99. C Vimentin. D WT-1. E desmin. F SMA. G CK. H ER. I PR. J Ki67. K CD56. L Calretinin (IHC × 10) Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 5 of 9 Table 2  Immunohistochemical biomarkers in the 11-patient cohort Abbreviations: + positive, − negative, NA not available Case CD99 Desmin WT-1 CK Vimentin SMA α-Inhibin Ki67 CD56 ER PR CR 1 NA  +   +   +  NA  +   −  1%  +   +   +   +  2  +   +   +   +   +   +   −  2% +   +   +   +  NA 3  +   +   +   +   +   +   +  1% +  NA NA NA  +  4  −   −  NA  −   +   −   −  10% +   +   −   −   +  5  −  NA  +   +  NA  +   +  10% +   +   +   +   +  6  +   +   +   +   −   −   +  10% +  NA  +   +  NA 7  +   +  NA  +  NA  +   −  2% NA  +   +   +  8  +   −  NA  +   +   −   −  NA NA NA NA NA 9  +   +   +   +  NA  +   −  30% +   +   +  NA  +  10 NA  +   +   +  NA  −   −  40%  +   +  NA NA 11  +   +   +   +   +  NA NA 10% +  NA  +   +   − Table 2  Immunohistochemical biomarkers in the 11-patient cohort Abbreviations: + positive, − negative, NA not available recorded that hysterectomy is the prior surgery approach in 71 out of 75 UTROSCT patients [40]. Discussion In 1976, Clement and Scully first reported certain kinds of rare uterine tumors with histologic resemblances to ovarian sex-cord tumors [4]. Those types of tumors could be divided into two groups. The first group is endometrial stromal tumors with sex cord-like elements (ESTSCLE) with a particular propensity to recur or metastasize, and UTROSCT is the other group [5]. UTROSCT is rare and usually recognized as a benign tumor. Clinical observa- tions and literature data indicated a better prognosis [6]. However, sporadic reports showed it could metastasize and recur sometimes. In 2017, Moore documented 34 UTROSCT cases, of which about 23.5% exhibited malig- nant behavior [7]. Due to the potential malignancy and limited experience, treatment recommendations have not been well established. Interventions Besides, most patients had good survival outcomes simply by remov- ing the uterine [21, 22, 40]. Moreover, some scholars claimed recurrence cases previously treated by hyster- ectomy [11, 12, 20] could still get prolonged survival by completely removing the recurrent mass. In our study, 6 out of 11 patients treated by hysterectomy did not experi- ence recurrence and metastasis after a median follow-up time of 26.5 months (range 5–125 months). However, we still need a longer follow-up time and more cases to make survival outcomes more convincing. Calretinin (Fig. 3L) are often used for differentiated diagnosis. Radical surgery for UTROSCTs with high risk of recurrence or metastasis UTROSCT has uncertain malignant potential due to its low recurrence rate [7]. Distant metastasis and local recurrence have been occasionally reported [11, 22, 28, 29]. Our review found that the recurrence rate was low (19/117) during the median follow-up time between 20  days and 32  years. Currently, predictive features of aggressive UTROSCTs are poorly understood. Risk fac- tors, such as myometrial invasion, serosal involvement, lymph-vascular space invasion (LVSI), GREB1-NCOA2 fusion, NCOA2 or NCOA3 [23, 41, 42], and high mitotic activity [40, 43]have been proven to be associated with tumor recurrence. Baris Boyraz concluded that high- risk UTROSCT showed more than three of the following five features: at least moderate cytologic atypia, tumor size > 5  cm, above 3 mitoses/10 at high power fields (HPFs), marginal infiltration, and necrosis [40].i Surgery is recommended as the preferred treatment for UTROSCT patients [8]. Hysterectomy or mass resection alone are possible options for its management [9]. A sys- tematic review of the literature on UTROSCTs’ treatment approaches is detailed in Table 3. With advancements in medicine and humanities, individualized-oriented tumor treatments have been deeply rooted in people’s hearts. We should pay more attention to patients with special needs or in different conditions, and then make person- alized plans to balance the therapy outcomes and their needs. i High-risk UTROSCT patients could benefit from radical surgery. Miho believed hysterectomy alone was associated with a higher rate of recurrence. Extended radical surgery, including bilateral salpingo-oophorectomy, lymphadenec- tomy, and omentectomy, may reduce the recurrence rate Hysterectomy for UTROSCTs in the middle and old age i High-risk UTROSCT patients could benefit from radical surgery. Miho believed hysterectomy alone was associated with a higher rate of recurrence. Extended radical surgery, including bilateral salpingo-oophorectomy, lymphadenec- tomy, and omentectomy, may reduce the recurrence rate Hysterectomy for UTROSCTs in the middle and old age Hysterectomy resection with or without salpingo-oopho- rectomy should be a primary option for middle-aged or older women with UTROSCT, especially in patients whose follow-up cannot be guaranteed [9]. Baris Boyraz Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 6 of 9 of UTROSCT with sarcomatous features [34]. Based on the review in Table 3, no recurrence or distant metastasis was observed among 8 UTROSCT patients treated with radical surgery, indicating radical surgery seemed to be the optimal choice for high-risk patients. However, it should be attention that high-risk UTROSCT is often detected Table 3  Different therapies and outcomes of UTROSCT patients reported in the literature (2014 ~ 2023) Abbreviations: Ned no evidence of disease. Conservative treatment: tumorectomy, myomectomy, and electrical resection, etc. Present cases No Year Age Outcome (follow-up) Hysterectomy ± bilateral salpingo-oophorectomy Gutierrez-Pecharroman, et al. [10] 1 2014 49 Ned (18 months) Bakula-Zalewska, et al. [6] 4 2014 50–63 Ned (3–14.5 years) Moore, et al. [7] 31 2017 26–86 7/31 recurrence (6–135 months) Kondo, et al. [11] 1 2018 63 1/1 recurrence (6 years) Croce, et al. [12] 1 2019 72 1/1 recurrence (17 months) Dubruc, et al. [13] 1 2019 56 Ned (4 months) Zhang, et al. [14] 2 2019 33, 64 Ned (12 days–1 year) Lee, et al. [15] 2 2019 34, 47, 59 Ned (116 months–20 years) Vilos, et al. [16] 2 2019 47, 52 Ned (1–3 years) Dimitriadis, et al. [17] 1 2020 46 1/1 recurrence (2 years) Grither, et al. [18] 1 2020 69 Ned (8 months) Chang, et al. [19] 1 2020 57 1/1 recurrence (30 months) Bennett, et al. [20] 3 2020 32, 37, 54 3/3 recurrence (7–32 years) Kaur, et al. [21] 5 2020 42–50 1/5 recurrence (1 month–2 years) Goebel, et al. [22] 10 2020 34–74 1/10 recurrence (1–319 months) Zhou, et al. [8] 1 2021 51 Ned (58 months) Devereaux, et al. [23] 1 2021 42 1/1 recurrence (6 months) Shibahara, et al. [24] 1 2022 77 Ned (12 months) Wang, et al. [3] 1 2022 42 Ned (2 months) Pang, et al. Hysterectomy for UTROSCTs in the middle and old age Oriana Marrucci reported a rare vagi- nal vault recurrence case 5 years after total hysterectomy with bilateral salpingo-oophorectomy. She had a favorable prognosis with only a second surgery [44]. Shigeaki Umeda also represented a case with epiploic appendix metasta- sis, who was treated with surgery and has been followed up for 8 years without evidence of recurrence [28]. How- ever, Michelle presented an unusual case of an aggressive UTROSCT who underwent neoadjuvant chemotherapy followed by optimal cytoreductive surgery and adjuvant chemotherapy and still died 15  months after her initial diagnosis [31]. Therefore, the selection of auxiliary treat- ments needs to consider the specific situations of UTRO- SCT patients. resection. After a comprehensive examination without signs of residue tumor, case 2 opted for surveillance. The menstruation returned to normal after conservative ther- apy. She has been followed up for two months with dis- ease-free. In case 3, a PET/CT showed a max SUV value of 4.5 in the myometrium, which cannot be confirmed as postoperative changes or residue tumors. After exhaus- tive discussions with patients, she still chose to observe due to her eager solid of fertility preservation. Surpris- ingly, there are no signs of recurrence or metastasis after a follow-up of 54 months. Case 1 had a myomectomy and showed no sign of recurrence. She strongly desired to preserve her organs and decided to be closely observed. A conservative, uterus-preserving treatment appears jus- tified in those whose close follow-up can be guaranteed, even in high-risk ones. Once recurrence, patients could still get a favorable prognosis after salvage surgery [7]. Further investigations are needed to prove the safety of organ-preserving strategy in UTROSCT. UTROSCT is somehow inert and quickly got the atten- tion of irregular uterine bleeding. All patients in our research were diagnosed in the early stages. A hysterec- tomy seems enough. However, a 40-year-old female still firmly chose to have extra salpingo-oophorectomy due to psychological fear of tumor recurrence, even after being fully educated on UTROSCT. Moreover, pre-surgery examinations, such as MRI images and curettage pathol- ogy, could sometimes be confusing, leading to over- treatment for oncological safety. Clinicians should focus more on aggressive UTROSCT features patients shared, such as tumor size, myometrial invasion, LVSI, and gene fusion, to determine the surgical approaches. Working with an experienced pathologist is necessary. Still, the role of hormone therapy is also controversial. Sabrina M. Hysterectomy for UTROSCTs in the middle and old age [25] 2 2022 42, 46 Ned (4–35 months) Ye, et al. [2] 4 2022 40–70 Ned (uncertain) Zhou, et al. [26] 1 2023 49 Ned (1 months) Lu, et al. [27] 11 2023 37–60 Ned (11–141 months) Radical surgery Umeda, et al. [28] 2 2014 38, 57 Ned (11 months–8 years) Mačák, et al. [29] 1 2015 53 Ned (10 months) Cetinkaya, et al. [30] 1 2016 52 Ned (17 months) Kuznicki, et al. [31] 1 2017 49 Death (15 months) Fan, et al. [32] 1 2018 62 Ned (20 days) Segala, et al. [33] 1 2019 62 Ned (5 years) Sato, et al. [34] 1 2020 57 Ned (3 years) Conservative treatment Bakula-Zalewska, et al. [6] 2 2014 24,25 Ned (4.5–7 years) Watrowski, et al. [35] 1 2015 22 Ned (28 months) Jeong, et al. [36] 1 2015 32 Ned (47 months) De Franciscis, et al. [37] 1 2016 38 Ned (60 months) Moore, et al. [7] 2 2017 12, 36 Ned (27–63 months) Schraag SM, et al. [38] 2 2017 24, 28, 72 2/2 recurrence (6–39 months) Lee, et al. [15] 1 2019 34 Ned (49 months) Goebel, et al. [22] 1 2020 38 Ned (50 months) Carbone, et al. [39] 2 2021 25, 30 Ned (96 months,16 months) Ye, et al. [2] 1 2022 40 Ned (uncertain) Lu, et al. [27] 6 2023 27–50 Ned (17–55 months) Table 3  Different therapies and outcomes of UTROSCT patients reported in the literature (2014 ~ 2023) radical surgery, indicating radical surgery seemed to be the optimal choice for high-risk patients. However, it should be attention that high-risk UTROSCT is often detected of UTROSCT with sarcomatous features [34]. Based on the review in Table 3, no recurrence or distant metastasis was observed among 8 UTROSCT patients treated with of UTROSCT with sarcomatous features [34]. Based on the review in Table 3, no recurrence or distant metastasis was observed among 8 UTROSCT patients treated with Lin et al. World Journal of Surgical Oncology (2024) 22:42 Lin et al. World Journal of Surgical Oncology Page 7 of 9 incidentally after a hysterectomy surgery, which makes the initial treatment insufficient. For this reason, the roles of adjuvant therapies should be carefully considered. Surpris- ingly, the literature showed adjuvant treatments such as chemotherapy, hormone therapy, or radiotherapy were still in debate for the UTROSCTs with high risks of recurrence or metastasis [35]. Conservative therapy for UTROSCTs with fertility desire Conservative therapy for UTROSCTs with fertility desire With the development of minimally invasive surgery, tumorectomy by hysteroscopy or laparoscopy could be expected in young UTROSCTs who desire to preserve their organs and fertility. Many scholars reported suc- cessful pregnancies in young UTROSCT women treated with fertility-preserving surgery and had no sign of recurrence [38, 39], even in a myometrial invasion case [36]. Only one relapsed case after 20  months of con- servative surgery [38]. In our review, all UTROSCTs with conservative therapy had favorable prognoses. Most researchers believed UTROSCT patients could main- tain fertility without affecting the survival rate. In our study, three cases (Nos. 1, 2, and 3) were done with mass Hysterectomy for UTROSCTs in the middle and old age Schraag recorded that surgery and endo- crine treatment were applied for a young UTROSCT with recurrence. She was disease-free 34  months after the last surgery and was still on a monitor [38]. High- dose progesterone therapy was also applied after surgery in a UTROSCT with pelvic lymph node metastasis, and no recurrence was observed [28]. While Daisuke Endo pointed out that hormone therapy may be ineffective for recurrent tumors [45]. Some scholars even believed hor- mone therapy such as tamoxifen progressed this disease [33]. In our study, none patients received additional treat- ments, and no recurrences were observed. To be pointed out, two cases received radical surgery due to malignancy features shown in MRI images and pathology ambigu- ity. Based on the above, we believe the choice of surgi- cal approaches should be carefully considered based on whether she is a high-risk UTROSCT. As far as we know, few articles have summarized and compared the different therapy approaches to UTROSCT. Our study gave a comprehensive knowledge of UTRO- SCT therapy methods. However, the main limitation of our study is that it was designed retrospectively. Specific information about related cases is lacking, the follow-up time for the young UTROSCT needs longer, and genetic alterations and molecular detection remain to be explored. However, we still contributed 11 UTROSCTs to the medi- cal literature, which might aid clinical decision-making. References 1. Hillard JB, Malpica A, Ramirez PT. Conservative management of a uterine tumor resembling an ovarian sex cord-stromal tumor. Gynecol Oncol. 2004;92(1):347–52. 21. Kaur K, Rajeshwari M, Gurung N, Kumar H, Sharma MC, Yadav R, Kumar S, Manchanda S, Singhal S, Mathur SR. Uterine tumor resembling ovarian sex cord tumor: A series of six cases displaying varied histo- pathological patterns and clinical profiles. Indian J Pathol Microbiol. 2020;63(Supplement):S81-s86. 2. Ye S, Wu J, Yao L, He J. Clinicopathological characteristics and genetic variations of uterine tumours resembling ovarian sex cord tumours. J Clin Pathol. 2022;75(11):776–81. 3. Wang XY, Zhang MC, Chen J, Huang JH. Uterine tumor resembling ovarian sex cord tumor: A rare case report. Medicine (Baltimore). 2022;101(35):e30414. 22. Goebel EA, Hernandez Bonilla S, Dong F, Dickson BC, Hoang LN, Hardis- son D, Lacambra MD, Lu FI, Fletcher CDM, Crum CP, et al. Uterine Tumor Resembling Ovarian Sex Cord Tumor (UTROSCT): A Morphologic and Molecular Study of 26 Cases Confirms Recurrent NCOA1-3 Rearrange- ment. Am J Surg Pathol. 2020;44(1):30–42. 4. Clement PB, Scully RE. Uterine tumors resembling ovarian sex-cord tumors. A clinicopathologic analysis of fourteen cases. Am J Clin Pathol. 1976;66(3):512–25. 4. Clement PB, Scully RE. Uterine tumors resembling ovarian sex-cord tumors. A clinicopathologic analysis of fourteen cases. Am J Clin Pathol. 1976;66(3):512–25. 23. Devereaux KA, Kertowidjojo E, Natale K, Ewalt MD, Soslow RA, Hodgson A. GTF2A1-NCOA2-associated uterine tumor resembling ovarian sex cord tumor (UTROSCT) shows focal rhabdoid morphology and aggressive behavior. Am J Surg Pathol. 2021;45(12):1725–8. 5. McCluggage WG, Singh N, Gilks CB. Key changes to the World Health Organization (WHO) classification of female genital tumours introduced in the 5th edition (2020). Histopathology. 2022;80(5):762–78. 5. McCluggage WG, Singh N, Gilks CB. Key changes to the World Health Organization (WHO) classification of female genital tumours introduced in the 5th edition (2020). Histopathology. 2022;80(5):762–78. 6. Bakula-Zalewska E, Danska-Bidzinska A, Kowalewska M, Piascik A, Nasier- owska-Guttmejer A, Bidzinski M. Uterine tumors resembling ovarian sex cord tumors, a clinicopathologic study of six cases. Ann Diagn Pathol. 2014;18(6):329–32. 6. Bakula-Zalewska E, Danska-Bidzinska A, Kowalewska M, Piascik A, Nasier- owska-Guttmejer A, Bidzinski M. Uterine tumors resembling ovarian sex cord tumors, a clinicopathologic study of six cases. Ann Diagn Pathol. 2014;18(6):329–32. 24. Shibahara M, Kurita T, Murakami M, Harada H, Tsuda Y, Hisaoka M, Kagami S, Matsuura Y, Yoshino K. Uterine tumor resembling ovarian sex cord tumor: a case report. J Uoeh. 2022;44(2):161–6. 25. Declarations 15. Lee CH, Kao YC, Lee WR, Hsiao YW, Lu TP, Chu CY, Lin YJ, Huang HY, Hsieh TH, Liu YR, et al. Clinicopathologic Characterization of GREB1-rearranged Uterine Sarcomas With Variable Sex-Cord Differentiation. Am J Surg Pathol. 2019;43(7):928–42. Conclusion UTROSCT patients are often diagnosed in the early stage. Hysterectomy with or without salpingo-oopho- rectomy should be the primary treatment for UTROSCT patients. Radical surgery is a favored choice for patients with invasive features. Mass resection is safe for young fertility women in clinical surveillance. Further studies are needed to validate these findings. Page 8 of 9 Lin et al. World Journal of Surgical Oncology (2024) 22:4 Availability of data and materials The data used or analyzed in the current study are available from the cor- responding author upon reasonable request. 14. Zhang X, Zou S, Gao B, Qu W. Uterine tumor resembling ovarian sex cord tumor: a clinicopathological and immunohistochemical analysis of two cases and a literature review. J Int Med Res. 2019;47(3):1339–47. Ethics approval and consent to participate This retrospective study was approved by the Ethics Committee of Fujian Medical Cancer Hospital and Fujian Union Hospital and adhered to the Helsinki Declaration. Oral consent was obtained from both patients for the publication of their information and images. 16. Vilos AG, Zhu C, Abu-Rafea B, Ettler HC, Weir MM, Vilos GA. Uterine tumors resembling ovarian sex cord tumors identified at resectoscopic endometrial ablation: report of 2 cases. J Minim Invasive Gynecol. 2019;26(1):105–9. The authors declare no competing interests. The authors declare no competing interests. 17. Dimitriadis GK, Wajman DS, Bidmead J, Diaz-Cano SJ, Arshad S, Bakhit M, Lewis D, Aylwin SJB. Ectopic hyperprolactinaemia due to a malignant uterine tumor resembling ovarian sex cord tumors (UTROCST). Pituitary. 2020;23(6):641–7. Acknowledgements 8. Zhou FF, He YT, Li Y, Zhang M, Chen FH. Uterine tumor resembling an ovarian sex cord tumor: A case report and review of literature. World J Clin Cases. 2021;9(23):6907–15. The author Jie Lin thanks her husband Deliang Li for the unconditional support. 9. Blake EA, Sheridan TB, Wang KL, Takiuchi T, Kodama M, Sawada K, Matsuo K. Clinical characteristics and outcomes of uterine tumors resembling ovarian sex-cord tumors (UTROSCT): a systematic review of literature. Eur J Obstet Gynecol Reprod Biol. 2014;181:163–70. Funding This work was sponsored by the Startup Fund for scientific research, Fujian Medical University, China (Grant number: 2021QH1141), Joint Funds for the Innovation of Science and Technology, Fujian Province (Grant number 2021Y9209), the Fujian Provincial Health Technology Project (Grant number 2021QNA043) and the Natural Science Foundation of Fujian Province (Grant number 2023J011257). 12. Croce S, Lesluyes T, Delespaul L, Bonhomme B, Pérot G, Velasco V, Mayeur L, Rebier F, Ben Rejeb H, Guyon F, et al. GREB1-CTNNB1 fusion transcript detected by RNA-sequencing in a uterine tumor resembling ovarian sex cord tumor (UTROSCT): A novel CTNNB1 rearrangement. Genes Chromo- somes Cancer. 2019;58(3):155–63. 13. Dubruc E, Alvarez Flores MT, Bernier Y, Gherasimiuc L, Ponti A, Mathevet P, Bongiovanni M. Cytological features of uterine tumors resembling ovar- ian sex-cord tumors in liquid-based cervical cytology: a potential pitfall. Report of a unique and rare case. Diagn Cytopathol. 2019;47(6):603–7. Received: 3 November 2023 Accepted: 23 January 2024 20. Bennett JA, Lastra RR, Barroeta JE, Parilla M, Galbo F, Wanjari P, Young RH, Krausz T, Oliva E. Uterine Tumor Resembling Ovarian Sex Cord Stromal Tumor (UTROSCT): A Series of 3 Cases With Extensive Rhabdoid Differen- tiation, Malignant Behavior, and ESR1-NCOA2 Fusions. Am J Surg Pathol. 2020;44(11):1563–72. Authors’ contributions Yang Sun designed the present study. Jie Lin and Linying Liu wrote the manu- script. Jie Lin checked and revised the manuscript. Linghua Wang helped to reviewed and collected all data, Ning Ma reviewed, collected and checked MRI data, Kailin Zhang reviewed, collected and checked pathology data, Ning Xie, Haijuan Yu and Sufang Deng contributed to collecting and reviewing materials. 10. Gutierrez-Pecharroman A, Tirado-Zambrana P, Pascual A, Rubio-Marin D, García-Cosío M, Moratalla-Bartolomé E, Palacios J. A uterine tumor resembling ovarian sex cord tumor associated with tamoxifen treatment: a case report and literature review. Int J Gynecol Pathol. 2014;33(2):151–5. 11. Kondo Y, Sakaguchi S, Mikubo M, Naito M, Shiomi K, Ohbu M, Satoh Y. Lung metastases of a uterine tumor resembling ovarian sex-cord tumor: report of a rare case. Diagn Cytopathol. 2018;46(1):88–91. Author details 1 1 Department of Gynecology, Clinical Oncology School of Fujian Medical Uni- versity, Fujian Cancer Hospital, Fuzhou, Fujian Province, China. 2 Department of Radiology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, Fujian Province, China. 3 Department of Pathology, Fujian Medical University Union Hospital, Fuzhou, China. 18. Grither WR, Dickson BC, Fuh KC, Hagemann IS. Detection of a somatic GREB1-NCOA1 gene fusion in a uterine tumor resembling ovarian sex cord tumor (UTROSCT). Gynecol Oncol Rep. 2020;34:100636. 19. Chang B, Bai Q, Liang L, Ge H, Yao Q. Recurrent uterine tumors resembling ovarian sex-cord tumors with the growth regulation by estrogen in breast cancer 1-nuclear receptor coactivator 2 fusion gene: a case report and literature review. Diagn Pathol. 2020;15(1):110. Received: 3 November 2023 Accepted: 23 January 2024 Received: 3 November 2023 Accepted: 23 January 2024 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 28. Umeda S, Tateno M, Miyagi E, Sakurai K, Tanaka R, Tateishi Y, Tokinaga A, Ohashi K, Furuya M. Uterine tumors resembling ovarian sex cord tumors (UTROSCT) with metastasis: clinicopathological study of two cases. Int J Clin Exp Pathol. 2014;7(3):1051–9. 29. Mačák J, Dundr P, Dvořáčková J, Klát J. Uterine tumors resembling ovarian sex cord tumors (UTROSCT). Report of a case with lymph node metasta- sis. Cesk Patol. 2014;50(1):46–9. 30. Cetinkaya N, Bas S, Cuylan ZF, Erdem O, Erkaya S, Gungor T. Uterine tumors resembling ovarian sex cord tumors: a case report and literature review. Oncol Lett. 2016;11(2):1496–8. 31. Kuznicki ML, Robertson SE, Hakam A, Shahzad MM. Metastatic uterine tumor resembling ovarian sex cord tumor: a case report and review of the literature. Gynecol Oncol Rep. 2017;22:64–8. 32. Fan LL, Shen Y, Chanda K, Ren ML. Uterine tumors resembling ovarian sex-cord tumor: A case report and literature review. J Cancer Res Ther. 2018;14(Supplement):S1209-s1212. 32. Fan LL, Shen Y, Chanda K, Ren ML. Uterine tumors resembling ovarian sex-cord tumor: A case report and literature review. J Cancer Res Ther. 2018;14(Supplement):S1209-s1212. 33. Segala D, Gobbo S, Pesci A, Martignoni G, Santoro A, Angelico G, Arciu- olo D, Spadola S, Valente M, Scambia G, et al. Tamoxifen related uterine tumor resembling ovarian sex cord tumor (UTROSCT): a case report and literature review of this possible association. Pathol Res Pract. 2019;215(5):1089–92. 34. Sato M, Yano M, Sato S, Aoyagi Y, Aso S, Matsumoto H, Yamamoto I, Nasu K. Uterine tumor resembling ovarian sex-cord tumor (UTROSCT) with sarcomatous features without recurrence after extended radical surgery: a case report. Medicine (Baltimore). 2020;99(11):e19166. 35. Watrowski R, Jäger C, Möckel J, Kurz P, Schmidt D, Freudenberg N. Hyst- eroscopic treatment of uterine tumor resembling ovarian sex cord-like tumor (UTROSCT). Gynecol Endocrinol. 2015;31(11):856–9. 36. Jeong KH, Lee HN, Kim MK, Kim ML, Seong SJ, Shin E. Successful delivery after conservative resectoscopic surgery in a patient with a uterine tumor resembling ovarian sex cord tumor with myometrial invasion. Obstet Gynecol Sci. 2015;58(5):418–22. 37. De Franciscis P, Grauso F, Ambrosio D, Torella M, Messalli EM, Cola- curci N. Conservative resectoscopic surgery, successful delivery, and 60 months of follow-up in a patient with endometrial stromal tumor with sex-cord-like differentiation. Case Rep Obstet Gynecol. 2016;2016:5736865. 38. Schraag SM, Caduff R, Dedes KJ, Fink D, Schmidt AM. References Pang L, Dai Y, Ren F, Peng X, Guo Z. Uterine tumor resembling ovarian sex cord tumors (UTROSCT): two case reports of the rare uterine neoplasm with literature review. Curr Med Imaging. 2022;18(10):1125–31. 7. Moore M, McCluggage WG. Uterine tumour resembling ovarian sex cord tumour: first report of a large series with follow-up. Histopathology. 2017;71(5):751–9. 7. Moore M, McCluggage WG. Uterine tumour resembling ovarian sex cord tumour: first report of a large series with follow-up. Histopathology. 2017;71(5):751–9. Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 9 of 9 45. Endo D, Todo Y, Okamoto K, Suzuki H. A case of recurrent group II uterine tumor resembling ovarian sex-cord tumors, against which two hormonal agents were ineffective. Taiwan J Obstet Gynecol. 2016;55(5):751–3. 26. Zhou Y, Chen YH, Wang YN, Sun L. Uterine tumor resembling ovarian sex cord tumor with an abnormal increase in CA125: a case report. Asian J Surg. 2023;46(6):2641–42. 27. Lu B, Xia Y, Chen J, Tang J, Shao Y, Yu W. NCOA1/2/3 rearrangements in uterine tumor resembling ovarian sex cord tumor: a clinicopathological and molecular study of 18 cases. Hum Pathol. 2023;135:65–75. Publisher’s Note Uterine tumors resembling ovarian sex cord tumors - treatment, recurrence, pregnancy and brief review. Gynecol Oncol Rep. 2017;19:53–6. 39. Carbone MV, Cavaliere AF, Fedele C, Vidiri A, Aciuolo D, Zannoni G, Scambia G. Uterine tumor resembling ovarian sex-cord tumor: conserva- tive surgery with successful delivery and case series. Eur J Obstet Gyneco Reprod Biol. 2021;256:326–32. 40. Boyraz B, Watkins JC, Young RH, Oliva E. Uterine tumors resembling ovar- ian sex cord tumors: a clinicopathologic study of 75 cases emphasizing features predicting adverse outcome and differential diagnosis. Am J Surg Pathol. 2023;47(2):234–47. 41. Dickson BC, Childs TJ, Colgan TJ, Sung YS, Swanson D, Zhang L, Anto- nescu CR. Uterine tumor resembling ovarian sex cord tumor: a distinct entity characterized by recurrent NCOA2/3 gene fusions. Am J Surg Pathol. 2019;43(2):178–86. 42. Chen Z, Lan J, Chen Q, Lin D, Hong Y. A novel case of uterine tumor resembling ovarian sex-cord tumor (UTROSCT) recurrent with GREB1- NCOA2 fusion. Int J Gynaecol Obstet. 2021;152(2):266–8. 43. Yin X, Wang M, He H, Ru G, Zhao M. Uterine tumor resembling ovarian sex cord tumor with aggressive histologic features harboring a GREB1- NCOA2 fusion: case report with a brief review. Int J Gynecol Pathol. 2023;42(1):54–62. 44. Marrucci O, Nicoletti P, Mauriello A, Facchetti S, Patrizi L, Ticconi C, Sesti F, Piccione E. Uterine tumor resembling ovarian sex cord tumors type II with vaginal vault recurrence. Case Rep Obstet Gynecol. 2019;2019:5231219.
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Risk factors associated with Dengue incidence in Bandung, Indonesia: a household based case-control study
Health Science Journal of Indonesia
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Risk factors associated with Dengue incidence in Bandung, Indonesia Risk factors associated with Dengue incidence in Bandung, Indonesia 45 Vol. 11, No. 1, June 2020 Unit of Health Research and Development of Pangandaran District, West Java Province, Ministry of Health of Republic of Indonesia Unit of Health Research and Development of Pangandaran District, West Java Province, Ministry of Health of Republic of Indonesia Corresponding author: Hubullah Fuadzy Email: hubullah_fy@yahoo.com Corresponding author: Hubullah Fuadzy Email: hubullah_fy@yahoo.com Received: Desember 9, 2019; Revised: Januari 20, 2020; Accepted: May Received: Desember 9, 2019; Revised: Januari 20, 2020; Accepted: May 19, 2020. Abstrak Latar belakang: Bandung memiliki daerah perkotaan dengan kualitas bangunan rumah yang memadai, namun masih memiliki kasus endemik Demam Berdarah Dengue (DBD) yang tinggi. Penelitian ini bertujuan untuk mengetahui karakteristik kejadian demam berdarah di tingkat rumah tangga. Metode: Data dianalisis dari 781 rumah tangga yang terdiri dari 261 kasus dan 522 kontrol. Pemilihan sampel menggunakan metode kasus kontrol berpasangan dengan rasio 1:2. Tahapan penelitian terdiri dari pengamatan status rumah menggunakan form ceklis pemeriksaan dan wawancara menggunakan kuesioner tertutup. Analisis data korelasi pairwise spearmen, kemudian regresi logistik biner digunakan untuk prediksi faktor risiko. Hasil: Faktor risiko usia produktif dan rendahnya tingkat pendidikan kepala keluarga, toilet yang kotor, dan status rumah tidak sehat memiliki pengaruh yang signifikan terhadap peningkatan kasus demam berdarah di Kota Bandung ((p<0.05). Faktor determinan adalah usia produktif kepala keluarga (31 - 60 tahun) dimana memiliki kemungkinan 2,53 (95%CI 1.34-4.78;p<0.05) kali lebih besar untuk memiliki anggota rumah tangga yang menderita DBD di Bandung. Kesimpulan: Usia dan pendidikan kepala rumah tangga, kebersihan toilet dan status rumah sehat memiliki peran penting dalam mempengaruhi kejadian demam berdarah. Dengan demikian, petugas kesehatan perlu melakukan promosi kesehatan mengenai DBD secara intensif kepada kepala rumah tangga. (Health Science Journal of Indonesia 2020;11(1):45-51) Kata kunci: Demam Berdarah Dengue (DBD), kepala rumah tangga, usia, sanitasi rumah, Kota Bandung Keywords: Dengue, head of household, ages, house sanitation, Bandung City DOI: dx.doi.org/10.22435/hsji.v11i1.3150 DOI: dx.doi.org/10.22435/hsji.v11i1.3150 Hubullah Fuadzy, Mutiara Widawati, Endang P. Astuti, Heni Prasetyowati, Joni Hendri, Rohmansyah W. Nurindra, Dewi N. Hodijah. Abstract Background: Bandung have urban areas with adequate housebuilding quality, yet still has high Dengue endemic cases. This study aims to investigate the characteristics of dengue incidence at the household level. Methods: Data analyzed from 781 households consisted of 261 cases and 522 controls. We applied matched case-control samples with a ratio of 1:2 (case: controls). The stages of the research consisted of a house status observation using a form inspection checklist and interviews using a closed-ended questionnaire. The data analyzed by pairwise spearmen correlation and binary logistic regression for risk factor prediction. Results: Risk factors for productive age and low level of education of family heads, dirty toilets, and unhealthy house status have a significant effect on the increase of dengue cases in Bandung (p<0.05). The determinant factor is the productive age of the head of the family (31 - 60 years), it means that the family who has a family head in a productive age is 2.53 (95% CI 1.34-4.78; p <0.05) times more likely to have a household member suffering from DHF in Bandung. Conclusion: The age and the level of education of the household heads, toilet hygiene, and healthy home status have an essential role in influencing dengue fever. Therefore, health workers need to perform an intensive health promotion regarding DHF to the household heads. (Health Science Journal of Indonesia 2020;11(1):45-51) Keywords: Dengue, head of household, ages, house sanitation, Bandung City Health Science Journal of Indonesia Fuadzy et al. 46 Dengue haemorrhagic fever (DHF) is a mosquito-borne disease caused by Dengue virus and transmitted by Aedes aegypti and Ae. albopictus. Dengue infection has become a major public health problem worldwide, especially in Asia. In recent decades, the disease outbreak has been reported in 128 countries with more than 3.79 billion people are at risk, mostly in urban and semi-urban areas.1 In Southeast Asia, Dengue is endemic in 12 countries. An estimated 2.9 million dengue infections occur annually with 5,906 among them died, thus impacting the annual economic burden of US $ 1.65 (the US $ 1.06 - the US $ 2.41) per capita.2 efforts were also closely related to the knowledge and attitudes of the community. Several studies have suggested that proper education and beliefs about dengue are related to the prevention of dengue.6–8 These determinant factors significantly affect the ongoing transmission of dengue in several regions. Abstract The region with the highest risk of increasing DHF cases is the area in the center of Bandung, with peak cases from January to August. Many people are at risk of catching the dengue fever in the center of Bandung due to socio-economic conditions, especially slum housing conditions, population density, relatively high mobility, and low education levels.9 Respati’s research (2018) states that the most decisive risk factors in the prevention and control of dengue in Bandung are community participation, housing, environmental factors, and climate.10 The government of Indonesia has conducted various efforts to prevent dengue transmission by physical, biology, chemical, empowering of community, and integrated management control, but dengue morbidity keeps increasing. Indonesia has reported that the morbidity rate of dengue still increases to 700 times in the last 45 years, whereas the mortality rate decreases to 56.16 times.3dengue vector-density and human mobility cause rapid spread of dengue virus in Indonesia. We investigated the changes in dengue haemorrhagic fever (DHF However, Indonesia has not succeeded in preventing and controlling Ae. Aegypti from dengue virus transmission in both rural and urban settlements. In Bandung, most of the citizens use containers for collecting and storing water for daily needs. These conditions will trigger an increase in the population of Aedes and the potential for the Dengue transmission. In 2010, at least of 3,430 Dengue cases were reported in this city. However, in the following two years (2013), the dengue cases were increased to 5,736 cases.11 The free larvae index was also increased and close to the national target at 93.38%.12 In this study, we have investigated the characteristic of the dengue incidence at the household level in Bandung, Indonesia Household activities may play a role in the transmission of Dengue Fever. Various factors, such as house sanitation, room construction, occupants’ behavior, management of water containers4sanitation and hand hygiene in low- and middle-income settings and provide an overview of the impact on other diseases.\\n\\nMETHODS: For estimating the impact of water, sanitation and hygiene on diarrhea, we selected exposure levels with both sufficient global exposure data and a matching exposure-risk relationship. Global exposure data were estimated for the year 2012, and risk estimates were taken from the most recent systematic analyses. We estimated attributable deaths and disability-adjusted life years (DALYs, socio-economic conditions and knowledge about dengue fever have the potential to influence the incidence of dengue fever. Abstract Communities with less stable socio-economics condition tend to have inadequate or unhygienic houses. The previous study about risk factor analysis showed that residents in rented houses are more at risk of dengue than those who live in their own houses (AOR = 2.2, 95% CI= 1.1 - 4.6; p < 0.05). In addition, residents of unhygienic dwellings were more likely to get dengue compared to residents in hygienic houses (AOR = 3.4, 95% CI = 1.0 – 11.7; p < 0.05).5 The behavior of increasing house hygiene and vector control Study Subjects Bandung is the capital of the West Java Province, located at coordinates of 107o E and 6o55’ S. Bandung has 75 PHCs (Primary Health Care/Pusat Kesehatan Masyarakat), as a unit in sub-district level that responsible for the community health efforts, consisting of public health activities for the population within their working area. This study has been carried out in 4 PHCs areas, i.e. Dago, Sekejati, Kopo, and Cipamokolan. The location of this study was determined based on the highest dengue case. This study also refers to research on Determining Risk Factors for Residential Sanitation in Dengue Hemorrhagic Fever (DHF) in Bandung in 2016.13 This study aims to explain what factors cause the number of healthy home coverage continues to show an increasing trend every year, despite the increasing trend of dengue fever as well. Risk factors associated with Dengue incidence in Bandung, Indonesia Vol. 11, No. 1, June 2020 47 variables were analyzed through binary logistic regression using Minitab version 19. This research is observation research with a matched case-control design with a ratio of 1:2 so that the confounding factor in both groups can be controlled. The case group population is a house that has household members who have had DHF in 2015 in Bandung, while the control population is a house where household members have never had DHF in Bandung. The sample size was calculated using the odds ratio estimation formula, and a total sample of 783 households was obtained, consisting of 261 case groups and 522 control groups. The criteria for selecting a research location were if the puskesmas has a high number of dengue cases in 2015 in the city of Bandung. The sample selection of case group households uses a systematic random sampling technique, while the control group uses purposive sampling techniques with the criteria of a control group household being at a radius of 100 meters from the sample cases. Data Collection Data were collected through observation and inter­ views. The observation using the Ministry of Health’s standard questionnaire viz healthy housing inspec­ tion checklist to determine the house status based on the regulation of the Ministry of Health, Republic of Indonesia No. 829/Menkes/VII/1999. There were four observed places, such as living/family room, bed­ room, bathroom, and kitchen. This study evaluated all room construction (ceilings, walls, floors, windows, vents, chimney, and lighting), and sanitation facili­ ties (water facilities, toilets, wastewater disposal, and garbage cans). After observation on four rooms, KAP interviews using structural questionnaire were con­ ducted with some question to respondents about vec­ tor, transmission, and control of Dengue Result of the head of household characteristic component for case and control groups showed that the majority of households considered as a settled family with the indicator of age included into the productive period (95.02% and 88.70%), level of education ranged from high school to higher education (65.52% and 56.90%), and income (70.11% and 65.33%) was more than the regional minimum wage of Bandung (2,310,000 IDR), respectively. There are different age period and education level of head of household between case and control group (p<0.05) (Table 1). Overall, the value of the house component consisting of the construction of houses, and sanitation facilities, both in case and control group was relatively similar. For a few different house components, namely the ventilation, and chimney, (p<0.05) (Table 1). Characteristics of Dengue Incidences The data samples of 783 respondents were successfully collected, which distributed to four Public Health Centers. As for the distribution consisted of 67 cases and 134 controls in Dago PHCs, 60 cases and 120 controls in Sekejati PHCs, 72 cases and 144 controls in Kopo PHCs, also 62 cases and 124 controls in Cipamokolan PHCs. Ethics Approval As this research involving humans as subjects, the research ethics approval was required. Therefore, research ethics approval letter has been published by the National Institute of Health Research and Development of Indonesia with Registration Number LB.02.01/5.2/KE.056/2016. The respondents gave a statement written in informed consent. Data Analysis Status of a healthy house determined by calculating the weight of each component. The result of the calculation compared with the average standard value. The value for room construction is 44, and the value for sanitation facilities is 31. Based on the Arikunto criteria (2003), a 75% cut-off14 can be used to determine the status of healthy and unhealthy houses. Then the house is characterized as a healthy house if the total amount achieved is 641 - 855 and an unhealthy house if the total amount obtained is less than 641. Then, the data analyzed by bivariate analysis using pairwise Spearman correlation. After that, multivariate analyses were performed to determine the dominant risk factor in affecting the incidence of Dengue in Bandung. Later, those House status showed that the majority of the Bandung citizen still included in the category of an unhealthy house, both in case and control group (83.14% and 89.08%). Moreover, the significantly different result was related to the house status to the incidence of Dengue in Bandung (p<0.05) (Table 2).The majority of Bandung citizen had inadequate knowledge (75.48% and 72.99%) regarding the transmission and eradication of Dengue, yet they had a right attitude (93.87% and 91.76%), and action (60.15% and 66.48%) concerning the prevention of Dengue. No significant different results related to knowledge, attitudes, and practices to the incidence of Dengue (p>0.05) (Table 3). Health Science Journal of Indonesia Fuadzy et al. 48 Fu 48 48 Table 2. Association of house status with Dengue incidences No House Status Houses p-value Case, n=261 Control, n=522 1 Houses Healthy 44(16.86) 57(10.92) 0.019* Unhealthy 217(83.14) 465(89.08) > Houses status are revealed healthy when whole of the house components assessment has value more than 641.25 based on Decree of the Minister of Health of the Republic of Indonesia No.829/ Menkes/VII/1999 concerning the requirements of healthy housing Table 3. Association of knowledges, attitudes, and practices with Dengue incidences No Variables Houses p-value Case, n=261 Control, n=522 1 Knowledge 0.456 Good 64(24.52) 141(27.01) Poor 197(75.48) 381(72.99) 2 Attitudes 0.293 Positive 245(93.87) 479(91.76) Negative 16(6.13) 43(8.24) 3 Practices 0.082* Good 157(60.15) 347(66.48) Poor 104(39.85) 175(33.52) * Candidates for Multivariate Analysis Table 2. Association of house status with Dengue incidences Table 1. Assosiation between house components with Dengue incidences Table 1. Determinants of Dengue The goodness of fit on logistic regression analysis showed that p-value of Hosmer Lemeshow was 0.499, indicated that the model is fit to be postulated. The value of concordant is 65.1, which means that probably there is only 65.1% risk factor variable correlated with the incidence of Dengue in Bandung. Risk factors that influence the increase in DHF are the age and the education of the household head, toilet, and the status of the healthy house (p<0.05). As for the most dominant risk factor of the spread of Dengue in Bandung is the age of household head which is still productive which leads to a tendency of dengue infection risk with OR 2.53 (95% CI, 1.34- 4.78). Data Analysis Assosiation between house components with Dengue incidences No House Components Houses p-value Case, n=261 Control, n=522 1 Household head characteristics Age of household head 0.004* Productive ( 16-60 Year) 248(95.02) 463(88.70) Not Productive (>60 Year) 13(4.98) 59(11.30) Education of household head 0.020* ≥ Senior High School 171(65.52) 297(56.90) < Senior High School 90(34.48) 225(43.10) Income of household head` 0.180* ≥ 3,000,000 IDR 183(70.11) 341(65.33) < 3,000,000 IDR 78(29.89) 181(34.67) 2 House constructions Ceiling 0.477 Healthy 203(77.78) 394(75.48) Unhealthy 58(22.22) 128(24.52) Wall 0.635 Healthy 233(89.27) 460(88.12) Unhealthy 28(10.73) 62(11.88) Floor 0.789 Healthy 237(90.80) 477(91.38) Unhealthy 24(9.20) 45(8.62) Bedroom window 0.956 Healthy 184(70.50) 367(70.31) Unhealthy 77(29.50) 155(29.69) Living room window 0.206* Healthy 230(88.12) 475(91.00) Unhealthy 31(11.88) 47(9.00) Ventilation 0.045* Healthy 140(53.64) 319(61.11) Unhealthy 121(46.36) 203(38.89) Chimney 0.011* Healthy 161(61.68) 369(70.69) Unhealthy 100(38.32) 153(29.31) Sunlight 0.076* Healthy 155(59.39) 275(52.68) Unhealthy 106(40.61) 247(47.32) 3 Sanitation facilities Clean water 0.858 Healthy 201(77.01) 399(76.44) Unhealthy 60(22.99) 123(23.56) Toilet 0.234* Healthy 152(58.24) 327(62.64) Unhealthy 109(41.76) 195(37.36) Waste disposal 0.946 Healthy 218(83.52) 437(83.72) Unhealthy 43(16.48) 85(16.28) Garbage disposal 0.559 Healthy 193(73.95) 396(75.86) Unhealthy 68(26.05) 126(24.14) > House components is Part of Physical building inside of house and householder behaviours, and was revealed of healthy when it’s still functioning well and could prevent the disease’s transmission; DISCUSSIONS In this study, the majority of cases of dengue infection in Bandung in 2016 lived in urban areas with good socioeconomic status, but they lived in houses with poor sanitation and lack of practice and behavior in vector control. This study also revealed that dirty toilets, bathtubs that are rarely drained, frequent use of insecticides, and lack of vector control efforts will Risk factors associated with Dengue incidence in Bandung, Indonesia Vol. 11, No. 1, June 2020 49 Table 4. Binary Logistic Regression Analysis on determinants of dengue incidence characteristics No Variables Odds Ratio 95% CI, p-value 1 Age of household head 2.53 (1.34-4.78) 0.004** 2 Education of household head 1.39 (1.00-1.93) 0.048* 3 Living room window 0.65 (0.39-1.77) 0.095 4 Chimney 0.73 (0.52-1.03) 0.078 5 Toilet 0.68 (0.48-0.96) 0.027* 6 Practices 0.73 (0.53-1.00) 0.055 7 Healthy house 1.72 (1,05-2.83) 0.031* increase the risk of family members infected with dengue fever / DHF. In Kediri-Indonesia it was found that the activity to prevent Dengue through mosquito breeding sites eradication in the form of water container management has a significant correlation with the increase in Dengue cases.15 Previous studies also reported that the condition of toilets in parts of Kuala Lumpur16 and Thailand17 related to the existence of Aedes spp; as well as in Venezuela. It stated that the practice of vector prevention was negatively associated with dengue cases.18 Table 4. Binary Logistic Regression Analysis on determinants of dengue incidence characteristics The attitudes and actions of the people of Bandung City have shown functional categories in efforts to control the vector of DHF, but it is not supported by good knowledge as well. The impact is the community makes efforts to clean the house environment as daily tasks, but the city has not made an effort to eradicate mosquitoes routinely and precisely. Some people of the City of Bandung admit that attempts to drain water are often but not routine and not accompanied by brushing actions, so the possibility of mosquito eggs attached to the walls of water containers can grow into adult mosquitoes. Therefore, comprehensive knowledge about Dengue should also be taken into consideration since inadequate knowledge will not have an impact on the behaviors of mosquito breeding sites eradication in total.19–21 Important information in this study is that the majority of DHF patients and their household heads are in the phase where they are productive. DISCUSSIONS They occupy a sturdy house but pay less attention to aspects of vector control of DHF. This phenomenon is probably due to the lack of complete community knowledge about DHF. This phenomenon can result in the spread of DHF from the workplace to the residential environment, and transmit DHF to household members who live at home, to reduce family productivity. The mean number of workdays lost for adult DHF patients was 7.6 (SD 3.1) days;27 as well as the head of the family who must treat patients while in hospital. Therefore, serious efforts are needed from the Bandung local government to provide systematic education on the control of DHF vectors at the household level to the head of the household, so that they are motivated to make efforts to eradicate mosquito nests regularly and appropriately. The hope is that the head of the house can invite all members of his family to start a clean and healthy life, especially in preventing dengue transmission. The existence of water containers in the toilet can become a potential habitat for Aedes aegypti breeding such as bathtubs and buckets.22,23 Stagnant water in containers that are not drained and brushed for more than nine days can be a habitat that is loved by Aedes aegypti to complete the life cycle of mosquitoes ranging from eggs, larvae, pupae, to adults.24 Aedes aegypti adults infected with dengue virus can become dengue vectors during their lifetime between 15-65 days 25; as long as its blood needs are met, Aedes aegypti will always be active inside the house. Therefore, in dengue-endemic areas, it was reported that household members who live at house with dengue patient have the opportunity to become infected with dengue 3.2 times (95% CI 1.8-5.7).26 The limitation of this study was that this study did not do in-depth interviews related to the explanation of why the people of Bandung City were low in knowledge but had good vector control attitudes and actions, though the means to access media information about DHF is very easy to obtain. Besides, no more digging about the mobility of household heads in maintaining the socioeconomic status of the community in the city of Bandung. The results of the binary logistic regression analysis showed that the productive age of the head of the household had a dominant role in dengue transmission in the city of Bandung (Table 4). REFERENCES 1 Brady OJ, Gething PW, Bhatt S, Messina JP, Brownstein JS, Hoen AG, et al. Refining the global spatial limits of Dengue Virus transmission by evidence-based consensus. PLoS Negl Trop Dis. 2012; 6. doi:10.1371/ journal.pntd.0001760. 15 Hakim L, Yanuwiadi B, Hariyono, Suyadi, Hakim L, Yanuwiadi B. The role of environmental and behavior factors to dengue fever incidents. J Appl Environ Biol Sci. 2016;6:1–8. 16 Mahmud MAF, Mutalip MH, Lodz NA, Shahar H. Study on key Aedes spp breeding containers in dengue outbreak localities in Cheras district, Kuala Lumpur. Int J Mosq Res. 2018;5:23–30. j p 2 Shepard DS, Undurraga EA, Halasa YA. Economic and disease burden of Dengue in Southeast Asia. PLoS Negl Trop Dis. 2013; 7: e2055. 3 Karyanti MR, Uiterwaal CSPM, Kusriastuti R, Hadinegoro SR, Rovers MM, Heesterbeek H, et al. The changing incidence of Dengue Haemorrhagic Fever in Indonesia: a 45-year registry-based analysis. BMC Infect Dis. 2014; 14. doi:10.1186/1471-2334-14-412. 17 Waewwab P, Sungvornyothin S, Okanurak K, Soonthornworasiri N, Potiwat R. Characteristics of water containers influencing the presence of Aedes immatures in an ecotourism area of Bang Kachao Riverbend, Thailand. J Heal Res. 2019;33:398–407. 4 Prüss-Ustün A, Bartram J, Clasen T, Colford JM, Cumming O, Curtis V, et al. Burden of disease from inadequate water, sanitation and hygiene in low- and middle-income settings: a retrospective analysis of data from 145 countries. Trop Med Int Heal. 2014; 19: 894– 905. 18 Elsinga J, Schmidt M, Lizarazo EF, Vincenti-gonzalez MF, Velasco-salas ZI, Arias L, et al. Knowledge, attitudes, and preventive practices regarding Dengue in Maracay, Venezuela. Am J Trop Med Hyg. 2018;99:195–203. 19 Jeelani S, Sabesan S, Subramanian S. Community knowledge, awareness and preventive practices regarding dengue fever in Puducherry - South India. Public Health. 2015;129:790–6. 5 Toan DTT, Hoat LN, Hu W, Wright P, Martens P. Risk factors associated with an outbreak of dengue fever/dengue haemorrhagic fever in Hanoi, Vietnam. Epidemiol Infect. 2015; 143: 1594–8. 20 Elyas TB, Elsidig EM, Aseri A, Break A, Salim H, Adil R, et al. Knowledge, attitudes and preventive practices of household regarding Dengue Fever in the rural areas of Jazan Region, Saudi Arabia. Int J Prev Med Res. 2016;2:8–12. 6 Harapan H, Bustamam A, Radiansyah A, Angraini P, Fasli R, Salwiyadi S, et al. Dengue prevention: confirmatory factor analysis of relationships between economic status, knowledge, attitudes and practice, vaccine acceptance and willingness to participate in a study. Acknowledgments We would like to thanks to the National Institute of Health Research and Development of the Ministry of Health, Indonesia for funding this research, and also Health Department of Bandung for providing facilities to researchers thus this research can be well completed. This research is fully supported by the Pangandaran Unit of Vector-borne Diseases Control. Therefore, no conflicts of interest in this original research. g 12 Bandung Health Office. Bandung City Health Profile 2011. (Unpublished). Bandung; 2011. 13 Fuadzy H, Astuti EP, Prasetyowati H, Hendri J, Rohmansyah, Hodijah DN. Determining risk factors for residential sanitation in Dengue Hemorrhagic Fever (DHF) in Bandung in 2016. Final Report. Pangandaran Unit of Health Research and Development, Ministry of Health of Indonesia. Pangandaran, 2016. 14 Arikunto S. Prosedur penelitian suatu pendekatan praktik. Rineka Cipta:Jakarta;2010. Indonesian. DISCUSSIONS This finding explains that the activity of the head of the household outside the home all day has caused the lack of time and ability to coordinate other family members to carry out DHF control efforts. In conclusion, the description above explains that the incidence of Dengue caused by are the age and the education of the household head, toilet condition, and the status of healthy house. Therefore, it is necessary to establish cadres to promote the information about Dengue to the household heads. And also, the cadres must inspect and clean houses where larvae of Aedes spp. are found. Health Science Journal of Indonesia Fuadzy et al. 50 50 11 West Java Provincial Health Office. Report on Dengue Hemorrhagic Fever Case 2009-2013. [Unpublished]. Bandung; 2014.fii 26 Chien Y, Huang H, Ho T, Tseng F, Ko N, Ko W, et al. Seroepidemiology of dengue virus infection among adults during the ending phase of a severe dengue 25 Chakraborty T, Alcamo IE. Dengue fever and other hemorrhagic viruses (deadly diseases & epidemics). New York: Infobase Publishing. 2008. 25 Chakraborty T, Alcamo IE. Dengue fever and other hemorrhagic viruses (deadly diseases & epidemics). New York: Infobase Publishing. 2008. 26 Chien Y, Huang H, Ho T, Tseng F, Ko N, Ko W, et al. Seroepidemiology of dengue virus infection among adults during the ending phase of a severe dengue REFERENCES Southeast Asian J Trop Med Public Health. 2017; 48: 297–305. 21 Alobuia WM, Missikpode C, Aung M, Jolly PE. Knowledge, attitude, and practices regarding vector- borne diseases in Western Jamaica. Ann Glob Heal. 2015;81:654–63. 7 Rao, Minhat, Hayati. Predictors of practices related to dengue fever prevention among international students in Universiti Putra Malaysia, Serdang. Int J Public Heal Clin Sci. 2016; 3: 36–47. 22 Prasetyowati H, Ipa M, Widawati M. Pre-adult survey to identify the key container habitat of Aedes Aegypti (L.) in Dengue Endemic areas of Banten Province, Indonesia. Southeast Asian J Trop Med Public Health. 2018;49:23–31. 8 Fuadzy H, Widawati M, Hodijah DN, Wahono T. The Role of Information Channels to Encourage Mosquito Breeding Site Eradication for Community of Bandung City. Bul Penelit Sist Kesehat. 2019; 22: 27–33. 23 Wijayanti SPM, Octaviana D, Nurlaela S. Mosquito indices in outdoor spatial spraying treated area, Banyumas Regency, Indonesia. IOP Conf Ser Earth Environ Sci. 2019;255:0120033. 9 Jaya IGNM, Folmer H. Bayesian spatiotemporal mapping of relative dengue disease risk in Bandung, Indonesia. J Geogr Syst. 2020;22:105–142. 24 Collett GC, Graham JE, Schoof HF, Quarterman KD, Johnson DR, Barnes AM, et al. Vector Control in International Health. World Health Organization:Switzerland; 1972. 10 Respati T, Feriandi Y, Ndoen E, Djuhaeni H, Sofyan A, Dale D. A qualitative eco-health model of Dengue Fever (DF) in Bandung, Indonesia. Int J Trop Dis. 2018;1. Risk factors associated with Dengue incidence in Bandung, Indonesia Vol. 11, No. 1, June 2020 51 epidemic in southern Taiwan, 2015. BMC Infec. 2019;19:1–9. 25 Chakraborty T, Alcamo IE. Dengue fever and other hemorrhagic viruses (deadly diseases & epidemics). New York: Infobase Publishing. 2008. 27 Tozan Y, Ratanawong P, Sewe MO, Wilder- A, Kittayapong P. Household costs of hospitalized dengue illness in semi-rural Thailand. PLoS Negl Trop Dis. 2017; 11: e0005961.
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Feasibility of computerized adaptive testing evaluated by Monte-Carlo and post-hoc simulations
Annals of Computer Science and Information Systems
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Proceedings of the Federated Conference on Proceedings of the Federated Conference on Computer Science and Information Systems pp. 359–367 DOI: 10.15439/2020F197 ISSN 2300-5963 ACSIS, Vol. 21 Feasibility of computerized adaptive testing evaluated by Monte-Carlo and post-hoc simulations Patrícia Martinková Institute of Computer Science of the Czech Academy of Sciences Pod Vodárenskou vˇeží 2, Praha 8 Faculty of Education, Charles University, Myslíkova 7, Praha 1 martinkova@cs.cas.cz Lubomír Štˇepánek Institute of Biophysics and Informatics First Faculty of Medicine, Charles University Salmovská 1, Praha 2 lubomir.stepanek@lf1.cuni.cz Lubomír Štˇepánek Institute of Biophysics and Informatics First Faculty of Medicine, Charles University Salmovská 1, Praha 2 lubomir.stepanek@lf1.cuni.cz tests [3] on the other hand involve assigning blocks of items adaptively depending on the ability estimate from the previous test section. Abstract—Computerized adaptive testing (CAT) is a modern alternative to classical paper and pencil testing. CAT is based on an automated selection of optimal item corresponding to current estimate of test-taker’s ability, which is in contrast to fixed predefined items assigned in linear test. Advantages of CAT include lowered test anxiety and shortened test length, increased precision of estimates of test-takers’ abilities, and lowered level of item exposure thus better security. Challenges are high technical demands on the whole test work-flow and need of large item banks. Basic principles of computerized adaptive test are presented in Figure 1. select and show the first item test-taker’s response their ability estimation select the next item termination criteria met? exit and output the final ability estimate yes no Fig. 1. Computerized adaptive testing flowchart In this study, we analyze feasibility and advantages of comput- erized adaptive testing using a Monte-Carlo simulation and post- hoc analysis based on a real linear admission test administrated at a medical college. We compare various settings of the adaptive test in terms of precision of ability estimates and test length. We find out that with adaptive item selection, the test length can be reduced to 40 out of 100 items while keeping the precision of ability estimates within the prescribed range and obtaining ability estimates highly correlated to estimates based on complete linear test (Pearson’s ρ .= 0.96). We also demonstrate positive effect of content balancing and item exposure rate control on item composition. termination criteria met? I. INTRODUCTION M M ULTI-ITEM assessment instruments find their use in number of areas including admission or other educa- tional tests, psychological measurement, health-related ques- tionnaires, and other behavioral measurements. A usual way to perform achievement testing is by assigning a fixed set of items which are supposed to measure construct of interest, such as knowledge of biology, level of depression, fatigue, or respondent’s quality of life. Fig. 1. Computerized adaptive testing flowchart An adaptive test is initialized by the selection and adminis- tration of the first item. The first item can be selected randomly or based on prior ability estimate of the respondent. Average ability can be used as an uninformed estimate, alternatively, initial estimate may be based on respondent’s answers to one or a small number of pre-test items. Given that the abilities may greatly differ across test-takers, the respondents with higher levels of ability may be bored by easier items, while those with lower levels of ability might experience inconvenient stress. An effective and appropriate selection of items which suit the best the test-takers of a given ability can thus be more convenient for respondents, may save time and moreover provide estimates of better precision than fixed tests of the same length. Depending on the answer to the first item, the test-taker ability estimate is updated. If the termination criterion (such as number of administered items or precision of the estimate) is not met, the updated ability estimate is used to select the next optimal item. This cycle is repeated until the a priori specified termination criterion is met; then, eventually, the test is stopped and final estimate of the test-taker ability is provided as an output. Adaptive tests [1], [2] have been an alternative to linear tests for decades. The most complex version of adaptive tests is the one in which the item selection is done after each item administration depending on the current estimate of test-taker’s ability which is iteratively updated. Multistage PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 360 (IRT) model was fitted to describe the probability of a correct answer given applicant’s ability [12], tests have been demonstrated in areas of educational testing [4], testing of psychological distress [5], [6], as well as health-related measurements such as in mobility surveys [7], and testing general disability [8]. While the adaptive tests are usually shorter in terms of number of items and overall time needed to complete the test, they also enable to estimate test-taker’s ability with better precision than linear tests of sim- ilar length. The lower level of item exposure usually implies also better security when items are administered adaptively. pi (θp) = Pr (Upi = 1|θp, ξi) = Ψ [ai (θp −bi)] = exp [ai (θp −bi)] 1 + exp [ai (θp −bi)], (1 (1) where θp is the ability of subject p ∈{1, 2, . . . , N}, vector ξi = (ai, bi)T stands for set of item parameters (discrimination and difficulty, respectively) for item i ∈{1, 2, . . . , I}, and Ψ(•) is the logistic function. However, since the adaptive testing is more complex it re- quires higher technical facility and support of trained experts. The initial setting of the adaptive test may provide number of options which may have crucial impact on functioning of the adaptive test. Therefore, feasibility and optimal setting of CAT with respect to the given item bank and population of test takers need to be analyzed in order to apply the adaptive test effectively and profitably. In the item calibration phase, the item parameters (ai, bi)T were estimated. To estimate the item parameters, we used marginal maximum likelihood (MML) as follows [13]. Let us assume local independence, i. e. independence of item responses for the same subject given their ability θp (within subject). Then the probability Pr (up|θp, ξ) of response pattern up of subject p follows the form In this work, we use Monte Carlo simulations and post-hoc analysis based on real data of admission test administrated at a medical college with the aim to derive the optimal setting of adaptive test. We also compare the precision of different settings and estimate the correlation between the adaptively estimated ability and estimates based on answers to complete set of 100 items. We discuss results for different levels of precision, and various test termination criteria. PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 We also implement content balancing and item exposure rate control to see how it affects performance and properties of the adaptive test. We discuss the findings in context of the admission testing and other educational testing at medical faculties. Pr (up|θp, ξ) = IY i=1 Pr (Upi = upi|θp, ξi) . (2) (2) Supposing there is no cooperation between subjects, we can also assume independence between subjects (in-between). Let’s further denote ξ = (ξ1, . . . , ξI) the matrix of parameters for all items i. Then the marginal likelihood function takes form L (ξ, µ, σ; U) = N Y p=1 Pr (up|ξ, µ, σ) (3) (3) with The paper proceeds as follows. We firstly describe the data and introduce all necessary background theory, including underlying models, settings of adaptive tests and design of the simulation studies in the Research Methodology section. We then present results of the post-hoc analysis and Monte Carlo simulation in Section Results. Finally, discussion and final remarks are provided in Conclusion section. Pr (up|ξ, µ, σ) = = Z . . . Z Pr (up|θp, ξ) g (θp|µ, σ) dθp, where µ and σ are the expected value and the variance of respondent ability θp. With this approach, abilities θp are treated as stochastic variables with normal distribution, θp ∼N (µ, σ) and are integrated out [14]. The first-order derivatives with respect to ability parameters θp result into the likelihood equations [15] that could be numerically estimated using Expectation-Maximization (EM) algorithm [16], producing the desired estimates of item pa- rameters ˆξ =  ˆξ1, . . . , ˆξI  . A. Data and item calibration We used data from a real fixed admission test administrated to 2372 test-takers (applicants) at First Faculty of Medicine, Charles University, Prague in 2015 [9], also see [10]. Interac- tive presentation of psychometric properties of the admission test is available in R package ShinyItemAnalysis [11]. A. Comparison of linear and adaptive testing Both the linear and adaptive test scenario have their advan- tages and disadvantages, respectively. Advantages of adaptive IEEE Catalog Number: CFP2085N-ART ©2020, PTI 359 LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING 361 the maximum likelihood estimates (MLE) and their standard errors [18]. Content balancing. Balancing of an adaptive test content is usually treated as a combinatorial constrained optimization problem [22]. Alternatively, it is based on a shadow-test approach by projection of rest of the test at the current moment (after k −1 items are administrated), which is a nonlinear program using maximum-information rule and constrained by domain attributes and other conditions [22]. While we used MLE to estimate ability in most results shown here, other methods are available. In case the ability is only unidimensional, a popular approach is the weighted like- lihood estimator (WLE) [19]; which maximizes equation (4) weighted by a function w(θ), thus In the post-hoc analysis described in this paper, we used one of the combinatorial designs, where we initially set desired proportions of expected administration rate to each of the three domains (genetics, taxonomy, human biology). The items were selected in a way to minimize differences between the currently observed and initially set proportions. L (θ; up) = w(θ)L (θ|up) . (5) (5) Finally, Bayesian ability estimation [20] specifies prior ability distribution p (θp|µ, σ) and maximizes posterior dis- tribution of θp given up of the following form: p (θp|up, ξ, µ, σ) = Pr (up|θp, ξ) p (θp|µ, σ) R . . . R Pr (up|θp, ξ) p (θp|µ, σ) dθp . Item exposure rate control. The rates of how many times each item is administrated to one or more of test-takers throughout one adaptive test session may be controlled to minimize their unwanted leakage outside the tested population. Hetter-Sympson experiment is commonly applied to face this problem and was also used in our simulation study [23]. The algorithm was run before the optimally selected item was administrated, output of which was a decision either to administer the item, or to pass and select the next best item at the current estimate of ability ˆθp,k. The administered items were removed from the item pool. Hetter-Symspon experiment is based on evaluation of joint conditional probabilities of item administration; thus cumbersome and usually must be numerically simulated. Item selection. We used likelihood-based item selec- tion [17], i. e. LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING in each step, the next (k-th) item was selected to maximize the observed Fisher information ik ≡arg max j n IUk−1,Uj  ˆθk−1 o (6) at θp = ˆθp,k−1 given a subject p, where Uk−1 is an answer pattern up to the (k −1)-th item [17]. This rule is also known as the maximum-information rule in adaptive testing. Other item selection procedures include naive approach such as Urry’s criterion picking always an item with diffi- culty closest to the current ability estimate [21]. In Bayesian framework, the posterior distribution of θp after the preceding item serves as the prior distribution for the selection of the next item. If the posterior distribution after k −1 items has density p (θ|uk−1), then the k-th item is selected such that the posterior distribution There are also some alternatives – an experiment determin- ing which items are eligible for subjects and which not [24]. If an item is eligible, it remains in the pool for the subject p; otherwise it is removed. This works as a principle of "self-adjustment"; when an item was highly exposed within previous p −1 subjects, it is likely not to be eligible for the p-th subject. p (θ|uk−1, Uik) ∝p (θ|uk−1) p (Uik = uik|θ) is optimized in some sense [20]. the maximum errors [18]. While we shown here, o only unidimen lihood estima weighted by Finally, Ba ability distrib tribution of θ p (θp|up, ξ Item selec tion [17], i. e to maximize at θp = ˆθp,k− pattern up to as the maxim Other item such as Urry culty closest framework, th item serves a next item. If density p (θ|u posterior distr p (θ|u is optimized i Termination ability estima IUp,k−1(ˆθp,k− where Uk−1 (inclusively) ˆθp,k−1 is S LUBOM´IR ˇSTˇE LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING B. Settings of adaptive tests Initialization. Initial item was selected as the one maximiz- ing observed Fisher information at ability θ0 = 0, see [17]. Ability estimation. Test-taker’s ability is iteratively updated whenever the respondent answers to a given item and the answer is collected. Beginning with the equation (2), the likelihood is as follows The test consisted of 100 dichotomously scored items covering different Biology topics. For the purpose of this analysis, items were classified into three general domains – genetics, taxonomy, and human biology, respectively. The mutual proportions of these three domains were of nearly equal size. L (θ; up) = IY i=1 P (Upi = upi|θ, ξi) (4) (4) and is maximized with respect to θ. Then, the first-order and second-order partial derivatives are needed to compute To evaluate psychometric properties of the items, unidi- mensional two-parameter logistic (2PL) item-response theory C. Post-hoc analysis {E} ; Algorithm 2: Investigation of average adaptive test length depending on the z-score from the original linear test Data: data of the real test Result: boxplots of average test lengths for groups based on z-scores from the original linear test 1 δ = 0.05 // neighbourhood around z∗; 2 {A} = ∅ // list of vectors of lengths; 3 // for different z-scores; 4 {Z} // list of original z-scores; 5 {Z∗} = ∅// list of z∗-scores; 6 for j = 1 : 17 do 7 z∗= −2.00 + 0.25 · j; 8 {Z∗} = {Z∗} ∪{z∗}; 9 {D} = ∅; 10 for all subjects with z ∈Z such that |z −z∗| ≤δ do 11 run an adaptive test for the subject with stopping criterion SE(θ)max = 0.30 and save its length as d; 12 {D} = {D} ∪{d}; 13 end 14 {A} = {A} ∪{D}; 15 end 16 make a boxplot of {A} vs. {Z∗} ; Algorithm 1: Investigation of adaptive test length depending on the precision of ability estimate Data: data of the real test Result: boxplots of test lengths for CAT with different standard errors of ability estimates Result: boxplots of average test lengths for groups based on z-scores from the original linear test 1 δ = 0.05 // neighbourhood around z∗; 2 {A} = ∅ // list of vectors of lengths; 3 // for different z-scores; 4 {Z} // list of original z-scores; 5 {Z∗} = ∅// list of z∗-scores; 1 δ = 0.05 // neighbourhood around z∗; 2 {A} = ∅ // list of vectors of lengths; 3 // for different z-scores; 4 {Z} // list of original z-scores; 5 {Z∗} = ∅// list of z∗-scores; r j = 1 : 17 do z∗= −2.00 + 0.25 · j; {Z∗} = {Z∗} ∪{z∗}; {D} = ∅; { } for all subjects with z ∈Z such that |z −z∗| ≤δ do { } for all subjects with z ∈Z such that |z −z∗| ≤δ do 1 run an adaptive test for the subject with stopping criterion SE(θ)max = 0.30 and save its length as d; its length as d; 12 {D} = {D} ∪{d}; 3 end 4 {A} = {A} ∪{D}; d {A} = {A} ∪{D}; d 17 make a boxplot of {A} vs. {E} ; 17 make a boxplot of {A} vs. {E} ; end make a boxplot of {A} vs. C. Post-hoc analysis {Z∗} ; δ = 0.05 and z∗∈{−2.00, −1.75, −1.50, . . . , +1.75, +2.00} were supposed to virtually take the adaptive test, keeping the SE(θ)max = 0.30 for equation (7) constant. The z∗neighbour- hood δ = 0.05 was chosen empirically, but consequently, one can realize that δ = 0.125 would cover continuously the entire range of all z-scores. For each z∗, a vector of all the adaptive tests’ lengths was displayed in the final boxplot. The schema of the simulation is provided in Algorithm 2. mismatch rate for adaptive tests with stopping criteria SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. While the best fifth for the linear test was calculated using the z-scores, the MML ability estimates were used for the adaptive test. D. Monte Carlo simulation studies Whereas the post-hoc analysis requires real data from an administrated test, the Monte-Carlo simulation study starts from the scratch – it generates abilities of "virtual" test-takers usually following normal distribution and responses based on selected model (e. g. the 2PL IRT model) with given item parameters. We first simulated the linear test, then, based on the simulated answers, the adaptive scenario was simulated. We then correlated ability estimates from the adaptive test with the true ability values. Finally, we displayed lengths of adaptive test and we correlated the ability estimate with the true ability. Other comparisons and analyses are possible (length with respect to the true ability score, etc.), but not presented here. The algorithm of the simulation is technically described in Algorithm 3. Similarly, the effect of content balancing and item exposure rate control was analyzed. When an adaptive test was admin- istered to each test-taker from a randomly selected subset, we counted how many times individual items occur in the tests. Absolute numbers of the items’ occurrences were then counted up for different scenarios – besides the situation when neither the content balancing nor the item exposure was applied, the case of (only) the content balancing and (only) the item exposure rate controlling was taken into account. Eventually, using the fact, the items were classified into three domains (Genetics, Taxonomy, Human Biology), their counts could be clearly plotted using boxes in a boxplot. Finally, to study the impact of adaptive test with different settings on the admission process, we enumerated the admission mismatch rate between linear and adaptive tests. We assumed the best fifth of all the applicants would be admitted and we calculated the mismatch rate as the ratio of students who would be admitted based on their score in the linear test but not based on the score in adaptive test and vice versa. We then compared the admission Analyses were performed in R programming language and environment [25] using the package mirtCAT [26]. C. Post-hoc analysis {E} ; h ent s; s; ing h Algorithm 2: Investigation of average adaptive test length depending on the z-score from the original linear test Data: data of the real test Result: boxplots of average test lengths for groups based on z-scores from the original linear test 1 δ = 0.05 // neighbourhood around z∗; 2 {A} = ∅ // list of vectors of lengths; 3 // for different z-scores; 4 {Z} // list of original z-scores; 5 {Z∗} = ∅// list of z∗-scores; 6 for j = 1 : 17 do 7 z∗= −2.00 + 0.25 · j; 8 {Z∗} = {Z∗} ∪{z∗}; 9 {D} = ∅; 10 for all subjects with z ∈Z such that |z −z∗| ≤δ do 11 run an adaptive test for the subject with stopping criterion SE(θ)max = 0.30 and save its length as d; 12 {D} = {D} ∪{d}; 13 end 14 {A} = {A} ∪{D}; 15 end 16 make a boxplot of {A} vs. {Z∗} ; Algorithm 1: Investigation of adaptive test length depending on the precision of ability estimate Data: data of the real test Result: boxplots of test lengths for CAT with different standard errors of ability estimates 1 {S} // set or subset of respondents; 2 // of the real test; 3 {A} = ∅ // list of vectors of lengths; 4 // for different standard; 5 // errors; 6 {E} = ∅ // list of standard errors; 7 for j = 1 : 7 do 8 SE = 0.15 + 0.05 · j; 9 {E} = {E} ∪{SE}; 10 {D} = ∅; 11 for p ∈S do 12 run an adaptive test for subject p with stopping criterion SE(θ)max = SE and save its length as d; 13 {D} = {D} ∪{d}; 14 end 15 {A} = {A} ∪{D}; 16 end 17 make a boxplot of {A} vs. C. Post-hoc analysis Termination criteria. In our simulation studies, we used ability estimate precision as a stopping rule. Assuming the IUp,k−1(ˆθp,k−1) is observed Fisher information [17] at ˆθp,k−1 where Uk−1 is an answer pattern until the (k −1)-th item (inclusively) given a subject p, then standard error of ability ˆθp,k−1 is In post-hoc analysis, the item parameters and the response patterns of the respondents were used to rerun the test under adaptive conditions. By doing this, the properties of the adaptive test (such as the test length, precision of estimated abilities etc.) were "post-hoc" evaluated and compared to the original linear test. SE(ˆθp,k−1) = 1 r IUp,k−1  ˆθp,k−1 . (7) (7) Considering the dataset of test-takers taking the real test, we varied the maximal allowed standard error of the ability estimates and ran the adaptive version of the test for each of the test-takers investigating how many items were needed to complete the test. The pseudocode of this simulation is provided in Algorithm 1. For the adaptive test, we specified the maximal allowed standard error SE(θ)max of the ability estimate based on the distribution of standard errors of the ability estimates from the full 100-item test. For subject p, the test was terminated just after administration of the k-th item if SE(ˆθp,k) ≤SE(θ)max and SE(ˆθp,k−1) > SE(θ)max. Otherwise, the test was stopped if the length of 100 items was reached and all available items were used. Similarly, we calculated the z-score for each subject using the test scores from the real test, z-score = xp −¯x sx , Whenever the termination (stopping) criterion is met, the adaptive test is ended and final estimate of test-taker’s ability is provided. where xp is a test score of a subject p, ¯x is an average test score and sx is a standard deviation of all test scores. All test-takers having their z-scores in the interval of |z −z∗| ≤δ, where where xp is a test score of a subject p, ¯x is an average test score and sx is a standard deviation of all test scores. All test-takers having their z-scores in the interval of |z −z∗| ≤δ, where PROCEEDINGS OF THE FEDCSIS. C. Post-hoc analysis SOFIA, 2020 362 Algorithm 2: Investigation of average adaptive test length depending on the z-score from the original linear test Data: data of the real test Result: boxplots of average test lengths for groups based on z-scores from the original linear test Algorithm 2: Investigation of average adaptive test length depending on the z-score from the original linear test Data: data of the real test Result: boxplots of average test lengths for groups based on z-scores from the original linear test 1 δ = 0.05 // neighbourhood around z∗; 2 {A} = ∅ // list of vectors of lengths; 3 // for different z-scores; 4 {Z} // list of original z-scores; 5 {Z∗} = ∅// list of z∗-scores; 6 for j = 1 : 17 do 7 z∗= −2.00 + 0.25 · j; 8 {Z∗} = {Z∗} ∪{z∗}; 9 {D} = ∅; 10 for all subjects with z ∈Z such that |z −z∗| ≤δ do 11 run an adaptive test for the subject with stopping criterion SE(θ)max = 0.30 and save its length as d; 12 {D} = {D} ∪{d}; 13 end 14 {A} = {A} ∪{D}; 15 end 16 make a boxplot of {A} vs. {Z∗} ; Algorithm 1: Investigation of adaptive test length depending on the precision of ability estimate Data: data of the real test Result: boxplots of test lengths for CAT with different standard errors of ability estimates 1 {S} // set or subset of respondents; 2 // of the real test; 3 {A} = ∅ // list of vectors of lengths; 4 // for different standard; 5 // errors; 6 {E} = ∅ // list of standard errors; 7 for j = 1 : 7 do 8 SE = 0.15 + 0.05 · j; 9 {E} = {E} ∪{SE}; 10 {D} = ∅; 11 for p ∈S do 12 run an adaptive test for subject p with stopping criterion SE(θ)max = SE and save its length as d; 13 {D} = {D} ∪{d}; 14 end 15 {A} = {A} ∪{D}; 16 end 17 make a boxplot of {A} vs. A. Post-hoc analysis Post-hoc analysis used the real test-takers data to simulate the results under scenario of an adaptive test with selected parameters. As an example, Fig. 5 demonstrates iteratively estimated ability estimates and order of items in which they would be administered to the 1-st subject under adaptive scenario with terminating criterion SE(θ)max = 0.30. We can see that the initial item would be item number 81, the last item would be item number 70. The width of the grey belt stands for precision of the ability estimate at each step k, equal to two standard errors 2SE(ˆθ)p,k of the ability estimate of person p. The belt becomes more narrow as the test-taker answers more and more items. Note that the standard error after 18 administered items is SE(ˆθ)1,18 ≤0.30 while after 17 administered items it is SE(ˆθ)1,17 > 0.30. ∀i ∈{1, 2, . . . , 100}, resulting in a spectrum of the item information curves. q P(q) 0.0 0.2 0.4 0.6 0.8 1.0 -6 -4 -2 0 2 4 6 Fig. 2. Item characteristic curves of the linear test estimated using 2PL IRT model. q P(q) 0.0 0.2 0.4 0.6 0.8 1.0 -6 -4 -2 0 2 4 6 , As a result of Algorithm 1, Fig. 6 presents how the number of items needed to stop the adaptive test depends on the termination criterion. We can see that the higher the maximal standard error is applied as the termination criterion, the lower the number of items is needed to terminate the adaptive test. As a result of simulation described with Algorithm 2, Fig. 7 illustrates how the respondent ability (estimated with a z-score) affects the number of items needed to stop the adaptive test. The size of maximal allowed standard error of the ability estimates as the stopping criterion was set to SE(θ)max = 0.30 based on the distribution of the standard As a result of Algorithm 1, Fig. 6 presents how the number of items needed to stop the adaptive test depends on the termination criterion. We can see that the higher the maximal standard error is applied as the termination criterion, the lower the number of items is needed to terminate the adaptive test. Fig. 2. Item characteristic curves of the linear test estimated using 2PL IRT model. III. RESULTS 10 for p = 1 : n do p 11 apply 2PL IRT model on p-th ability of {S} and simulate an answer pattern ; p 13 {A} = {A} ∪ˆθp; 14 {D} = {D} ∪{d}; d Fig. 4. Histogram of standard errors of the ability estimates. 17 make a scatterplot of {A} vs. {S}, calculate a correlation of {A} and {S} ; III. RESULTS All items of the linear test were calibrated using the 2PL IRT model as described by equation (1). Item characteristic curves and item information curves are plotted in Fig. 2 and Fig. 3. All items have positive discrimination ai > 0 for LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING 363 Algorithm 3: Investigation of ability estimates based on adaptive tests using a Monte-Carlo simulation Data: generated abilities following N(0, 12), item parameters (estimated from real data), adaptive test’s stopping criterion SE(θ)max = 0.30, item selection using maximum-information rule Result: a list of ability estimates based on adaptive test 1 n = 300 // number of generated; 2 // abilities; 3 {S} // list of n generated; 4 // abilities; 5 // following N(0, 12); 6 {A} = ∅ // list of adaptive-based; 7 // ability estimates; 8 {D} = ∅// list of lengths; 9 // of adaptive tests; 10 for p = 1 : n do 11 apply 2PL IRT model on p-th ability of {S} and simulate an answer pattern ; 12 use the answer pattern and run an adaptive test for p-th ability and save its length as d and ability estimate as ˆθp ; 13 {A} = {A} ∪ˆθp; 14 {D} = {D} ∪{d}; 15 end 16 make a boxplot of {D} ; 17 make a scatterplot of {A} vs. {S}, calculate a correlation of {A} and {S} ; Algorithm 3: Investigation of ability estimates based on adaptive tests using a Monte-Carlo simulation q I(q) 0.0 0.2 0.4 0.6 0.8 -6 -4 -2 0 2 4 6 Fig. 3. Item information curves of the linear test estimated using 2PL IRT model. q I(q) 0.0 0.2 0.4 0.6 0.8 -6 -4 -2 0 2 4 6 1 n = 300 // number of generated; 2 // abilities; 3 {S} // list of n generated; 4 // abilities; 5 // following N(0, 12); 6 {A} = ∅ // list of adaptive-based; 7 // ability estimates; 8 {D} = ∅// list of lengths; 9 // of adaptive tests; Fig. 3. Item information curves of the linear test estimated using 2PL IRT model. standard error of ability estimates absolute frequency 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0 200 400 600 800 Fig. 4. Histogram of standard errors of the ability estimates. A. Post-hoc analysis errors, displayed in Fig. 4. We can see that the closer the z-score is to zero, the lower number of items is needed to complete the adaptive test while meeting the required ability estimate precision defined by SE(θ)max = 0.30. This corresponds to the fact that the information functions for majority of items have the maxima for ability around zero as demonstrated in Fig. 3. Contrary, for z-scores far from zero, the observed Fisher information is small for most of the items, thus a larger number of items is needed to meet the stopping criterion, and often not even meeting it using all 100 items available. Table I provides mismatch matrices for linear and adaptive tests with different stopping criteria SE(θ)max. As expected, the mismatch rate increases with increased allowed standard error applied as a stopping criterion in the adaptive test. The mismatch rate is 0.036, 0.083, 0.102 and 0.118 for adaptive tests with stopping rules SE(θ)max = 0.20, 0.30, 0.40 and 0.50, respectively. A. Post-hoc analysis A plot of progress of 1-st subject in an adaptive test with the terminating criterion set to maximal allowable standard error of the ability estimates of SE(θ)max = 0.30. Fig. 7. Number of items needed to stop the adaptive test in respondents of different ability levels. is employed, there is no visible change in comparison to no application of the exposure control. 0.20 0.25 0.30 0.35 0.40 0.45 0.50 20 40 60 80 100 standard error of θ # of items needed to stop the adaptive test 0.20 0.25 0.30 0.35 0.40 0.45 0.50 20 40 60 80 100 standard error of θ # of items needed to stop the adaptive test Fig. 6. Number of items needed to stop the adaptive test versus a size of standard error of the ability estimate as the stopping criterion. 0 10 20 30 40 50 # of times item is administered neither balancing nor control content balancing exposure control genetics taxonomy human biology Fig. 8. Number of items belonging to the domains genetics, taxonomy, human biology, respectively, as were administrated with application of neither content balancing nor item exposure control, with application of content balancing, and with application of item exposure rate control only. 0 10 20 30 40 50 # of times item is administered neither balancing nor control content balancing exposure control genetics taxonomy human biology Fig. 6. Number of items needed to stop the adaptive test versus a size of standard error of the ability estimate as the stopping criterion. Fig. 8. Number of items belonging to the domains genetics, taxonomy, human biology, respectively, as were administrated with application of neither content balancing nor item exposure control, with application of content balancing, and with application of item exposure rate control only. errors, displayed in Fig. 4. We can see that the closer the z-score is to zero, the lower number of items is needed to complete the adaptive test while meeting the required ability estimate precision defined by SE(θ)max = 0.30. This corresponds to the fact that the information functions for majority of items have the maxima for ability around zero as demonstrated in Fig. 3. Contrary, for z-scores far from zero, the observed Fisher information is small for most of the items, thus a larger number of items is needed to meet the stopping criterion, and often not even meeting it using all 100 items available. A. Post-hoc analysis When applying the 2PL IRT model on the data from the linear test, we get, besides other, also standard errors of the ability estimates for each test-taker. Histogram of these standard errors in in Fig. 4. Range of the standard errors of the ability estimates is between 0.20 to 0.50, with majority of values within the interval ⟨0.20, 0.30⟩. As a result of simulation described with Algorithm 2, Fig. 7 illustrates how the respondent ability (estimated with a z-score) affects the number of items needed to stop the adaptive test. The size of maximal allowed standard error of the ability estimates as the stopping criterion was set to SE(θ)max = 0.30 based on the distribution of the standard PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 364 CAT Standard Errors Item θ −1.0 −0.5 0.0 0.5 1.0 0 81 72 57 82 73 76 18 78 56 74 90 30 80 53 89 37 70 Fig. 5. A plot of progress of 1-st subject in an adaptive test with the terminating criterion set to maximal allowable standard error of the ability estimates of SE(θ)max = 0.30. CAT Standard Errors Item θ −1.0 −0.5 0.0 0.5 1.0 0 81 72 57 82 73 76 18 78 56 74 90 30 80 53 89 37 70 Fig. 5. A plot of progress of 1-st subject in an adaptive test with the terminating criterion set to maximal allowable standard error of the ability estimates of SE(θ)max = 0.30. −2.00 −1.50 −1.00 −0.50 0.00 0.50 1.00 1.50 2.00 20 40 60 80 100 z−score # of items needed to stop the adaptive test standard error of θ = 0.3 Fig. 7. Number of items needed to stop the adaptive test in respondents of different ability levels. CAT Standard Errors Item θ −1.0 −0.5 0.0 0.5 1.0 0 81 72 57 82 73 76 18 78 56 74 90 30 80 53 89 37 70 CAT Standard Errors −2.00 −1.50 −1.00 −0.50 0.00 0.50 1.00 1.50 2.00 20 40 60 80 100 z−score # of items needed to stop the adaptive test standard error of θ = 0.3 Fig. 7. Number of items needed to stop the adaptive test in respondents of different ability levels. −2.00 −1.50 −1.00 −0.50 0.00 0.50 1.00 1.50 2.00 20 40 60 80 100 z−score # of items needed to stop the adaptive test standard error of θ = 0.3 Fig. 5. TABLE I MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND ADAPTIVE TEST WITH STOPPING CRITERION SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. TABLE I MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND ADAPTIVE TEST WITH STOPPING CRITERION SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND ADAPTIVE TEST WITH STOPPING CRITERION SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. SE(θ)max = 0.20 admitted by adaptive test no yes admitted by linear test no 1842 38 yes 48 435 SE(θ)max = 0.30 admitted by adaptive test no yes admitted by linear test no 1787 93 yes 103 380 SE(θ)max = 0.40 admitted by adaptive test no yes admitted by linear test no 1762 118 yes 123 360 SE(θ)max = 0.50 admitted by adaptive test no yes admitted by linear test no 1737 143 yes 136 347 Fig. 10. A scatterplot of the generated abilities and their estimates based on the adaptive tests. The blue line stands for an axis of the first quadrant of the plot. 75% shortening as compared to the linear test, while keeping the same precision of ability estimates for most respondents. When even larger standard errors of the ability estimates are tolerated, the length of the test could be reduced even more, e. g. to only 10 items per one test, as was shown in the post-hoc analysis of the average adaptive test length with varying stopping criterion. 20 40 60 80 100 adaptive test # of items needed to stop the adaptive test # of items needed to complete the linear test The post-hoc simulation also demonstrated that an average length for adaptive tests is shorter for average ability levels. While the content balancing with the combinatorial ap- proach showed a significant improvement in test domain equalizing, an effect of the item exposure rate did not seem to be so eminent under our setting. The lower the tolerated standard error as a stopping criterion of the adaptive test is, the lower is the mismatch error rate when using an adaptive test instead of the linear one. The mismatch rate was less than 10% for adaptive test with stopping criterion of SE(ˆθ)max = 0.30. The Monte-Carlo simulation study also indicated that abil- ity estimates provided by the adaptive tests can be tightly correlated with their true (generated) values; thus, although the shortened length, the adaptive test can provide precise estimates of the respondent abilities. adaptive test Fig. 9. TABLE I MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND ADAPTIVE TEST WITH STOPPING CRITERION SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. A boxplot of number of items needed to be answered to complete the adaptive test based on Monte-Carlo simulated test-takers’ abilities. The blue dashed line shows a length of the linear test (100 items). To conclude, usage of adaptive testing seems to be a promis- ing alternative to classic linear tests and offers many advan- tages as showed by the simulations. Pearson’s correlation between the generated abilities and their estimates based on the adaptive tests is about ρ .= 0.960, which is depicted also in Fig. 10. IV. CONCLUSION Research was supported by Charles University grant PRIMUS/17/HUM/11. Research was supported by Charles University grant PRIMUS/17/HUM/11. Both the post-hoc analysis and Monte-Carlo simulation study showed that average test lengths can be shortened with adaptive tests, while keeping the standard error of the ability estimates at the same level for most of the respondents. The shortening of the test within the adaptive test with SE(ˆθ)max = 0.30 was by about 75 % percent, i. e. while the original linear test had 100 items, the adaptive one was ended on average after answering 25 items only. B. Monte-Carlo simulation study In Fig. 8, we plot numbers of occurrences of items in all individual adaptive tests for randomly selected 50 test-takers, considering that each item belongs to one of the following three domains – either to genetics, taxonomy, or human biol- ogy, respectively. While the proportions of the three domains of items as they were administrated vary a lot in Fig. 8 where neither the content balancing nor the item exposure rate control is applied, these numbers are near equal when the content balancing is applied. When the item exposure rate control As a result of the Monte-Carlo simulation study described by Algorithm 3, Fig. 9 provides a boxplot illustrating the mean length of the adaptive test for the set of test-takers with the generated abilities. While each test-taker has to answer to all (100) items within the linear fashion, they would only have to answer about 25 % of items to finish the simulated adaptive test with the termination criterion SE(θ)max = 0.30. The length of the test using this adaptive scenario provides LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING 365 -2 -1 0 1 2 -2 -1 0 1 2 generated abilities abilities estimated based on the adaptive tests Fig. 10. A scatterplot of the generated abilities and their estimates based on the adaptive tests. The blue line stands for an axis of the first quadrant of the plot. -2 -1 0 1 2 -2 -1 0 1 2 generated abilities abilities estimated based on the adaptive tests PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 366 [3] David Magis, Duanli Yan, and Alina A Von Davier. Computerized adaptive and multistage testing with R: Using packages catr and mstr. Springer, 2017. [14] Yoshio Takane and Jan de Leeuw. “On the relationship between item response theory and factor analysis of discretized variables”. In: Psychometrika 52.3 (Sept. 1987), pp. 393–408. DOI: 10.1007/bf02294363. URL: https://doi.org/10.1007/bf02294363. [4] David J Weiss and G Gage Kingsbury. “Application of computerized adaptive testing to educational problems”. In: Journal of Educational Measurement 21.4 (1984), pp. 361–375. [15] Cees A. W. Glas. “Modification indices for the 2-PL and the nominal response model”. In: Psychometrika 64.3 (Sept. 1999), pp. 273–294. DOI: 10.1007/bf02294296. URL: https://doi.org/10.1007/bf02294296. [5] Jan Stochl, Jan R Böhnke, Kate E Pickett, et al. “Com- puterized adaptive testing of population psychological distress: simulation-based evaluation of GHQ-30”. In: Social psychiatry and psychiatric epidemiology 51.6 (2016), pp. 895–906. [16] A. P. Dempster, N. M. Laird, and D. B. Rubin. “Max- imum likelihood from incomplete data via the EM algorithm”. In: Journal of the Royal Statistical Society, Series B 39.1 (1977), pp. 1–38. [6] [6] Jan Stochl, Jan R Böhnke, Kate E Pickett, et al. “An evaluation of computerized adaptive testing for general psychological distress: combining GHQ-12 and Affectometer-2 in an item bank for public mental health research”. In: BMC medical research methodology 16.1 (2016), p. 58. [17] Hua-Hua Chang and Zhiliang Ying. “Nonlinear sequen- tial designs for logistic item response theory models with applications to computerized adaptive tests”. In: The Annals of Statistics 37.3 (June 2009), pp. 1466– 1488. DOI: 10.1214/08-aos614. URL: https://doi.org/10. 1214/08-aos614. [7] Dagmar Amtmann, Alyssa M Bamer, Jiseon Kim, et al. “A comparison of computerized adaptive testing and fixed-length short forms for the Prosthetic Limb Users Survey of Mobility (PLUS-MTM)”. In: Prosthetics and orthotics international 42.5 (2018), pp. 476–482. [18] Daniel O. Segall. “Multidimensional adaptive testing”. In: Psychometrika 61.2 (June 1996), pp. 331–354. DOI: 10.1007/bf02294343. URL: https://doi.org/10.1007/ bf02294343. [19] Thomas A. Warm. “Weighted likelihood estimation of ability in item response theory”. In: Psychometrika 54.3 (Sept. 1989), pp. 427–450. DOI: 10.1007/bf02294627. URL: https://doi.org/10.1007/bf02294627. [8] Karon F Cook, Seung W Choi, Paul K Crane, et al. “Letting the CAT out of the bag: comparing com- puter adaptive tests and an eleven-item short form of the Roland-Morris Disability Questionnaire”. In: Spine 33.12 (2008), p. 1378. [20] Frederic Lord. Applications of item response theory to practical testing problems. Hillsdale, N.J: L. REFERENCES [1] Wim J Linden, Wim J van der Linden, and Cees AW Glas. Computerized adaptive testing: Theory and prac- tice. Springer, 2000. [2] Howard Wainer, Neil J Dorans, Ronald Flaugher, et al. Computerized adaptive testing: A primer. Routledge, 2000. PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 Erlbaum Associates, 1980. ISBN: 978-0898590067. [9] Patricia Martinková, Lubomír Štˇepánek, Adéla Drabi- nová, et al. “Semi-real-time analyses of item character- istics for medical school admission tests”. In: Proceed- ings of the 2017 Federated Conference on Computer Science and Information Systems. Ed. by M. Ganzha, L. Maciaszek, and M. Paprzycki. Vol. 11. Annals of Computer Science and Information Systems. IEEE, 2017, pp. 189–194. DOI: 10 . 15439 / 2017F380. URL: http://dx.doi.org/10.15439/2017F380. [21] Frank L. Schmidt, John E. Hunter, and Vern W. Urry. “Statistical power in criterion-related validation stud- ies.” In: Journal of Applied Psychology 61.4 (1976), pp. 473–485. DOI: 10.1037/0021-9010.61.4.473. URL: https://doi.org/10.1037/0021-9010.61.4.473. [22] Wim J. van der Linden and Richard M. Luecht. “Observed-score equating as a test assembly problem”. In: Psychometrika 63.4 (Dec. 1998), pp. 401–418. DOI: 10.1007/bf02294862. URL: https://doi.org/10.1007/ bf02294862. [10] [10] ˇCestmír Štuka, Patrícia Martinková, Karel Zvára, et al. “The prediction and probability for successful com- pletion in medical study based on tests and pre- admission grades”. In: New Educational Review 28 (2012), pp. 138–52. [23] [23] Rebecca D. Hetter and J. Bradford Sympson. “Item exposure control in CAT-ASVAB.” In: Computerized adaptive testing: From inquiry to operation. American Psychological Association, 1997, pp. 141–144. DOI: 10. 1037/10244-014. URL: https://doi.org/10.1037/10244- 014. [11] Patrícia Martinková and Adéla Drabinová. “ShinyIte- mAnalysis for Teaching Psychometrics and to Enforce Routine Analysis of Educational Tests.” In: R Journal 10.2 (2018). [24] Martha L. Stocking and Charles Lewis. “Controlling Item Exposure Conditional on Ability in Computerized Adaptive Testing”. In: Journal of Educational and Be- havioral Statistics 23.1 (1998), p. 57. DOI: 10.2307/ 1165348. URL: https://doi.org/10.2307/1165348. [12] Wim J. van der Linden and Cees A.W. Glas. “25 Statistical Aspects of Adaptive Testing”. In: Handbook of Statistics. Elsevier, 2006, pp. 801–838. DOI: 10.1016/ s0169-7161(06)26025-5. URL: https://doi.org/10.1016/ s0169-7161(06)26025-5. [13] [13] [25] [25] R Core Team. R: A Language and Environment for Sta- tistical Computing. R Foundation for Statistical Com- puting. Vienna, Austria, 2017. URL: https://www.R- project.org/. [13] R. Darrell Bock and Murray Aitkin. “Marginal maxi- mum likelihood estimation of item parameters: Appli- cation of an EM algorithm”. In: Psychometrika 46.4 (Dec. 1981), pp. 443–459. DOI: 10.1007/bf02293801. URL: https://doi.org/10.1007/bf02293801. [26] R. Philip Chalmers. “Generating Adaptive and Non- Adaptive Test Interfaces for Multidimensional Item Re- sponse Theory Applications”. In: Journal of Statistical LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING 367 [26] R. Philip Chalmers. “Generating Adaptive and Non- Adaptive Test Interfaces for Multidimensional Item Re- sponse Theory Applications”. In: Journal of Statistical
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Analyse der Mitgliederentwicklung sowie weiterer Kennzahlen der bundesunmittelbaren Krankenkassen ohne und mit Zusatzbeitrag bzw. Prämie
Gesundheits- und Sozialpolitik
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THEMA THEMA 1 Dieser Beitrag gibt die persönlichen Auffas- sungen der Verfasser wieder. Maximilian GaSSner, Frank Otto, Eva Ludwig Dr. Maximilian Gaßner ist Präsident des Bundesversiche- rungsamts (BVA) in Bonn Dr. Maximilian Gaßner ist Präsident des Bundesversiche- rungsamts (BVA) in Bonn Frank Otto ist Referatsleiter im Referat Finanzaufsicht in der Krankenversicherung im BVA Eva Ludwig ist Referentin im Referat Finanzaufsicht in der Krankenversicherung im BVA Die nachfolgende Untersuchung be- schäftigt sich mit der Frage der Mit- gliederentwicklung sowie weiterer Kennzahlen der bundesunmittelbaren Krankenkassen ohne und mit Zusatz- beitrag bzw. Prämie. Die Ergebnisse müssen als statistisch nicht signifikante Zwischenergebnisse interpretiert wer- den. Endgültige Feststellungen verbie- ten sich in Folge der geringen Zahl der Kassen und der besonderen Situation im betrachteten Zeitraum (01.01.2009 bis heute). Einfluss auf die Zahlen dürften zudem politische Interpretationen der gesetzlichen Regelungen, aber auch sonstige exogene Ereignisse, wie z. B. die Intervention des Bundeskartellamtes ge- gen die gemeinsame Pressekonferenz der betroffenen Kassen zur Erhebung des Zusatzbeitrages gehabt haben, sodass die Zahlen nicht nur auf die Wirkungen des Preissignals zurückzuführen sind. Analyse der Mitgliederentwicklung sowie weiterer Kennzahlen der bundesunmittelbaren Krankenkassen ohne und mit Zusatzbeitrag bzw. Prämie1 Mit Einführung des Gesundheitsfonds im Jahr 2009 wurden die individuellen Beitragssätze der gesetzlichen Krankenkassen durch ein System mit einheitlichem Beitragssatz, Zusatzbeiträgen und Prämien ersetzt. Einige Krankenkassen haben seitdem Zusatzbeiträge eingeführt und wieder abgeschafft. Andere Krankenkassen waren in der Lage, Prämien an ihre Mitglieder auszuzahlen. Welche Erfahrungen haben diese Krankenkassen mit den neuen Instrumenten gemacht? Eine erster Überblick. Maximilian GaSSner, Frank Otto, Eva Ludwig Dr. Maximilian Gaßner ist Präsident des Bundesversiche- rungsamts (BVA) in Bonn Frank Otto ist Referatsleiter im Referat Finanzaufsicht in der Krankenversicherung im BVA Eva Ludwig ist Referentin im Referat Finanzaufsicht in der Krankenversicherung im BVA https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig Einleitung Soweit der Finanzbedarf einer Kranken- kasse durch die Zuweisungen aus dem Gesundheitsfonds nicht gedeckt ist, hat sie gem. § 242 Abs. 1 SGB V einen »ein- kommensunabhängigen Zusatzbeitrag« zu erheben. Der Zusatzbeitrag setzt ein klares Preissignal, das von vielen Ge- sundheitsökonomen und den Vertretern von Prämienmodellen zur Verschärfung des Wettbewerbs immer gefordert wur- de. Dieses Preissignal ist von den Mit- gliedern der gesetzlichen Krankenversi- cherung auch »gut« verstanden worden. Viele haben die Kassen gewechselt. Zu- mal dieser Kassenwechsel durch ein Son- derkündigungsrecht gem. § 175 Abs. 4 S. 5 SGB V vom Gesetzgeber auch noch erleichtert wird. Die Transparenz wird noch verstärkt durch die Verpflichtung der Krankenkasse gem. § 175 Abs. 4 S. 6 SGB  V, ihre Mitglieder auf das Sonderkündigungsrecht ausdrücklich hinzuweisen. G+S 5/2012 G+S 5/2012 52 THEMA Tabelle 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Zusatzbeitrag Bundesunmittelbare Kranken­ kassen mit ZB Höhe* Von Bis Mitglieder­ veränderung im ZB-Zeit­ raum Unter 200.000 Mitglieder Ungewichteter  Durchschnitt 8,83 € Feb 2010 Okt 2011 -20,45% Gewichteter   Durchschnitt 9,23 € Mrz 2010 Sep 2011 -29,48% Über 200.000 Mitglieder Ungewichteter  Durchschnitt 8,00 € Jun 10 Feb 12 -12,08% Gewichteter   Durchschnitt 8,00 € Nov 10 Jun 12 -7,75% Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet. * Ohne prozentuale Zusatzbeiträge Tabelle 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Zusatzbeitrag Im Zeitraum vom 01.01.2009 bis heute (Juli 2012) gab es insgesamt 14 (9,6 %) bundesunmittelbare Kranken- kassen mit Zusatzbeitrag und 7 (4,8 %) mit einer Prämie. Hinzu kommen weitere 3 (2,1 %) landesunmittelbare Krankenkassen mit Zusatzbeitrag und 3 (2,1 %) landesunmittelbare Kranken- kassen mit Prämie. 2 Vgl. Eibich, Schmitz und Ziebarth im DIW Wochenbericht Nr. 51+52, 2011, S. 8. 1. Entwicklung und Kennzahlen der Krankenkassen mit Zusatzbeitrag Übersicht und Mitglieder­ entwicklung Der Zusatzbeitrag wurde in Höhe von 6,50 Euro bis 15 Euro erhoben sowie in Höhe von 1 % des beitragspflichtigen Einkommens. Die folgende Tabelle zeigt eine Übersicht der Durchschnittswerte für bundesunmittelbare Krankenkassen mit Zusatzbeitrag. Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet. * Ohne prozentuale Zusatzbeiträge Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet. * Ohne prozentuale Zusatzbeiträge nige bis gar keine Mitgliederverluste. 2 Vgl. Eibich, Schmitz und Ziebarth im DIW Wochenbericht Nr. 51+52, 2011, S. 8. 3 Vgl. ebd., S. 9. nige bis gar keine Mitgliederverluste. se übereinstimmt, sind die Zahlen nicht vergleichbar. buchen. Dieses Konto wird nach dem Soll-Prinzip der zeitlichen Rechnungs- abgrenzung geführt, dieses sieht die Ver- buchung in jener Rechnungsperiode vor, für die die Rechnungsstellung erfolgt ist. Die Differenz dieser beiden Posten lässt sich damit als Unterschiedsbetrag zwischen dem maximal zu erreichenden Zusatzbeitragseinnahmen und den tat- sächlichen Rechnungsstellungen für den Zusatzbeitrag interpretieren. nige bis gar keine Mitgliederverluste. Thema Zusatzbeitrag vor. Die Autoren berechneten, dass die Einführung eines Zusatzbeitrags im Durchschnitt zu Ver- sichertenverlusten von –7,61 % führt.2 Die individuelle Wahrscheinlichkeit für einen Krankenkassenwechsel verdop- pelt sich durch den Zusatzbeitrag, so die Autoren.3 Da die Art und Weise der Berechnung nicht mit der dieser Analy- Die Mitgliederentwicklung der Kran- kenkassen über 200.000 Mitglieder mit Zusatzbeitrag zeigt ein homogeneres Bild, als das der kleineren Krankenkas- sen. Es wird deutlich, dass sich der Zu- satzbeitrag je nach Krankenkassegröße unterschiedlich stark auf die Mitglieder- entwicklung auswirkt. Der Anstieg im Januar 2012 ergibt sich aus einer Fusion. Die Erhebung des Zusatzbeitrags wirkte sich auf die Mitgliederzahl un- terschiedlich aus. Die folgende Abbil- dung zeigt die Mitgliederentwicklung der bundesunmittelbaren Krankenkas- sen mit Zusatzbeitrag. Im Wochenbericht Nr. 51+52/2011 vom 21. Dezember 2011 legte das Deutsche Institut für Wirtschaftsfor- schung (DIW) eigene Analysen zum Diagramm1 Die Krankenkassen mit dem stärksten Mitgliederverlust finden sich im Bereich der mittleren Mitgliederstärke. Kleine geschlossene Krankenkassen hatten we- 53 5/2012 G+S Abbildung 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Zusatzbeitrag Page 1 Abbildung 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Zusatzbeitrag 0 200.000 400.000 600.000 800.000 1.000.000 1.200.000 1.400.000 1.600.000 1.800.000 2.000.000 2010.01 2010.02 2010.03 2010.04 2010.05 2010.06 2010.07 2010.08 2010.09 2010.10 2010.11 2010.12 2011.01 2011.02 2011.03 2011.04 2011.05 2011.06 2011.07 2011.08 2011.09 2011.10 2011.11 2011.12 2012.01 2012.02 2012.03 2012.04 2012.05 2012.06 0 20.000 40.000 60.000 80.000 100.000 120.000 140.000 160.000 180.000 200.000 Durchschnitt; über 200.000 Mitglieder Durchschnitt; unter 200.000 Mitglieder https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Page 1 https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 53 THEMA Tabelle 2: Rückstandsquoten des Zusatzbeitrags Tabelle 2: Rückstandsquoten des Zusatzbeitrags Theoretische Soll- Einnahmen ZB 2010 Soll-Einnahmen ZB gemäß KJ1 2010 Forderungen auf ZB 2010 Differenz zu den Soll-Einnahmen KJ1 in % 2010 Quelle: KM1 2010 Kt. 2500, KJ1 2010 Kt. 0210, KJ1 2010 Eig. Berechnung Unter 200.000 Mitglieder Ungewichteter Durchschnitt 3.019.165 € 2.949.699 € 664.386 € 22,52% Gewichteter Durchschnitt 7.943.505 € 7.875.940 € 2.022.810 € 25,68% Über 200.000 Mitglieder Ungewichteter Durchschnitt 136.196.266 € 126.138.097 € 16.937.853 € 13,43% Gewichteter Durchschnitt 282.397.428 € 265.393.676 € 30.024.052 € 11,31% Summe für alle bundesunmittelbare Krankenkassen mit Zusatzbeitrag 699.096.320 € 648.388.682 € 88.675.582 € 13,68% Als Gewicht wurde die Anzahl der Mitglieder verwendet. Ohne Krankenkassen mit prozentualem Zusatzbeitrag. Verpflichtungen auf Zusatzbeitrag wurden hier nicht berücksichtigt. 3. Vergleich der Mitgliederveränderungen bei Krankenkassen ohne bzw. mit Zusatzbeitrag oder Prämie Bei den Einnahmen gemäß KJ1 wur- de die Schlüsselnummer 2500 herange- zogen. Sie entspricht dem Konto 2500, auf welchem die Krankenkassen ihre Einnahmen aus dem Zusatzbeitrag ver- Im Vergleich der Mitgliederverände- rung (Abbildung 3) wird deutlich, dass Krankenkassen mit Zusatzbeitrag im Durchschnitt Mitglieder verloren haben, während Krankenkassen mit Prämie im Durchschnitt Mitglieder gewonnen haben. Betrachtet man die prozentua- le Mitgliederänderung bezogen auf die Ausgangswerte im Januar 2009, sind nur für die Krankenkassen mit Zusatz- beitrag extrem negative Veränderungs- raten zu beobachten. Tabelle 3: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie Höhe Von Bis Mitgliederzuwachs im Prämien-Zeitraum Bundesunmittelbare Krankenkassen mit Prämie Ungewichteter Durchschnitt 5,38 € Mai. 10 Fast alle bis heute 12,06% Gewichteter Durchschnitt 4,70 € Apr. 10 10,48% Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet Tabelle 3: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie Höhe Von Bis Mitgliederzuwachs im Prämien-Zeitraum Bundesunmittelbare Krankenkassen mit Prämie Ungewichteter Durchschnitt 5,38 € Mai. 10 Fast alle bis heute 12,06% Gewichteter Durchschnitt 4,70 € Apr. 10 10,48% Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet Tabelle 3: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie Die Daten wurden für den Boxplot auf den aktuellen Fusionsstand (Juli 2012) gebracht. Diese Vorgehensweise Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Rückstands- und Realisierungsquoten des Zusatzbeitrags Die ersten Prämien gab es bereits ab dem 01.01.2009. Von den 7 bundesunmittel- baren Krankenkassen, die seither eine Prämie auszahlen, sind 3 geschlossene BKKen, die ausgezahlten Prämien bewe- gen sich in einer Spanne von 2,50 Euro bis 10 Euro im Monat. Tabelle 3 zeigt die Durchschnittswerte für die Auszah- lung von Prämien. Tabelle 2 zeigt eigene Berechnungen des BVA auf Basis der amtlichen Statistik für das Jahr 2010. Die Soll-Einnahmen wurden für je- den Monat mit Zusatzbeitrag berechnet, indem der erhobene Zusatzbeitrag mit der Mitgliederzahl multipliziert wurde. Dieses Vorgehen führt allerdings nur zu einer näherungsweisen Lösung, da die Mitglieder, die vom Zusatzbeitrag be- freit sind, nicht berücksichtigt werden. Hierbei ist insbesondere zu beachten, dass Mitglieder, die bis zum Zeitpunkt der erstmaligen Fälligkeit kündigen, den Zusatzbeitrag nicht zahlen. In der folgenden Spalte ist die Schlüs- selnummer 0210 abgebildet. Unter dem korrespondierenden Konto verbuchen die Krankenkassen die Forderungen auf den Zusatzbeitrag, d. h. die in Rechnung gestellten, aber nicht eingegangenen Zahlbeträge. Da laut Kontenrahmen Forderungen jedoch gemäß ihres Aus- fallrisikos bewertet werden müssen, ist es möglich, dass hier bereits mit dem Ausfallrisiko wertberichtigte Beträge verbucht wurden. Die Differenz zwi- schen den Soll-Einnahmen gemäß KJ1 und den Forderungen auf Zusatzbeitrag kann somit mit Einschränkungen als Rückstandsquote interpretiert werden. In Abbildung 2 sind die Mitglieder- entwicklungen der Krankenkassen mit Prämie über den gesamten Zeitraum von 01.01.2009 bis heute abzulesen. G+S 5/2012 54 THEMA Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie 72.000 74.000 76.000 78.000 80.000 82.000 84.000 86.000 88.000 90.000 92.000 2009.01 2009.02 2009.03 2009.04 2009.05 2009.06 2009.07 2009.08 2009.09 2009.10 2009.11 2009.12 2010.01 2010.02 2010.03 2010.04 2010.05 2010.06 2010.07 2010.08 2010.09 2010.10 2010.11 2010.12 2011.01 2011.02 2011.03 2011.04 2011.05 2011.06 2011.07 2011.08 2011.09 2011.10 2011.11 2011.12 2012.01 2012.02 2012.03 2012.04 2012.05 2012.06 Mitglieder Durchschnitt Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenka haben bzw. hatten. Das System »Zusatz- beiträge und Prämien« ist damit aktu- ell nicht vollständig verwirklicht. Ein Zusatzbeitrag wird von den Versicher- ten und den Krankenkassen noch nicht als reguläres Finanzierungsinstrument der gesetzlichen Krankenversicherung wahrgenommen und eine tiefere (Preis-) Differenzierung hat nicht stattgefunden. Bei den Krankenkassen, die einen Zusatzbeitrag erhoben haben, hat dies teilweise zu starken Mitgliederverlusten geführt, die weit über den bekannten Mitgliederschwankungen bei Beitrags- satzanpassungen lagen. Dieses Verhal- ten deutet auf die politisch gewünschte höhere Transparenz des Preissignals hin. Insbesondere ab einer bestimmten kann zu Verzerrungen führen, wenn Krankenkassen mit und ohne Zusatz- beitrag fusioniert haben, da die Daten dieser Krankenkassen vermischt wer- den. Verzerrungen der Steigerungsraten aufgrund von Fusionen werden durch diese Vorgehensweise jedoch vermieden. Die Boxen entsprechen dem Bereich, in dem 50 % der Daten, d. h. der Verände- rungsraten liegen. Die Antennen stellen die restlichen Werte (ca. 45 %) außer- halb der Box dar, die übrigen (ca. 5 %) werden als Extremwerte bzw. Ausrei- ßer bezeichnet und sind im Boxplot aus Datenschutzgründen nicht dargestellt. Die Linie innerhalb der Boxen stellt den Median der Veränderungsraten mit dem jeweiligen Kriterium (ohne Prämie/Zu- satzbeitrag, mit Prämie und mit Zusatz- beitrag) dar. Abbildung 3: Veränderung der Mitgliederzahlen in Prozent Anmerkung: Bei geschlossenen Krankenkassen wurde die Mitgliederzahl zum Schlie- ßungszeitpunkt herangezogen. Abbildung 3: Veränderung der Mitgliederzahlen in Prozent Besonders extreme Werte kommen durch den langen Betrachtungszeitraum zustande, der im Januar 2009 beginnt und im Juni 2012 bzw. mit Schließungs- zeitpunkt endet. 4. Fazit Zusammenfassend lässt sich festhal- ten, dass relativ wenige Krankenkassen einen Zusatzbeitrag oder eine Prämie Anmerkung: Bei geschlossenen Krankenkassen wurde die Mitgliederzahl zum Schlie- ßungszeitpunkt herangezogen. 55 5/2012 G+S https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 55 THEMA H di sie bi lag de Au M Di ke zu tu be »Z Ve be di ke di sp fü lic sin Patientenversorgung im internationalen Vergleich Comparing Chronic Disease Patterns between Germany and the US International Comparisons of Treatment Patterns, Disease Interactions and Healthcare Use and Costs Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen 2012, 179 S., brosch., 24,– € ISBN 978-3-8329-7477-0 (Beiträge zum Gesundheitsmanagement, Bd. 35) Comparing Chronic Disease Patterns between Germany and the US Norbert Klusen/Frank Verheyen (eds.) International Comparisons of Treatment Patterns, Disease Interactions, and Healthcare Use and Costs Beiträge zum Gesundheitsmanagement 35 Nomos Nomos Höhe des Zusatzbeitrags überraschten die extremen Mitgliederbewegungen, da sie weit über den Erfahrungswerten des bisherigen »Beitragssatz-Wettbewerbs« lagen. Comparing Chronic Disease Patterns between Germany and the US International Comparisons of Treatment Patterns, Disease Interactions and Healthcare Use and Costs Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen 2012, 179 S., brosch., 24,– € ISBN 978-3-8329-7477-0 (Beiträge zum Gesundheitsmanagement, Bd. 35) International Comparisons of Treatment Patterns, Disease Interactions and Healthcare Use and Costs Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen 2012, 179 S., brosch., 24,– € ISBN 978-3-8329-7477-0 (Beiträge zum Gesundheitsmanagement, Bd. 35) Ländervergleich USA-Deutschland: Welche Unterschiede existieren in der Versorgung chronischer Krankheiten? Worin unterscheiden sich Qualität und Kosten? Was können wir voneinander lernen? Ausgehend von Hunderttausenden an Abrechnungsdaten diesseits und jenseits des Atlantiks werden erstmals Behandlungsmuster und -ergebnisse miteinander verglichen. Weitere Informationen: www.nomos-shop.de/14664 Weitere Informationen: www.nomos-shop.de/14664 Patientenversorgung im internationalen Vergleich Schließlich konnte in der vorliegen- den Analyse gezeigt werden, dass die Auszahlung einer Prämie weniger starke Mitgliederbewegungen nach sich zieht. Comparing Chronic Disease Patterns between Germany and the US Norbert Klusen/Frank Verheyen (eds.) International Comparisons of Treatment Patterns, Disease Interactions, and Healthcare Use and Costs Beiträge zum Gesundheitsmanagement 35 Nomos Nomos Die Analyse hat gezeigt, dass die Auszahlung einer Prämie weniger starke Mitgliederbewegungen als die Erhebung eines Zusatzbeitrages nach sich zieht. Dies kam den vorwiegend kleinen Kran- kenkassen mit Prämie zugute, die einen zu großen Mitgliederansturm verwal- tungstechnisch womöglich nicht hätten bewältigen können. g Infolge der Einführung des Systems »Zusatzbeiträge und Prämien« ist eine Verschärfung des Krankenkassenwett- bewerbs dergestalt zu beobachten, dass die Krankenkassen sich stark bemühen, keinen Zusatzbeitrag zu erheben bzw. diesen abzuschaffen. Die Anreize, Ein- sparungen zu realisieren und die Durch- führung der Krankenversicherung mög- lichst wirtschaftlich zu organisieren, sind deshalb sehr stark. Allerdings gibt es ebenso starke Anreize, die Entschei- dung über die Erhebung eines Zusatzbei- trags möglichst lange hinauszuzögern, auch wenn dadurch eine prekäre finan- zielle Situation entsteht, wenngleich die- se Gefahr aktuell durch die generell gute Finanzausstattung der Kassen durch den Gesundheitsfonds nicht so brisant ist.n Comparing Chronic Disease Patterns between Germany and the US International Comparisons of Treatment Patterns, Disease Interactions and Healthcare Use and Costs Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen 2012, 179 S., brosch., 24,– € ISBN 978-3-8329-7477-0 (Beiträge zum Gesundheitsmanagement, Bd. 35) Nomos https://doi.org/10.5771/1611-5821-2012-5-52 Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. G+S 5/2012 G+S 5/2012 56
https://openalex.org/W2159055716
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English
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A novel method to prepare L-Arabinose from xylose mother liquor by yeast-mediated biopurification
Microbial cell factories
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* Correspondence: mzxyjiang@163.com; zxdeng@sjtu.edu.cn 1Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800# Dongchuan Road, Shanghai, China 2School of Chemistry and Ecology Engineering, Guangxi University for Nationalities, Nanning, China Full list of author information is available at the end of the article © 2011 Cheng 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. Cheng et al. Microbial Cell Factories 2011, 10:43 http://www microbialcellfactories com/content/10/1/ Cheng et al. Microbial Cell Factories 2011, 10:43 http://www microbialcellfactories com/content/10/1/ Cheng et al. Microbial Cell Factories 2011, 10:43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 A novel method to prepare L-Arabinose from xylose mother liquor by yeast-mediated biopurification airong Cheng1, Hengwei Wang1, Jiyang Lv1, Mingguo Jiang2*, Shuangjun Lin1 and Zixin Deng1 Abstract Background: L-arabinose is an important intermediate for anti-virus drug synthesis and has also been used in food additives for diets-controlling in recent years. Commercial production of L-arabinose is a complex progress consisting of acid hydrolysis of gum arabic, followed by multiple procedures of purification, thus making high production cost. Therefore, there is a biotechnological and commercial interest in the development of new cost- effective and high-performance methods for obtaining high purity grade L-arabinose. Results: An alternative, economical method for purifying L-arabinose from xylose mother liquor was developed in this study. After screening 306 yeast strains, a strain of Pichia anomala Y161 was selected as it could effectively metabolize other sugars but not L-arabinose. Fermentation in a medium containing xylose mother liquor permitted enrichment of L-arabinose by a significant depletion of other sugars. Biochemical analysis of this yeast strain confirmed that its poor capacity for utilizing L-arabinose was due to low activities of the enzymes required for the metabolism of this sugar. Response surface methodology was employed for optimization the fermentation conditions in shake flask cultures. The optimum conditions were: 75 h fermentation time, at 32.5°C, in a medium containing 21% (v/v) xylose mother liquor. Under these conditions, the highest purity of L-arabinose reached was 86.1% of total sugar, facilitating recovery of white crystalline L-arabinose from the fermentation medium by simple methods. Conclusion: Yeast-mediated biopurification provides a dynamic method to prepare high purity of L-arabinose from the feedstock solution xylose mother liqour, with cost-effective and high-performance properties. inhibits the sucrase activity of intestinal mucosa [3-5]. It also suppresses increase of blood glucose after sucrose loading in a dose-dependent manner, but shows no effect after starch loading in mice [5]. These observa- tions have suggested the possibility of application of L-arabinose, mixed with small quantities of sucrose, in controlled diets such as those for weight-loss or for dia- betics [6]. Media and screening of yeast strains To obtain a yeast strain that could utilize xylose, xylitol, galactose but not L-arabinose from xylose mother liquor, 306 yeast strains from our laboratory stock were screened by inoculating each culture in solid 1% (w/v) yeast nitrogen base medium (YNB, Difco, Detroit, MI, USA) containing 2% (w/v) agar, supplemented with either 1% (w/v) L-arabinose (YNA), 1% (w/v) xylose (YNX), 1% (w/v) xylitol (YNXL) or 1% (w/v) galactose (YNGL) and cultured at 28°C for 5 days. Cultures which could grow on other media but not on YNA were inoculated into solid YNB medium supplemented with 30% (v/v) xylose mother liquor (YNXM) and cultured at 28°C for 7 days. The culture that showed the fastest growth rate based on the colony size was selected. This strain was adapted to a medium containing 1% (w/v) yeast extract, 1% (w/v) tryptone, 30% (v/v) xylose mother liquor, 2% (w/v) agar (YPX) by repeated cultur- ing for 30 cycles to further improve its adaptability in xylose mother liquor. Xylose mother liquor is an acid hydrolysate by-pro- duct in the preparation of xylose from corncob or sugar- cane bagasse and is rather abundant in China. It contains more than 350-400 g L-1 xylose and 150-180 g L-1 L-arabinose, as well as galactose and glucose. L-ara- binose is difficult to separate and crystallize by general procedures due to the chemical complexity. Simulated moving bed chromatography has been practiced to sepa- rate L-arabinose from the mixture [9], but it demands expensive equipment and high running costs and has poor separation efficacy, limiting its large-scale use in industry. Therefore, development of a simpler method to obtain L-arabinose from xylose mother liquor is crucial. For taxonomic identification of this yeast strain, geno- mic DNA was extracted according to the simple phenol lysis method [13] to amplify partial sequences of its 18S rDNA and cytochrome c oxidase subunit 2 genes. The primers used for amplification of 18 S rDNA were: 5’- ATC CTG CCA GTA GTC ATA TGC TTG TCT C-3’ and 5’-GAG GCC TCA CTA AGC CAT TCA ATC GGT A-3’; while those for the cytochrome c oxidase subunit 2 gene were: 5’-AATATAATGTTTTATTTAG- TATTAATA-3’ and 5’-TTTGATAGGCATCGCAC- TATGAGC-3’. PCR conditions were as follows: 95°C for 3 min, 30 cycles of denaturation at 94°C for 35 s, annealing at 61°C for 40 s, extension at 72°C for 90 s, and a final extension at 72°C for 10 min. Media and screening of yeast strains Each PCR pro- duct was independently ligated into T-vector (pMD18, Takara, Dalian, China) and sequenced. Homology search was performed using the Basic local alignment tool (BLAST) available from National Center for Biotechnol- ogy Information (NCBI, Bethesda, MD, USA) [14]. In recent times, biological removal and bio-purifica- tion have become increasingly attractive approaches for producing high-value compounds from crude sugar feedstocks [10,11]. Most recently, Cheng et al designed a strategy to produce xylitol from xylose mother liquor through biological removal of growth inhibitors, glucose and L-arabinose, and enrich xylose which was then reduced to xylitol by a yeast [12]. Thus, a possible approach for the efficient separation of L-arabinose from xylose mother liquor would be to biologically deplete other sugars such as xylose, glucose and galac- tose from the xylose mother liquor, thus enriching L- arabinose in this liquor and facilitating its crystallization by simple methods. In the present study, we identified Pichia anomala Y161 (China General Microbiological Culture Collection Center, accession no. 2480) that could grow well in a medium containing xylose, xylitol and galactose, but not L-arabinose, as a carbon source, and could also grow well in a medium containing 10-30% (v/v) xylose mother liquor. Therefore, this strain was used to biologically enrich L-arabinose from xylose mother liquor. Response Background The sugar, L-arabinose is named after gum arabic from which it was first isolated. It is a five-carbon sugar and is widely found in nature as a component of biopoly- mers such as hemicellulose and pectin. L-arabinose is traditionally used in the flavour industry in Maillard reaction and in culture media. Recently, L-arabinose has been used in food additives and as an intermediate in drug synthesis [1,2]. The effects of L-arabinose on intestinal absorption of sucrose have been investigated. Physiological experiments have revealed that L-arabinose Commercial production of L-arabinose consists of an initial step of acid hydrolysis of gum arabic, followed by its purification through multiple procedures such as neu- tralization reaction, ion exchange and other chromato- graphic separations. Recently, Lim et al. (2011) reported a new preparation method for L-arabinose from arabinan by the combination of endo-and exo-arabinanases, which yielded 16 g L-1 L-arabinose from 20 g l-1 arabinan (80% yield) [7]. Earlier, Ahmed et al. (1999) reported a novel Page 2 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 L-arabinose production method using Mycobacterium smegmatis to transform ribitol, first by oxidization to L- ribulose that is then isomerized into L-arabinose [8]. However, these methods need expensive raw materials and complex separation processes for purification of L- arabinose, which render these methods impractical on a large-scale. surface methodology and Box-Wilson central composite design (CCD) were used to optimize the fermentation conditions by P. anomala Y161. Optimal fermentation conditions were deduced and were applied in 3 L fer- mentors to enrich L-arabinose by depleting xylose and glucose and some of the galactose present. Crystallized L-arabinose was obtained from this enriched fermenta- tion medium by simple purification procedures. In 2010, China produced about 500 tons of L-arabi- nose at the cost of more than 70 USD per kg. This pro- hibitive cost for obtaining high grade L-arabinose limits its use in applications such as food additives and drug synthesis. Therefore, there is a biotechnological and commercial interest in the development of new cost- effective and high-performance methods for obtaining high purity grade L-arabinose, which has biotechnologi- cal applications as a valuable starting material for manu- facture of various life-saving drugs and other commercially important high-value products. Yeast seed culture Yeast pre-culture was prepared by transferring a loop of culture obtained as above to test tubes containing 10 mL seed culture medium (15% xylose mother liquor (v/v), 1% Page 3 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Page 3 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Experimental design and statistical analysis tryptone (w/v), 1% yeast extract (w/v)). The pre-culture was grown at 32°C on a rotary shaker at 200 rpm for 24 h and used to inoculate Erlenmeyer flasks (500 mL cap.) containing 90 mL of fermentation media with vary- ing concentrations of xylose mother liquor (containing 153 g L-1 L-arabinose, purchased from Jiahe Sugar Co. Ltd., Changyi City, Shandong Province, China). The cultural conditions were modified according to the experimental design. The speed of rotary shaker incuba- tor was set to 250 rpm. All tests were conducted in dupli- cate and results presented are the mean values of two independent experiments. p g y Based on the results of our preliminary experiments, the selected yeast strain was grown under various cultural conditions to obtain optimized purity of L-arabinose. In these trials, three parameters were kept constant: med- ium contained 2.5% (w/v) yeast extract, pH was 5.5 and aeration was by shaking at 250 rpm. Yeast extract has been shown to be a good nitrogen source, increasing aldose (xylose) reductase activity, thus enhancing the metabolism rate of xylose [15]. An aeration rate of 250 rpm improved significantly the assimilation and metabo- lism rate of xylitol presumably by providing more cofac- tor NAD, thus shortening the fermentation time to enrich L-arabinose. Three parameters, namely, fermenta- tion time (X1), temperature (X2) and concentration of xylose mother liquor in the medium (X3), were varied to obtain the optimized purity of L-arabinose according to the response surface methodology (RSM) (Design- Expert 7). A central composite design (CCD) was employed for fast multifactor screening to determine the most important independent factor [16]. A CCD at five levels was conducted with three independent variables of X1, X2 and X3 (Table 1). The complete design con- sisted of 20 experimental points (8 factorial points, 6 axial points and 6 center points) as shown in Table 2, and the experiments were carried out in a random order. Enzymatic assays Y161 culture was grown aerobically in 250 ml shaken flasks with 50 ml of medium containing 0.67% (w/v) YNB supplemented with 1% xylose (w/v), 1% xylitol (w/v), or 1% galactose (w/v), and incubated at 28°C on a rotary shaker at 250 rpm. 2 ml of cell suspension from each late-exponential phase culture (OD600 6-7) were har- vested by centrifugation at 5000 g for 5 min, washed three times with extraction buffer (50 mM Tris-Cl [pH7.5], 0.1 mM EDTA and 2 mM dithiothreitol) and centrifuged at 5000 g for 5 min. The pelleted cells (about 35 mg dry weight cells per tube) were re-suspended in 400 μl of the above extraction buffer and were sonicated using an ultrasonic homogenizer in an ice water bath for fifty, 5 s bursts, interrupted by 5 s cooling intervals at 600 Watt. Crude cell-free extracts were obtained by reco- vering the supernatant after spinning down cell debris. This preparation was used to determine enzyme activ- ities with L-arabinose, L-arabitol and L-xylulose as substrates and identify the enzymatic products by HPLC. For L-arabinose reductase (LAR, EC 1.1.1.21) and L-xylulose reductase (LXR, EC 1.1.1.10), activities were determined in Tris-HCl buffer (100 mM, pH 7.0), NADPH or NADH (0.2 mM) containing 1% (w/v, 66 mM) L-arabinose or 1% (v/v) L-xylulose. NAD- or NADP-dehydrogenase (EC 1.1.1.12) activities were assayed in glycine buffer (100 mM, pH 8.5), MgCl2 (50 mM), 1% (w/v, 66 mM) L-arabitol or 1% (w/v, 66 mM) xylitol containing either NAD or NADP (3.0 mM) respectively. All assays were carried out at 30°C for 5 min. One unit of enzyme activity was defined as the amount of activity necessary to convert 1 μmol of sub- strate to product (NADPH to NADP; or NADH to NAD; or NAD to NADH; or NADP to NADPH) per min. The protein concentration in crude extract was determined according to the Bradford method with bovine serum albumen (BSA) as a standard. The enzymatic reaction mixes (1 hour reaction) were extracted with phenol to remove proteins and 40 μL aliquots were applied to HPLC to identify the products Isolation and purification of L-arabinose h d l d d Isolation and purification of L arabinose The yeast seed culture prepared as described above was inoculated at 5% inoculum size into four 3 L capacity fer- mentors (Bioflo 110, New Brunswick Scientific, Edison, NJ, USA) each containing 2 L of the optimum fermenta- tion medium with 2.5% (w/v) yeast extract as nitrogen source at pH 5.5 and cultured under the optimum fer- mentation conditions obtained from response surface methodology. After fermentation, yeast cells were removed by centrifugation, and the supernatant was rotary decolorized with activated carbon at 80°C. Then the colorless fermentation broth thus obtained was clari- fied by removal of activated carbon via filtration, and was applied to ion-exchange resin columns for removal of metal ions, which was verified by a conductivity less than Table 1 Values of parameters used in the response surface design Factors Factor level -2 -1 0 1 2 Fermentation time (h) 50 60 70 80 90 Fermentation temperature (°C) 30 31 32 33 34 Xylose mother liquor (%, v/v) 15 20 25 30 35 Table 1 Values of parameters used in the response surface design Factors Factor level -2 -1 0 1 2 Fermentation time (h) 50 60 70 80 90 Fermentation temperature (°C) 30 31 32 33 34 Xylose mother liquor (%, v/v) 15 20 25 30 35 Table 1 Values of parameters used in the response surface design Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Page 4 of 11 Table 2 Results of experimental trials (20 trials) conducted as per the central composite design (CCD) for optimization of yield in terms of purity of L-arabinose obtained from xylose mother liquor Run No. Time (h) Temp. (°C) Xylose mother liquid (%, v/v) Purity degree of L-arabinose (%) 1 70 30 25 65.6 2 60 33 30 49.2 3 70 34 25 68.8 4 70 32 25 81.4 5 80 31 20 77.5 6 70 32 25 82.9 7 70 32 35 46.2 8 80 33 20 84.9 9 60 31 20 55.2 10 60 33 20 75.1 11 80 33 30 67.1 12 70 32 25 81.8 13 70 32 25 82.5 14 60 31 30 40.1 15 70 32 25 81.4 16 70 32 15 85.3 17 90 32 25 78.1 18 70 32 25 81.1 19 80 31 30 58.8 20 50 32 25 69.9 Materials and Methods. Isolation and purification of L-arabinose h d l d d Based on the relative colony sizes, one strain was found to grow faster than the other three and was selected for further experiments. After 30 successive cycles of adaptation to YPX medium contain- ing 30% (v/v) xylose mother liquor, the culture of this strain was found to grow faster (colony size 1.0-1.2 mm) than an unadapted culture (colony size 0.2-0.4 mm) on YPX medium after 5 days at 28°C. This yeast strain which utilizes well xylose, xylitol, galactose but not L- arabinose, grew fastest on YNXM and was adapted to YPX medium was designated as Y161. The growth char- acteristics of yeast Y161 on YNA, YNX, YNXL, YNGL and YPX solid medium are shown in Figure 1. The xylose mother liquor contains more than 350-400 g L-1 xylose and 150-200 g L-1 galactose plus glucose in addition to 150-180 g L-1 L-arabinose. The approach fol- lowed here for enriching L-arabinose was based on elim- ination of other sugars from the xylose mother liquor. Hence the screen applied was aimed at selecting a yeast strain with excellent properties for transport and rapid metabolism of glucose, galactose and xylose, and capa- city for adaptation to high concentration of xylose mother liquor. The yeast strain Y161 met these requirements and was adapted to a medium containing 30% (v/v) xylose mother liquor. q The taxonomic identity of Y161 was established based on cloning and sequencing of its 18S rDNA and cyto- chrome c oxidase subunit 2 genes (see Methods). The two nucleotide sequences were analyzed for homology by the BLAST search available from NCBI. The results of this analysis showed that the 1.7 kb 18S rDNA fragment from yeast Y161 (GenBank accession No. HQ901201) had 99% identity with that from Pichia anomala strain M34-1, strain HS054 and strain IFO 10213T [17], as well as with that from Pichia subpelliculosa NRRL Y-1683, Pichia sydo- wiorum NRRL Y-7130, Pichia ciferrii NRRL Y-1031, Can- dida silvicultrix NRRL Y-7789 [18]. Similarly, the 580 bp fragment of cytochrome c oxidase subunit 2 gene from yeast Y161 (GenBank accession No. HQ901202) showed 100% identity with a homologous sequence from Pichia anomala isolate WM 825 [19] and 99% identity to that from Wickerhamomyces anomalus NRRL Y-366 [20]. Both the sequenced DNA fragments showed less than 90% identity with corresponding sequences from other yeast strains. Thus, the yeast Y161 was identified as Pichia anomala. 20 μs/cm. Isolation and purification of L-arabinose h d l d d The solution was adjusted to contain 70% (w/v) of L-arabinose by concentrating, and cooled gradually from 70°C to 4°C to allow L-arabinose crystallized. White powdery crystals precipitated from solution, then were centrifuged and washed with 95% ethanol, and dried. The purity of L-arabinose was estimated by HPLC. Analytical methods L-arabinose, xylose, glucose, galactose and xylitol were estimated by resolving samples on prepacked analytical HPLC columns (Shodex SPO 810, 8 × 30 mm, Pb2+ cation exchange column), with distilled water as eluent, at a flow rate of 1.0 mL/min, at 70°C. Sugars were iden- tified using a refractive index detector (Shodex RI 101). Screening, selection and identification of yeast Pichia anomala Y161 Among 306 strains of yeasts that were screened on YNB-based media supplemented with different sugars, all strains could utilize galactose as they were capable of growth on solid YNGL medium. Of these, four strains grew well on solid YNX, YNXL medium but grew poorly on YNA. Cultures of these four yeast strains were diluted appropriately with sterilized water and spread onto solid YNXM medium as described in Growth pattern of Y161 in YPX liquid medium After 48 h, xylitol was exhausted as it was almost undetectable, while trace amounts of L-arabi- tol remained in the medium (less than 1.5 g L-1). At this point (48 h), the concentration of L-arabinose was 13.5 g L-1. Comparing to the starting fermentation med- ium which contains 15.3 g L-1 L-arabinose, 88.2% yield of L-arabinose was achieved in the final fermentation medium. Only 1.8 g L-1 L-arabinose was reduced to L-arabitol, indicating that yeast Y161 could not utilize well L-arabinose and showed a weak ability to reduce L-arabinose to L-arabitol (Figure 2F). After 48 h of fer- mentation, the degree of purity of L-arabinose increased more than 3-fold, from the initial 26.2% to 85.6% in terms of total sugars. YNA YNX YNGL YNXL YPX YPX Figure 1 Growth of yeast on solid medium containing different sugars. Screening of 306 yeast strains was performed by observing their growth on YNB medium supplemented with either L-arabinose (YNA), xylose (YNX), xylitol (YNXL) or xylose mother liquor (YPX). Black arrows indicate the sector inoculated with the selected strain that was capable of growth on all the above medium except YNA. After repeated culturing of this strain in YPX medium for 30 cycles, it appeared to have adapted as seen from the comparison of its growth (bottom left panel) with an unadapted culture (bottom right panel). The adapted strain was then designated as strain Y161. On YNA medium, Y161 showed poor growth in the first 3 days, and no further increase in colony size or number was observed in the next 4 days. YPX YPX Xylose is a major constituent of plant material such as corn cob and sugarcane bagasse. Xylose metabolic path- way is therefore well developed in microorganisms that live on decaying plant materials, including most strains of yeasts such as Pichia stiptis [21], Candida tropicalis [22], Candida shehatae [23] and H. anomala used in this study. The metabolism of xylose in yeasts has been described in detail in the literature [24-27]. Xylose transport across the plasma membrane is the first step in xylose metabolism pathway in yeasts. It is known that xylose is taken up by active xylose/proton symporter that mostly appeared to be associated with glucose transport and showed a much higher affinity for glucose than for xylose [28]. The uptake of xylose is highly influenced by the presence of glucose. Growth pattern of Y161 in YPX liquid medium Growth pattern of Y161 in YPX liquid medium p q Y161 cells were grown in 500 ml capacity flasks contain- ing 50 ml YPX liquid medium supplemented with 10% (v/v) xylose mother liquor and incubated at 32°C on an orbital shaker at 250 rpm. Samples were withdrawn per- iodically to estimate the content change of glucose, xylose, L-arabinose, galactose, L-arabitol and xylitol Page 5 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 http://www.microbialcellfactories.com/content/10/1/43 the external medium (Figure 2C). After 30 h of fermenta- tion, the amount of xylose and galactose decreased to 3.2 g L-1 and 2.5 g L-1, and the amount of xylitol increased to 12.3 g L-1. Presumably, xylose was metabo- lized and transformed to biomass and energy, while L-arabitol was still unutilized (Figure 2D). After 36 h, xylose from the medium had reached undetectable levels and the amount of xylitol started to decrease. Trace amounts of galactose and L-arabitol were found in med- ium (Figure 2E). After 48 h, xylitol was exhausted as it was almost undetectable, while trace amounts of L-arabi- tol remained in the medium (less than 1.5 g L-1). At this point (48 h), the concentration of L-arabinose was 13.5 g L-1. Comparing to the starting fermentation med- ium which contains 15.3 g L-1 L-arabinose, 88.2% yield of L-arabinose was achieved in the final fermentation medium. Only 1.8 g L-1 L-arabinose was reduced to L-arabitol, indicating that yeast Y161 could not utilize well L-arabinose and showed a weak ability to reduce L-arabinose to L-arabitol (Figure 2F). After 48 h of fer- mentation, the degree of purity of L-arabinose increased more than 3-fold, from the initial 26.2% to 85.6% in terms of total sugars. the external medium (Figure 2C). After 30 h of fermenta- tion, the amount of xylose and galactose decreased to 3.2 g L-1 and 2.5 g L-1, and the amount of xylitol increased to 12.3 g L-1. Presumably, xylose was metabo- lized and transformed to biomass and energy, while L-arabitol was still unutilized (Figure 2D). After 36 h, xylose from the medium had reached undetectable levels and the amount of xylitol started to decrease. Trace amounts of galactose and L-arabitol were found in med- ium (Figure 2E). Growth pattern of Y161 in YPX liquid medium During the fer- mentation of xylose mother liquor by the yeast Y161 in this study, xylose uptake was not observed until glucose was exhausted and xylitol was detected in medium only after 8 h (Figure 2B), indicating that glucose has a strong inhibitory effect on xylose uptake. Moreover, glucose was shown to be an inhibitor of the intracellular enzymes responsible for the catabolism of xylose, for example reduction to xylitol [29]. Once glucose has been con- sumed, enzymes for xylose metabolism are activated and xylitol accumulates intracellularly and is then secreted to the medium (Figure 2C-E). Extracellular xylitol concentra- tion reached a maximum value just before nearly all of the xylose was consumed (Figure 2D). The xylitol appears to have been utilized for generating biomass and energy dur- ing the later phases of fermentation, as seen from the sub- sequent decline in concentration of extracellular xylitol YPX YPX YPX Figure 1 Growth of yeast on solid medium containing different sugars. Screening of 306 yeast strains was performed by observing their growth on YNB medium supplemented with either L-arabinose (YNA), xylose (YNX), xylitol (YNXL) or xylose mother liquor (YPX). Black arrows indicate the sector inoculated with the selected strain that was capable of growth on all the above medium except YNA. After repeated culturing of this strain in YPX medium for 30 cycles, it appeared to have adapted as seen from the comparison of its growth (bottom left panel) with an unadapted culture (bottom right panel). The adapted strain was then designated as strain Y161. On YNA medium, Y161 showed poor growth in the first 3 days, and no further increase in colony size or number was observed in the next 4 days. during cultivation (Figure 2). The xylose mother liquor used in this study contained glucose, xylose, galactose, L-arabinose and an unknown substance (Figure 2A). The amount of xylose was approximately 2.5 fold higher than that of L-arabinose. Glucose was the first sugar to be metabolized by Y161, while trace amounts of L-arabitol were produced after 8 h indicating a low level reduction of arabinose (Figure 2B). After 20 h fermentation, the amount of xylose decreased significantly and was almost equal to that of L-arabinose. The xylose appeared to be first reduced to xylitol, of which 50% was secreted into Cheng et al. Microbial Cell Factories 2011, 10:43 http://www microbialcellfactories com/content/10/1/43 Cheng et al. Growth pattern of Y161 in YPX liquid medium However, Y161 grew poorly, if at all, on YNA agar/liquid medium, indicating that despite a detectable level of transport of this sugar, further metabolism may not be efficient due to low activities of enzymes involved in L-arabinose metabolism, such as L-arabinose specific reductase (LAR), LAD or L-xylulose reductase (LXR). and NAD-dependent activity, with a higher preference for NAD. An XDH activity of ~0.86 U mg-1 was found in galactose-grown cells, which increased to 5.0 and 5.2 U mg-1 when the cells were grown on xylose or xyli- tol, respectively. This suggests that XDH activity is more effectively induced by xylose or xylitol compared with the other three enzymes tested, which might explain why yeast Y161 utilizes xylose or xylitol efficiently (Figure 1). In contrast, the very low LAR, LAD and LXR activities inY161 might explain its poor ability to utilize L-arabinose (Figure 1). Through HPLC analysis, we also identified the enzy- matic reaction products with L-arabinose, L-arabitol and L-xylulose as substrates and NADPH or NAD as cofactors. These reactions were performed with crude cell free extracts of Y161 grown on medium containing galactose, xylose or xylitol as a carbon source. L-arabinose was reduced to L-arabitol by the above crude extracts of Y161 in the presence of NADPH as a cofactor (Figure 3 A2-A4). L-xylulose was formed with L-arabitol as substrate and NAD as cofactor, using the crude extracts of Y161 (Figure 3 B2-B4) while L-xylulose was the reduced to xyli- tol (Figure 3 C2-C4). It should be noted that the amount of L-xylulose oxidized from L-arabitol was significantly less than the amount of L-arabitol reduced from L-arabi- nose or xylitol reduced from L-xylulose (Figure 3), indicat- ing that the activity of dehydrogenase harboring LAD was very low, consistent with the results shown in Table 3. Y161 did not grow in a medium with L-arabitol as a car- bon source (data not shown), although the cells exhibited low LAD activity. There are a number of instances of a polyol oxidized by the cell-free crude extracts of a yeast, but not utilized for growth. Candida utilis could not uti- lize D-glucitol but it could be oxidized in crude extract, due to exogenous D-glucitol, which does not enter intact cells of this strain [32]. Growth pattern of Y161 in YPX liquid medium Only a small amount of L-ara- bitol (less than 1.2 g/L) was produced when Y161 was cultured in YPX medium containing 10% xylose mother liquor (Figure 2B-E). Subsequently, in the later stages of fermentation, like in the case of xylitol, the amount of extracellular L-arabitol decreased (Figure 2F). This indi- cates that a certain amount of L-arabinose could be transported across the plasma membrane and was meta- bolized. However, Y161 grew poorly, if at all, on YNA agar/liquid medium, indicating that despite a detectable level of transport of this sugar, further metabolism may not be efficient due to low activities of enzymes involved in L-arabinose metabolism, such as L-arabinose specific reductase (LAR), LAD or L-xylulose reductase (LXR). xylose metabolism pathway. Among these, Candida ara- binofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012 received most attentions [28,30,31]. L-arabi- nose transport across the yeast outer membrane is the first step in its metabolism. In yeast C. arabinofermen- tans PYCC 5603T and P. guilliermondii PYCC 3012 which have a high L-arabinose uptake rate, there are two L-arabinose transport systems, one is the L-arabi- nose/proton symporter with high-affinity transport com- ponents, and the other is a facilitated diffusion transport system with a relative low-affinity component. Once inside the cytoplasm, L-arabinose is reduced to L-arabi- tol by an unspecific NADPH-linked aldose reductase which could also reduce xylose to xylitol [10,28]. L-ara- bitol is then oxidized to L-xylulose by an NAD-depen- dent L-arabitol 4-dehydrogenase (LAD), and L-xylulose was reduced to xylitol, a common intermediate to the xylose metabolism pathway. Y161 could efficiently meta- bolize xylose and xylitol but showed a poor ability to metabolize L-arabinose, probably because of the low activities of the enzymes, L-arabinose reductase or L- arabitol 4-dehydrogenase. Only a small amount of L-ara- bitol (less than 1.2 g/L) was produced when Y161 was cultured in YPX medium containing 10% xylose mother liquor (Figure 2B-E). Subsequently, in the later stages of fermentation, like in the case of xylitol, the amount of extracellular L-arabitol decreased (Figure 2F). This indi- cates that a certain amount of L-arabinose could be transported across the plasma membrane and was meta- bolized. Growth pattern of Y161 in YPX liquid medium Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Page 6 of 11 g http://www.microbialcellfactories.com/content/10/1/43 until it was undetectable (Figure 2E-F) Ultimately L-ara- commonly found in hemicellulose such as L-arabinnans Figure 2 Sugar assimilation patterns during growth of Y161 in YPX medium containing 10% (v/v) xylose mother liquor. Aliquots were removed at different times during fermentation (A, 0 h; B, 8 h; C, 20 h; D, 30 h; E, 36 h; F, 48 h) and analyzed by HPLC to identify the residual sugars as indicated. Figure 2 Sugar assimilation patterns during growth of Y161 in YPX medium containing 10% (v/v) xylose mother liquor. Aliquots were removed at different times during fermentation (A, 0 h; B, 8 h; C, 20 h; D, 30 h; E, 36 h; F, 48 h) and analyzed by HPLC to identify the residual sugars as indicated. commonly found in hemicellulose such as L-arabinnans, L-arabino-xylans in corn fiber which is associated to xylose. Approximately a third of L-arabino-xylans in corn fiber is L-arabinose. Many types of yeast harbor the L-arabinose metabolism pathway as well as the until it was undetectable (Figure 2E-F). Ultimately, L-ara- binose was the predominant sugar left in the fermentation medium, thus reaching a high degree of purity. Similar to xylose, L-arabinose is also a natural sugar and a component of plant carbohydrates which is Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Page 7 of 11 xylose metabolism pathway. Among these, Candida ara- binofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012 received most attentions [28,30,31]. L-arabi- nose transport across the yeast outer membrane is the first step in its metabolism. In yeast C. arabinofermen- tans PYCC 5603T and P. guilliermondii PYCC 3012 which have a high L-arabinose uptake rate, there are two L-arabinose transport systems, one is the L-arabi- nose/proton symporter with high-affinity transport com- ponents, and the other is a facilitated diffusion transport system with a relative low-affinity component. Once inside the cytoplasm, L-arabinose is reduced to L-arabi- tol by an unspecific NADPH-linked aldose reductase which could also reduce xylose to xylitol [10,28]. L-ara- bitol is then oxidized to L-xylulose by an NAD-depen- dent L-arabitol 4-dehydrogenase (LAD), and L-xylulose was reduced to xylitol, a common intermediate to the xylose metabolism pathway. Y161 could efficiently meta- bolize xylose and xylitol but showed a poor ability to metabolize L-arabinose, probably because of the low activities of the enzymes, L-arabinose reductase or L- arabitol 4-dehydrogenase. Values presented are means of duplicates. Enzymatic activities and products Table 3 Specific activities of L-arabinose reductase (LAR), L-arabitol 4-dehydrogenase (LAD), L-xylulose reductase (LXR) and xylitol dehydrogenase (XDH) in crude cell free extracts of Y161 grown on different carbon sources Specific activity (U, mg of protein-1) Enzyme Cofactors Grown on galactose Grown on xylose Grown on xylitol LAR NADPH 0.032 0.16 0.12 NADH 0.002 0.014 0.011 LAD NAD 0.003 0.012 0.018 NADP ND ND ND LXR NADPH 0.004 0.021 0.024 NADH ND ND ND XDH NAD 0.86 5.0 5.2 NADP 0.007 0.18 0.26 Values presented are means of duplicates. ND not detectable Table 3 Specific activities of L-arabinose reductase (LAR), L-arabitol 4-dehydrogenase (LAD), L-xylulose reductase (LXR) and xylitol dehydrogenase (XDH) in crude cell free extracts of Y161 grown on different carbon sources In order to investigate the possible reasons for poor uti- lization of L-arabinose by Y161, activities of four known enzymes, namely, LAR, LAD LXR and XDH, involved in L-arabinose metabolism were assayed, in crude cell-free extracts, in the presence of appropriate cofactors (Table 3). In extracts of Y161 cultures grown on galactose as a carbon source the activity of LAR was 0.032 U mg-1 using NADPH as cofactor, and decreased to a tenth with NADH as cofactor, indicating a preference for NADPH. LXR activity was strictly NADPH-dependent. Weak LAD activity of ~0.003 U mg-1 was found in these galactose-grown cells, which increased to around 0.012 and 0.018 U mg-1 when the cells were grown on xylose and xylitol, respectively, suggesting LAD activity is less effectively induced by xylose or xylitol. Similarly, LAR and LXR were also less effectively induced by xylose or xylitol. In contrast, XDH showed both, NADP- Table 3 Specific activities of L-arabinose reductase (LAR), L-arabitol 4-dehydrogenase (LAD), L-xylulose reductase (LXR) and xylitol dehydrogenase (XDH) in crude cell free extracts of Y161 grown on different carbon sources Specific activity (U, mg of protein-1) Cheng et al. Microbial Cell Factories 2011, 10:43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Page 8 of 11 Figure 3 HPLC analysis of enzymatic reaction products. Crude cell free extracts were used for analysis of various enzyme activities. The reactions were allowed to run overnight and the solutions were extracted with phenol to remove proteins. Y = 80.27 + 5.32 × (X1) + 3.19 × (X2) −9.73 × (X3) −1.66 × (X1) × (X2) +0.56 × (X1) × (X3) −1.24 × (X2) × (X3) −2.75 × (X1)2 −4.45 × (X2)2 −4.82 × (X3)2 Enzymatic activities and products Aliquots (40 μl) from the various reactions were applied to HPLC column: A1, 1% (w/v) L-arabinose standard, no enzymatic reaction; A2-A4, 1% L-arabinose plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. B1, 1% (w/v) L-arabitol standard, no enzyme; B2-B4, L-arabitol plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. C1, 1% (v/v) L-xylulose standard; C2-C4, L-xylulose plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. Figure 3 HPLC analysis of enzymatic reaction products. Crude cell free extracts were used for analysis of various enzyme activities. The reactions were allowed to run overnight and the solutions were extracted with phenol to remove proteins. Aliquots (40 μl) from the various reactions were applied to HPLC column: A1, 1% (w/v) L-arabinose standard, no enzymatic reaction; A2-A4, 1% L-arabinose plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. B1, 1% (w/v) L-arabitol standard, no enzyme; B2-B4, L-arabitol plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. C1, 1% (v/v) L-xylulose standard; C2-C4, L-xylulose plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. Experimental design Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 http://www.microbialcellfactories.com/content/10/1/43 Figure 4 Response surface for the enrichment of L-arabinose by Pichia anomala Y161. Impact on the purity of L-arabinose (%) of the interactions between the different fermentation parameters was estimated: A: fermentation time (X1, h) and fermentation temperature (X2, °C); B: fermentation time (X1, h) and concentration of xylose mother liquor in the medium (X3, %, v/v); C: fermentation temperature (X2, °C) and the concentration of xylose mother liquor (X3, %, v/v). Figure 4 Response surface for the enrichment of L-arabinose by Pichia anomala Y161. Impact on the purity of L-arabinose (%) of the interactions between the different fermentation parameters was estimated: A: fermentation time (X1, h) and fermentation temperature (X2, °C); B: fermentation time (X1, h) and concentration of xylose mother liquor in the medium (X3, %, v/v); C: fermentation temperature (X2, °C) and the concentration of xylose mother liquor (X3, %, v/v). Table 4 Analysis of variance of the degree of purity of L- arabinose calculated with a quadratic equation obtained from experimental results Source Sum of Squares df Mean Square F-Value Probability > F Model 3093.963 9 343.7737 5.603423 0.0063 X1 452.6256 1 452.6256 7.377682 0.0217 X2 163.2006 1 163.2006 2.660129 0.1339 X3 1515.156 1 1515.156 24.69665 0.0006 (X1)(X2) 22.11125 1 22.11125 0.360408 0.5616 (X1)(X3) 2.53125 1 2.53125 0.041259 0.8431 (X2)(X3) 12.25125 1 12.25125 0.199692 0.6645 (X1)2 190.7719 1 190.7719 3.109534 0.1083 (X2)2 498.9091 1 498.9091 8.132091 0.0172 (X3)2 583.413 1 583.413 9.509483 0.0116 Residual 613.5065 10 61.35065 Lack of Fit 611.0115 5 122.2023 244.8944 < 0.0001 Pure Error 2.495 5 0.499 Total 3707.47 19 X1, X2 and X3 correspond to fermentation time, temperature and concentration of xylose mother liquor, respectively; df, degrees of freedom Table 4 Analysis of variance of the degree of purity of L- arabinose calculated with a quadratic equation obtained from experimental results In Figure 4B, the fermentation temperature was fixed at the 0 level (32°C). The purity degree of L-ara- binose increased when the concentration of the xylose mother liquor was reduced from 35% to 15%. The degree of purity of L-arabinose increased with the prolonged fermentation and reached the maximum value at 85 h. Figure 4C shows the 3-D response sur- face diagram and the contour diagram with varying ratios of xylose mother liquor and fermentation tem- perature at a fixed fermentation time (0 level, 70 h). X1, X2 and X3 correspond to fermentation time, temperature and concentration of xylose mother liquor, respectively; df, degrees of freedom Experimental design The statistical significance of the regression model was checked by F-test. The fit of the polynomial model (R2) was calculated to be 0.8345, indicating that 83.45% of the variability in the response could be explained by the model. Generally, a regression model having an R2 value higher than 0.8 is considered to have a high correlation [33]. Predicted R-squared was -0.3302, implying that the overall mean is a better predictor of response than the current model. The response equation represents a sui- table model for L-arabinose enrichment from xylose mother liquor. In general, the cost-effectiveness of any fermentation pro- cess is governed by several parameters, including the cost of the raw material and the efficacy of its conversion into the desired product; the cost of energy consumption, influ- enced by the time required to operate the fermentor, and the optimum temperature to provide the maximum yield. Here, we modeled optimization of the fermentation pro- cess based on three parameters, namely, time (X1), tem- perature (X2) and concentration of xylose mother liquor in the medium (X3). These three variables were tested in a total of 20 trial runs in the current CCD as depicted in the experimental design and the results were obtained from experiments (Table 2). Maximum purity of L-arabinose (85.3%) was achieved under the experimental conditions of fermentation time 70 h, fermentation temperature 32°C in a medium containing 15% (v/v) xylose mother liquor. By applying multiple regression analysis on the experimen- tal data, the response variable, Y, for purity of L-arabinose and the three test variables were related with the second- order polynomial equation (using the above codes) as given below: 3-D response surfaces plots were employed to determine the interaction of the fermentation conditions and the optimum levels of the parameters that have a significant impact on the purity of L-arabinose. In Figure 4A, when the 3-D response plot was developed for the enrichment of L-arabinose with varying fermentation time (X1) and fermentation temperature (X2) at a set concentration of xylose mother liquor (X3, 25%), the degree of purity of L- arabinose increased with increasing fermentation time, and was enhanced with increasing fermentation tempera- ture from 31 to 32.5°C, but then dropped at temperatures between 32.5°C and 34°C, indicating that the fermentation temperature has a crucial influence on the outcome. Cheng et al. Microbial Cell Factories 2011, 10:43 Page 9 of 11 Cheng et al. Authors’ contributions CHR and JMG designed research; CHR and WHH performed the yeast strain screening and molecular identification; LJY performed HPLC analysis and fermentation experiments; LSJ and DZX provided advice on organizing the manuscript and on editorial quality; CHR performed the literature review and drafted the manuscript. All authors have read and approved the final version of the manuscript. Purification of L-arabinose from fermentation medium The purity of L-arabinose in the fermentation medium using Y161 could reach around 86% of total sugars, which exceeds the purity requirement for crystalliza- tion of L-arabinose from a solution. From 8 L fermen- tation broth of Y161containing 1.6 L xylose mother liquor, 165 g white powdery crystals of L-arabinose was obtained, an yield of 103.1 g L-arabinose per L of xylose mother liquor. Theoretically, 240 g L-arabinose should be obtained from the 8 L fermentation medium containing 1.6 L xylose mother liquor (153 g L-arabi- nose/L). About 30 g L-arabinose was lost during the process of purification and there is a residual of 45 g L-arabinose in the fermentation medium which could not be recovered due to the increased conductivity and impurities which interfere with the L-arabinose crystallization. The final preparation of the purified white powder was dissolved in water and applied for HPLC analysis to confirm a purity of 99% (data not shown). Author details 1 1Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800# Dongchuan Road, Shanghai, China. 2School of Chemistry and Ecology Engineering, Guangxi University for Nationalities, Nanning, China. Acknowledgements Thi k This work was supported by grants 973 and 863 from the Ministry of Sciences and Technology, Shanghai Leading Academic Discipline Project B203, and Guangxi Science Foundation Grant No.0832011. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Recently, yeast strains, especially S.cerevisiae, have received intensive studies to act as cell factories in green chemistry. Many natural products used in phar- maceutical and nutraceutical industries such as isopre- noids, flavonoids, and other fine chemicals could be efficiently synthesized using S.cerevisiae as the green synthesis factory [34,35]. On the other hand, yeast cells could also be used as green decomposition factory to biodegrade or metabolize unwanted compounds to enrich the desired product [36,37]. Here, the yeast strain P. anomala Y161 was employed as a green decomposition factory to metabolize other low-valued sugars and enrich high-valued L-arabinose from com- positionally complex xylose mother liquor. To our knowledge, this is the first attempt to apply this bio- technological method for the preparation of L-arabi- nose from xylose mother liquor. Latter is a viscous and compositionally complex, low-value by-product of the xylose production industry. Further studies on the fea- sibility of this novel strategy for the scaled-up prepara- tion of L-arabinose from xylose mother liquor are currently underway. Received: 30 March 2011 Accepted: 7 June 2011 Published: 7 June 2011 Received: 30 March 2011 Accepted: 7 June 2011 Published: 7 June 20 Experimental design The concentration of xylose mother liquor had a negative effect on the degree of purity of L-arabinose. The latter increased with decreasing concentration of xylose mother liquor. Among the three fermentation parameters studied, concentration of xylose mother liquor in the medium had the highest impact on the degree of purity of L-ara- binose, followed by fermentation time and fermentation temperature, based on the regression coefficients in the quadratic polynomial model (Table 4). To verify the predicted results of the model for opti- mal purity of L-arabinose, experiments were performed Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Page 10 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 8. Ahmed Z, Shimonishi T, Bhuiyan SH, Utamura M, Takada G, Izumori K: Biochemical preparation of L-ribose and L-arabinose from ribitol: A new approach. J Biosci Bioen 1999, 88:444-448. Conclusion in duplicate under the optimized fermentation condi- tions: medium volume 100 mL, inoculation volume 5% (v/v), fermentation time 75 h, fermentation tempera- ture 32.5°C, 21% (v/v) xylose mother liquor, 2.5% yeast extract and pH 5.5 in 500 mL Erlenmeyer flask. The degree of purity of L-arabinose obtained from xylose mother liquor reached 86.1% by HPLC analysis (data not shown). The predicted value 87.05% is in good agreement with experimental values, proving the accu- racy of the model and the existence of optimum points. in duplicate under the optimized fermentation condi- tions: medium volume 100 mL, inoculation volume 5% (v/v), fermentation time 75 h, fermentation tempera- ture 32.5°C, 21% (v/v) xylose mother liquor, 2.5% yeast extract and pH 5.5 in 500 mL Erlenmeyer flask. The degree of purity of L-arabinose obtained from xylose mother liquor reached 86.1% by HPLC analysis (data not shown). The predicted value 87.05% is in good agreement with experimental values, proving the accu- racy of the model and the existence of optimum points. In this study, we described a novel biological method to obtain high purity L-arabinose from xylose mother liquor. A strain of P. anomala Y161 was recovered in a screen designed to identify a yeast strain with a poor capacity of utilizing L-arabinose but a good capacity for utilizing other sugars that are present in xylose mother liquor. Optimum fermentation conditions were deduced by evaluating the effect of three independent variables using RSM. Under the optimized conditions, a high degree of purity of L-arabinose (86.1%) was obtained, and white powdery L-arabinose could be crystallized from this fermentation medium by following standard, simple techniques. The method described here appears to be amenable for large scale biotechnological applications. References Akinterinwa O, Khanka R, Cirino PC: Metabolic engineering for bioproduction of sugar alcohols Curr Opin Biotechnol 2008 19:4 0. te a O, a a , C o C: etabo c e g ee g o bioproduction of sugar alcohols. Curr Opin Biotechnol 2008, 19:461-467. y y 34. Pscheidt B, Glieder A: Yeast cell factories for fine production. 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Microbial Cell Factories 2011, 10:43 http://www.microbialcellfactories.com/content/10/1/43 References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 30. Fonseca C, Spencer-Martins I, Hahn-Hägerdal B: L-arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation. Appl Microbiol Biotechnol 2007, 75:303-310. 31. 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Spermidine Suppresses Age-Associated Memory Impairment by Preventing Adverse Increase of Presynaptic Active Zone Size and Release
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OPEN ACCESS * stephan.sigrist@fu-berlin.de Citation: Gupta VK, Pech U, Bhukel A, Fulterer A, Ender A, Mauermann SF, et al. (2016) Spermidine Suppresses Age-Associated Memory Impairment by Preventing Adverse Increase of Presynaptic Active Zone Size and Release. PLoS Biol 14(9): e1002563. doi:10.1371/journal.pbio.1002563 Abstract Memories are assumed to be formed by sets of synapses changing their structural or func- tional performance. The efficacy of forming new memories declines with advancing age, but the synaptic changes underlying age-induced memory impairment remain poorly under- stood. Recently, we found spermidine feeding to specifically suppress age-dependent impairments in forming olfactory memories, providing a mean to search for synaptic changes involved in age-dependent memory impairment. Here, we show that a specific synaptic compartment, the presynaptic active zone (AZ), increases the size of its ultrastruc- tural elaboration and releases significantly more synaptic vesicles with advancing age. These age-induced AZ changes, however, were fully suppressed by spermidine feeding. A genetically enforced enlargement of AZ scaffolds (four gene-copies of BRP) impaired mem- ory formation in young animals. Thus, in the Drosophila nervous system, aging AZs seem to steer towards the upper limit of their operational range, limiting synaptic plasticity and contributing to impairment of memory formation. Spermidine feeding suppresses age- dependent memory impairment by counteracting these age-dependent changes directly at the synapse. Academic Editor: Heidi A. Tissenbaum, University of Massachusetts Medical School, UNITED STATES Academic Editor: Heidi A. Tissenbaum, University of Massachusetts Medical School, UNITED STATES Received: February 21, 2016 Accepted: August 26, 2016 Published: September 29, 2016 Copyright: © 2016 Gupta 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. SpermidineSuppresses Age-Associated Memory Impairment by Preventing Adverse Increase of Presynaptic Active Zone Size and Release Varun K. Gupta1,2, Ulrike Pech3, Anuradha Bhukel1,2, Andreas Fulterer1,2, Anatoli Ender1, Stephan F. Mauermann1,2, Till F. M. Andlauer4, Emmanuel Antwi-Adjei1, Christine Beuschel1, Kerstin Thriene5, Marta Maglione1,2, Christine Quentin1,2, Rene´ Bushow6, Martin Schwa¨rzel1, Thorsten Mielke6, Frank Madeo7,8, Joern Dengjel5, Andre´ Fiala3, Stephan J. Sigrist1,2* a1111 1 Institute for Biology/Genetics, Freie Universita¨t Berlin, Berlin, Germany, 2 NeuroCure, Charite´, Berlin, Germany, 3 Georg-August-Universita¨t Go¨ttingen, Molecular Neurobiology of Behavior, Go¨ttingen, Germany, 4 Max Planck Institute of Psychiatry, Munich, Germany, 5 Centre for Systems Biological Analysis, University of Freiburg, Freiburg, Germany, 6 Max Planck Institute for Molecular Genetics, Berlin, Germany, 7 Institute for Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria, 8 BioTechMed Graz, Graz, Austria Data Availability Statement: Underlying data for every analysis is shown in S1 Data. Funding: This work was supported by grants from the Bundesministerium fu¨r Bildung und Forschung (Smartage, 01GQ1420A) to SJS and CB, the Forschungszentrum fu¨r neurodegenerative Erkrankungen to SJS and AE, and the Deutsche Forschungsgemeinschaft to SJS and AB (Exc 257, TP A3 and A6 SFB 958; SFB 740 TP C09). UP and AF were supported by the German Research Foundation (SFB 889/B04) and the German RESEARCH ARTICLE * stephan.sigrist@fu-berlin.de Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Ministry of Research and Education via the Bernstein Center for Computational Neuroscience Go¨ttingen (grant number 01GQ1005A). FM is grateful to the Austrian Science Fund FWF for grants P23490-B12, P24381, P 27893, I1000 and grant ‘SFB Lipotox’ and to BMWFW and the Karl- Franzens University for grant ‘Unkonventionelle Forschung’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. functional changes during formation and retrieval of memories. Though alterations in synaptic performance are believed to accompany aging, the causal relationship between age-dependent memory impairment and synaptic changes remains largely unknown. Using the fly Drosophila melanogaster as a model, we found that feeding them spermidine —a polyamine compound—suppresses age-induced decline in olfactory memory, provid- ing us with a tool to further decipher mechanisms associated with age-dependent memory impairment. In this study, we investigated the relationship between synaptic changes and age-dependent memory impairment by studying the olfactory circuitry. We observedan age-related increase in the levels of the synaptic proteins Bruchpilot and Rim-binding pro- tein, which caused an enlargement of the presynaptic active zone—the complex of proteins that mediate neurotransmitter release—and enhanced synaptic transmission. Interest- ingly, feeding of spermidine was sufficient to abolish these age-associated presynaptic changes, further emphasizing the relationship between presynaptic performance and age- dependent memory impairment. Furthermore, flies engineered to express an excess of the core active zone protein Bruchpilot showed a premature impairment in memory forma- tion in young flies. Based on our data, aging plausibly steers the synapses towards the upper limit of their operational range, limiting synaptic plasticity and contributing to impairment of memory formation. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: AMI, age-dependent memory impairment; ARM, anesthesia-resistant memory; ASM, anesthesia-sensitive memory; AZ, active zone; BRP, Bruchpilot; EM, electron microscopy; GFP, green fluorescent protein; HDAC6, histone deacetylase-6; ITM, intermediate-term memory; KC, kenyon cell; MBON, mushroom body output neuron; MCH, 4-methylcyclehexanol; PN, projection neuron; RBP, rim-binding protein; STED, stimulated emission depletion microscopy; STM, short-term memory; SV, synaptic vesicle; Syb, Synaptobrevin; SynpH, SynaptopHluorin; 2xBRP, two-copy BRP; 3d, 3-day old flies; 3-Oct, 3- Octanol; 4xBRP, four-copy BRP; 30d, 30-day-old flies; 30dSpd, 30-day-old flies treated with spermidine. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Author Summary Neurons communicate by sending impulses, in the form of secretion of neurotransmitters, across small spaces called synapses. It is these synapses that undergo structural and 1 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Results It is known that the ability to acquire new memories declines with advancing age. Based on pre- vious study [10], one plausible explanation for this observation might be the increase in the threshold required for memory formation with age. In fact, when we analyzed olfactory condi- tioning in aged flies (30-d-old flies or 30d) we found that greater number of exposures to the unconditioned stimulus in order to attain saturated levels of memory scores, which, however, never reached the same maximal learning scores found in young flies (3-d-old flies or 3d), indi- cating that the dynamic range of memory formation is altered with advancing age (S1 Fig). Multiple lines of evidence suggest that presynaptic plasticity processes are responsible for form- ing olfactory associative memory in Drosophila [11–13]. Therefore, we set out to determine the role of age-induced changes in the organization and function of synapses in AMI, using dietary supplementation with spermidine as a tool to identify synaptic changes that can potentially contribute to AMI. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Therefore, we set out to determine the role of age-induced changes in the organization and function of synapses in AMI, using dietary supplementation with spermidine as a tool to iden- tify synaptic changes that can potentially contribute to AMI. To accomplish this, we analyzed age-induced changes in the ultrastructural,molecular, and functional organization of synapses within the olfactory system of flies by comparing aged flies fed with normal food to aged flies fed with spermidine-supplemented food. We found that aging is associated with an increase in the average size of active zone (AZ) scaffolds, structures recently shown to scale with synaptic vesicle (SV) release. Consistent with this, optophysiologi- cal analysis showed that more SVs are released in response to natural odor stimuli in aged flies. Interestingly, these age-associated changes were suppressed by spermidine feeding, indicating that these changes might be causally relevant to AMI. In fact, genetic manipulation provoking an increase of T-bar size in young animals was sufficient to induce a premature decline in memory performance. We suggest that a cumulative increase in the size and function of pre- synaptic AZ scaffolds might reduce the operational range of synaptic plasticity processes, and thus, hamper the formation of new memories with age. Additionally, levels of postsynaptic neurotransmitter receptors and postsynaptic Ca2+ signals remained largely unaffected with age, suggesting that homeostatic adaptations might be involved in increasing the threshold for memory formation with advancing age. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Introduction Age-dependent memory impairment (AMI), which is associated with both psychiatric and neurodegenerative disorders, starts in midlife and worsens with advancing age, suggesting that the greatest driving factor is age itself. The lack of effective treatments that prevent, halt, or reverse the condition is contributing to a diminishing quality of life for many senior citizens. Therefore, animal models that allow one to monitor physiological changes across their lifespan and to test for a causal character of age-induced changes might be helpful in exploring the mechanistic basis of AMI. D. melanogaster, with its short lifespan of around 60 d and advanced molecular genetic tools, provides an efficient experimental model to unravel mechanisms underlying AMI. Additionally, the olfactory nervous systems of insects and mammals exhibit many similarities, suggesting that the mechanisms for olfactory learning may be shared [1]. Moreover, aversive short-, intermediate-, and long-term olfactory memories have been found to be subject to age-induced decline in Drosophila, with an onset at about 10 d of age and pla- teaus at about 30 d of age [2–6]. Notably, we recently found a simple dietary supplementation of spermidine, a polyamine that specificallyprotects from AMI in Drosophila. External stimuli are believed to be represented in the brain as spatiotemporal patterns of neural activity within a set of neuronal connections. Changes in synaptic communication (“plasticity”) within certain neuron populations are meant to ultimately encode behavioral adaptations such as learning and memory. Thus, dysfunctioning of synaptic plasticity might well be relevant to age-dependent deterioration of learning and memory [7,8]. One of the fun- damental problems of studying AMI, however, is the inability to differentiate causative changes from adaptive or protective changes. Moreover, the brain undergoes changes at multiple levels with advancing age, including alterations in circuits, individual neurons, and single synapses, further complicating the situation [8]. Nonetheless, recent work has linked AMI to subtle syn- aptic alterations in the hippocampus and other cortical brain areas, rather than to the loss of neurons [7,9]. At the same time, the age-associated modulation of molecular entities underly- ing learning and memory that define and change synapse function remain poorly understood. 2 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes photon microscopy. Similar to our previous observations[3], we found no significant difference in the amplitude or time course of cytosolic GCamp3.0 signals of young (3d) and aged (30d) ani- mals (S3 Fig). Thus, in the context of odor information processing, odor-evoked action potential frequency or presynaptic Ca2+ influx remained rather unaffected by the age of the animal. Next, we asked whether the release of SVs was altered with advancing age and analyzed the odor-driven SVs release. To this end, we used SynaptopHluorin (SynpH), a pH-sensitive green fluorescent protein (GFP) fused to the luminal side of the SV membrane protein Synaptobrevin (Syb) [16]. SynpH is nonfluorescent at the acidic pH inside SVs; however, when SVs are released, SynpH is exposed to the neutral extracellular space, and the presynaptic terminal becomes brightly fluorescent. Following endocytosis,SVs become reacidified,and the cycle can start again [17]. SynpH was expressed within PNs, and the release of SVs in response to two odors was monitored, again, at PN-to-KC synapses (Fig 1A–1H). We found a profound increase in the amplitude of SynpH signals in aged (30d) animals when compared to young (3d) flies (Fig 1A–1H). In contrast, spermidine administration to 30d flies prevented this age- dependent increase of odor-induced SynpH signals (30dSpd; Fig 1A–1H). Alterations in the endocytoticclearance of newly released SVs might, per se, explain the increase in SynpH sig- nals observed;however, the decay constants of the poststimulus SynpH signal remained essen- tially unchanged with aging (S4 Fig), indicating that the endocyticclearance cannot be responsible for the difference in odor-driven SynpH signals observedin aged animals. In addi- tion, neither the basal expression of SynpH before odor stimulation nor the maximal SynpH signal determined by high-molar KCl treatment showed systematic differences between young and aged cohorts (S2B and S5 Figs). These experiments, thus, indicate that the exocytosis of SVs underlies the increase in SynpH response with advancing age. In addition to measuring the SV release at the PN presynaptic terminals within calyx, we also measured odor-evoked changes within the axonal projections of KCs within the mushroom body horizontal lobes by expressing SynpH using mb247-Gal4. Though relative signals were smaller (when signal was normalized to the whole mushroom body horizontal lobe), likely reflectingthe well-documentedsparse odor coding of KCs [18], we still observeda substantially higher ampli- tude of SynpH signals in aged (30d) than in young (3d) flies, and, again, spermidine administra- tion (30dSpd) protected from this age-dependent increase (S6 Fig). Thus, two major neuron populations of the olfactory system—PNs and KCs, showed an increase in odor-evoked fluores- cence changes in response to odor stimuli, indicating higher release of SVs in aged animals. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Age-Induced Increase of Odor-Driven Vesicle Release Is Suppressed by Spermidine Feeding In order to identify synaptic mechanisms plausibly contributing to AMI, we used opto-physio- logical assays to characterize overall neuronal responses in synaptic terminals of live intact flies. For these experiments, we focused on projection neuron (PN) to kenyon cell (KC) synap- ses within the mushroom body calyx of the olfactory system for two reasons: first, aversive olfactory learning involves coincidence detection of a conditioned stimulus (odor) with an unconditioned stimulus (electric shock), causing changes in the odor-specific synaptic activity of second order PNs and third order mushroom body KCs [1,14]; second, the superficial posi- tion of the calyx within the fly brain enabled us to perform efficient optical analysis [15], since sensor signals could be retrieved from discrete synaptic bouton areas. We started by expressing cytosolic GCamp3.0 in the PNs (using GH146-Gal4) and found the basal expression of GCamp3.0 to remain largely unchanged with age (S2A Fig). Next, we monitored the PN boutons for intracellular Ca2+ responses to two odors typically used for olfactory conditioning, 3-Octanol (3-Oct) and 4-methylcyclehexanol (MCH), through two- 3 / 34 Spermidine Feeding Specifically Blocks Age-Induced Increases of Core AZ Components Since Ca2+ influx into presynaptic terminals was apparently not responsible for the profound age-induced increase in SV release, presynaptic mechanisms downstream of Ca2+ signaling might be involved. In order to address the molecular and cellular basis of this age-associated increase in SV release, we started by analyzing proteins directly associated with SVs: Synapsin, Syb, and Synaptotagmin-1. Synapsin is a SV-associated phosphoprotein important for control- ling the number of SVs available for release [19], and Syb is a core component of SNARE com- plex that drives the exocytosis of SVs [20,21]. We found the levels of Synapsin as well as Syb to remain unchanged with advancing age (comparing aged flies: 30-days old or 30d with young flies: 3d), regardless of spermidine feeding (30dSpd; Fig 2A–2D and S7 Fig). Synaptotagmin-1 is a vesicular protein with a central role as a Ca2+ sensor for SNARE-dependent SV fusion [22]. Synaptotagmin-1 decreased slightly with age, and feeding with spermidine had no discernable influence on this age-dependent change (Fig 2E–2H), indicating that these moderate changes are seemingly not associated with AMI. 4 / 34 The release of neurotransmitters is a sophisticated process that requires SVs to be in close vicinity to voltage-gated Ca2+ channels, and this precise spacing is orchestrated by interplay Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the calyx neuropil (GH146 > SynpH). Scale bar: 10 μm. (b–c) False color-coded image of the SynpH activity within the presynaptic terminals of PNs in response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, and cold colors indicate low levels or no SynpH activity. The color scale on the right indicates changes in fluorescence (ΔF/F in %). (d) Odor-evoked release of SVs, measured by changes in fluorescence of SynpH of individual flies over time shown as false colors in presynaptic terminals of PN in the calyx region. The left panel is in response to the odorant 3-Oct and the right panel is in response to MCH (n = 6–7 flies). (e) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of PNs within the calyx neuropil of 3d, 30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies). Spermidine Feeding Specifically Blocks Age-Induced Increases of Core AZ Components (f) Maximum change in SynpH fluorescence (ΔF/F in %) in response to 3-Oct within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of SynpH activity induced by MCH in the presynaptic terminals of PNs within the calyx region of 3d, 30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies) (h) Maximum change in SynpH fluorescence (ΔF/F in %) in response to MCH within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g001 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes The release of neurotransmitters is a sophisticated process that requires SVs to be in close vicinity to voltage-gated Ca2+ channels, and this precise spacing is orchestrated by interplay Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the calyx neuropil (GH146 > SynpH). Scale bar: 10 μm. (b–c) False color-coded image of the SynpH activity within the presynaptic terminals of PNs in response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, and cold colors indicate low levels or no SynpH activity. The color scale on the right indicates changes in fluorescence (ΔF/F in %). (d) Odor-evoked release of SVs, measured by changes in fluorescence of SynpH of individual flies over time shown as false colors in presynaptic terminals of PN in the calyx region. The left panel is in response to the odorant 3-Oct and the right panel is in response to MCH (n = 6–7 flies). (e) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of PNs within the calyx neuropil of 3d, 30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies). Spermidine Feeding Specifically Blocks Age-Induced Increases of Core AZ Components (f) Maximum change in SynpH fluorescence (ΔF/F in %) in response to 3-Oct within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of SynpH activity induced by MCH in the presynaptic terminals of PNs within the calyx region of 3d, 30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies) (h) Maximum change in SynpH fluorescence (ΔF/F in %) in response to MCH within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. -KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the calyx neuropil (GH146 > SynpH). Scale bar: 10 μm. (b–c) False color-coded image of the SynpH activity within the presynaptic terminals of PNs in response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, and cold colors indicate low levels or no SynpH activity. The color scale on the right indicates changes in fluorescence (ΔF/F in %). (d) Odor-evoked release of SVs, measured by changes in fluorescence of SynpH of individual flies over time shown as false colors in presynaptic terminals of PN in the calyx region. The left panel is in response to the odorant 3-Oct and the right panel is in response to MCH (n = 6–7 flies). (e) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of PNs within the calyx neuropil of 3d, 30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies). doi:10.1371/journal.pbio.1002563.g001 Spermidine Feeding Specifically Blocks Age-Induced Increases of Core AZ Components Spermidine feeding suppresses age-associated increase in BRP and rim-binding protein (RBP) levels. (a–c) Adult brains 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synapsin. Scale bar: 50 μm. (d) Quantification of Synapsin intensity within the central brain region normalized to 3d flies (n = 9–10 independent brains; Kruskal-Wallis test). (e–g) Adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synaptotagmin-1 (Syt-1). Scale bar: 50 μm. (h) Quantification of signal intensity of Syt-1 in the central brain region normalized to 3d flies (n = 8–9 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i–k) Adult brains of 3d, 30d w1118, and 30dSpd w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. Scale bar: 50 μm (l–n) Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d flies (n = 14–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. Fig 2. Spermidine feeding suppresses age-associated increase in BRP and rim-binding protein (RBP) levels. (a–c) Adult brains 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synapsin. Scale bar: 50 μm. (d) Quantification of Synapsin intensity within the central brain region normalized to 3d flies (n = 9–10 independent brains; Kruskal-Wallis test). (e–g) Adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synaptotagmin-1 (Syt-1). Scale bar: 50 μm. (h) Quantification of signal intensity of Syt-1 in the central brain region normalized to 3d flies (n = 8–9 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i–k) Adult brains of 3d, 30d w1118, and 30dSpd w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. Scale bar: 50 μm (l–n) Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d flies (n = 14–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. Spermidine Feeding Specifically Blocks Age-Induced Increases of Core AZ Components doi:10.1371/journal.pbio.1002563.g002 among several proteins that form the AZ scaffold [23,24]. In flies, the ELKS-family protein Bruch- pilot (BRP) is an essential building block of the AZ scaffold and is neededto effectively cluster Ca2 + channels as well as regulate the release of SVs [25–28]. When whole-mount brains were stained for BRP using two different antibodies (BRPNc82 and BRPN-term), we observeda substantial increase in the levels of BRP with advancing age (Fig 2I, 2J, 2L and 2M). Similarly, Rim-binding protein (RBP) [29], another structurallyand functionallyimportant component of the AZ scaffold, was found to be significantly increased in brains of 30d flies compared to 3d animals (Fig 2I, 2J and 2N). Furthermore, flies analyzed at shorter intervals throughout their lifetime exhibited a pro- gressive increase in the levels of both BRP and RBP (S8 Fig). Notably, the age-dependent increase in BRP and RBP signals was suppressed in aged flies fed with spermidine (30dSpd; Fig 2I–2N). The staining efficacycould potentially be influenced by the sheer age of the tissue, e.g., due to differences in antibody penetration. To rule this out, flies expressing a GFP-tagged genomic BRP construct (rescuing the lethal brp null mutant [28]) were aged on normal food or food supplemented with spermidine. We found the endogenous GFP signals to be significantly increased in 30d flies in comparison to 3d flies, while feeding with spermidine again prevented this age-related increase (S9 Fig). Since the AZ scaffold has previously been reported to effectively cluster Ca2+ channels [26– 28], we asked whether the age-associated increase in levels of core AZ-proteins might influence synaptic levels of Ca2+ channels. To address this, we expressed a GFP-labeled genomic con- struct of α1 subunit Cacophony (Cac), which is the only representative of the mammalian Cav2.1/2.2 family present in Drosophila [28], and stained the flies for GFP and BRP. We found the levels of Cac (quantified using an antibody against GFP) to remain unchanged with aging (S10 Fig). Besides its role in Ca2+ channel clustering, the AZ scaffold has been suggested to cre- ate a stereotypic arrangement that defines SV release slots by clustering SV release machinery [28]. In fact, the levels of Unc13, a protein essential for priming SVs by rendering them fusion- competent [24], were also increased in brains of 30d flies compared to 3d flies (S11 Fig). Again, spermidine administration suppressed this age-dependent increase (30dSpd; S11 Fig). Spermidine Feeding Specifically Blocks Age-Induced Increases of Core AZ Components Taken together, our data suggest that synaptic levels of core AZ scaffold proteins, BRP and RBP, as well as the levels of critical release factor Unc-13 increased with advancing age. Spermidine Feeding Specifically Blocks Age-Induced Increases of Core AZ Components (f) Maximum change in SynpH fluorescence (ΔF/F in %) in response to 3-Oct within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of SynpH activity induced by MCH in the presynaptic terminals of PNs within the calyx region of 3d, 30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies) (h) Maximum change in SynpH fluorescence (ΔF/F in %) in response to MCH within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. 5 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 6 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 2. Spermidine feeding suppresses age-associated increase in BRP and rim-binding protein (RBP) levels. (a–c) Adult brains 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synapsin. Scale bar: 50 μm. (d) Quantification of Synapsin intensity within the central brain region normalized to 3d flies (n = 9–10 independent brains; Kruskal-Wallis test). (e–g) Adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synaptotagmin-1 (Syt-1). Scale bar: 50 μm. (h) Quantification of signal intensity of Syt-1 in the central brain region normalized to 3d flies (n = 8–9 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i–k) Adult brains of 3d, 30d w1118, and 30dSpd w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. Scale bar: 50 μm (l–n) Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d flies (n = 14–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. Fig 2. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes In order to allow for an unbiased quantification, we applied automated data collection to acquire more than a thousand transmission electron microscopic images covering nearly a whole calyx cross-section, which were then “stitched” together into a single high-magnification image (see Materials and Methods). As describedpreviously [30], PN boutons could be easily identified, and light-colored boutons containing clear-core SVs were used for analysis. We rec- ognized that plasma membranes between cellular elements were less aligned, with an increase in extracellular spacing between cellular elements, in aged (30d) flies when compared to young (3d) flies (S12 Fig). Spermidine feeding appeared to substantially alleviate this age-related change (S12 Fig). Driven by the finding that SV release is increased with age, we decided to analyze the AZs within PN boutons. We found aged animals (30d) to display reduced numbers of AZs per unit bouton-area in comparison to 3d flies, with no apparent influence of spermi- dine feeding on this age-dependent decline (Fig 3A–3E). The density of SVs in proximity to the AZ scaffold appeared unchanged in aged flies (30d as well as 30dSpd), when compared to young flies (3d; Fig 3F). Additionally, the number of SVs docked at the AZ plasma membrane appeared essentially unaltered with advancing age (Fig 3G). The AZ scaffold exhibits an electron-dense structure in electron microscopy (EM), and due to its T-shaped structure in Drosophila, this scaffold is often referred to as a T-bar [24,26,27]. We found the average size of the T-bars to be significantly increased in 30d animals in compar- ison to 3d flies (Fig 4A–4D). Feeding flies with spermidine suppressed this age-induced increase in T-bar size (30dSpd; Fig 4A–4D). We have previously introduced STED in the analy- sis of AZ suborganization [26–28]. At peripheral neuromuscular synapses of Drosophila larvae, STED allowed us to unmask the “nano-architecture” of AZs where BRP and RBP organize a scaffold that provides slots for SV release and concentrates Ca2+ channels in the AZ center [28,29]. When planar AZs were imaged using the BRP C-terminal epitopes at neuromuscular synapses, they display a ring-shaped structure whose diameter correlated with the EM-derived physical size of individual T-bar/AZ scaffold [32]. We applied STED to PN-to-KC synapses of the calyx and found ring-like BRP structures at planar-oriented AZs (S13 Fig). Subsequently, the analysis of these STED images revealed an increase in the ring diameter of BRP spots with advancing age, while spermidine treatment was able to suppress this age-associated increase (Fig 4E–4H). Finally, we performed coimmuno-EM labeling against BRP and RBP on calycal slices. The number of gold particles positive for BRP as well as RBP was found to increase in aged flies (30d) in comparison to both young (3d) flies and aged flies fed with spermidine (30dSpd; Fig 4I–4N). Taken together, the morphological EM, immuno-EM, and STED analysis consistently show that aged animals display larger AZ scaffolds, plausibly due to an increase in local amounts of the critical scaffold components: BRP and RBP. Recent in vivo analysis of larval Drosophila neuromuscular junctions has shown that the local amounts of BRP at a given AZ scale directly with the probability of evoked SV release [33–37]. Consistent with these studies, we found SV release to increase and AZ scaffolds to enlarge with age, while importantly both these age-related changes were suppressed by dietary supplementation with spermidine. Therefore, we next wanted to determine the influence of these synaptic changes on olfactory memory formation. Age-Induced Enlargement of AZ Scaffolds Is Suppressed by Spermidine Feeding Next, we asked whether the increase of both BRP and RBP labeling in aged brains reflects an increase in the number of AZs or just the increase in local amounts of these proteins at individ- ual AZs. To resolve this, we performed ultrastructuralanalysis on PN-to-KC synapses within the mushroom body calyx. In contrast to presynaptic terminals of KCs, presynaptic PN termi- nals within the calyx exhibit a well-definedmorphology [30,31], by which synapse types can be reliably identified in EM micrographs. Moreover, the superficiality of the calyx enabled us to perform stimulation emission depletion microscopy (STED) analysis (see below). 7 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 3. Ultrastructural analysis of PN-to-KC synapses within the mushroom body calyx. (a) Overview of the calyx neuropil, obtained by amalgamation of several images over a whole calyx cross-section of a 3d w1118 fly. Scale bar: 10 μm. (b–d) Higher magnification of PN boutons and dendritic claws of KCs within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 2 μm. The asterisk indicates the PN bouton, and the arrowhead indicates the dendritic claws of KCs. (e) Quantification of AZs normalized to bouton area (1/pm2) (total of 95–103 boutons across three independent animals, with at least 25 boutons per animal; Kruskal- Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (f) Quantification of total SVs within a shell of 150 nm surrounding the AZ scaffold (total of 92–100 electron-micrographs across four independent animals, with at least 20 electron-micrographs per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Quantification of SVs touching the presynaptic plasma membrane (total of 92–100 electron- micrographs across four independent animals, with at least 20 electron micrographs per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g003 doi:10.1371/journal.pbio.1002563.g003 whether genetically provoking an artificial enlargement of AZ scaffolds, independent of the aging process, might affect memory formation. Since BRP is a major essential building block of the AZ scaffold in Drosophila [26–28,32], we decided to increase the gene copy number of BRP from two to four copies by combining two additional genomic copies of brp [28] with two endogenous copies. As a result, BRP signals increased substantially in 3d flies expressing four- copy BRP (4xBRP) when compared to 3d flies expressing two-copy BRP (2xBRP; Fig 5A–5E). Additionally, RBP levels also increased concomitantly with BRP (Fig 5A–5D and 5F), consis- tent with the suggested role of BRP to operate as a “master molecule” in shaping the size (and functional performance) of the AZ scaffold [28,29,36]. In order to confirm that the increase in BRP levels resulted in an increase of the average size of AZ scaffolds, we took advantage of STED imaging. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 “Early” Memory Impairment after Genetically Enforced Enlargement of AZ Scaffolds Presynaptic plasticity processes have been reported to be critical for forming olfactory associa- tive memory in Drosophila [11–13]. Based on our findings, we suggest that the scale-up in the size and function of AZ scaffolds is likely to change the “operational range” of synaptic plastic- ity processes and thus change the threshold for memory formation. Thus, we wanted to test 8 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 9 / 34 Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d) Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (e–g) STED images of BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of 3d w1118 flies. Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d) Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (e–g) STED images of BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of 3d w1118 flies. Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test p-values were subject to Bonferroni correction) * p < 0 05 ** p g p y y pp g y p g Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d) Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 (e–g) STED images of BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of 3d w1118 flies. Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. doi:10 1371/journal pbio 1002563 g004 o-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs 18 Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d) Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (e–g) STED images of BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of 3d w1118 flies. Again, a considerable increase in the ring diameter of BRP spots was observed in 2xBRP flies with advancing age (Fig 5G–5K). Meanwhile, we found young flies (3d) express- ing 4xBRP to have increased BRP ring diameters when compared to age-matched control flies (2xBRP), and the ring diameter of BRP spots in 4xBRP flies remained rather unchanged with age (Fig 5G–5K). Having created a genetic state wherein levels of AZ core scaffold proteins increased prema- turely in young animals, we decided to investigate the influence of this manipulation on mem- ory formation. Before doing so, however, we wanted to ascertain whether the innate behavior was affected in 4xBRP flies. Thus, we measured naïve odor response and shock reactivity and found 4xBRP flies to show odor avoidance and shock reactivity scores that were indistinguish- able from 2xBRP age-matched control flies (2xBRP; S1 Table). Subsequently, we started by measuring short-term memory (STM), and found 4xBRP flies to exhibit lower memory scores “already” at a young age (3d), and their memory scores declined only negligibly with age (Fig 5L). In contrast, control flies (2xBRP) exhibited normal AMI (Fig 5L). As mentioned earlier, intermediate-term memory (ITM) has also been reported to decline with age [2–4]. Consistently, we found that 30d 2xBRP flies show substantially reduced ITM scores (measured 3-h post-training) when compared to 3-d 2xBRP flies (Fig 5M). By contrast, the 4xBRP flies showed lower ITM scores at a young age (3 d) and, again, the ITM scores did not decrease further in 30-d 4xBRP flies (Fig 5M). In fact, the learning performance of 3-d 4xBRP flies was comparable to that of 30-d 2xBRP flies. Based on distinct genetic mutants and specific pharmacological sensitivities [2,4,38,39], the ITM can be dissected into anesthesia-sen- sitive memory (ASM) and anesthesia-resistant memory (ARM) components. The ASM, unlike the ARM, has been shown to be strongly impaired with aging [3,4]. The ASM can be calculated by subtracting ARM scores, measured after amnestic cooling, from ITM. Consistent with previ- ous studies [2–4,40], we found ARM in 2xBRP and 4xBRP flies to remain relatively unaffected with age (Fig 5M). In contrast, ASM was nearly absent in 30-d 2xBRP flies when compared to 3-d 2xBRP flies. Reaffirmingour idea, we found the young (3-d) 4xBRP flies to show lower ASM scores in comparison to age-matched control (2xBRP) flies, while their ASM scores PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 10 / 34 (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g004 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 11 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory impairment. (a–d) Adult brains of 3d and 30d flies expressing 4xBRP together with age-matched controls brp (2xBRP), immunostained for BRP (using Nc82 and BRP N-terminal antibody) and RBP. Scale bar: 50 μm. (e, f) Quantification of BRP (using N-terminal antibody) as well as RBP intensity within the central brain region normalized to 3d flies (n = 12–13 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g–j) STED images of BRP label within the calyx region of 3d and 30d flies expressing 4xBRP as well as 2xBRP. Ring-shaped structures are indicated (arrowheads). Scale bar: 500 nm. (k) Quantification of BRP ring diameter in 3d and 30d 4xBRP flies along with age-matched 2xBRP flies (total of 47–68 BRP rings across eight independent animals, with at least six BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (l) Aversive associative memory performance 3 min after training (short-term memory; STM) markedly reduced in 3d 4xBRP flies in comparison to 3d wild-type 2xBRP flies (n = 10–16; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (m) Aversive associative memory performance at 3 h after training (intermediate-term memory; ITM), anesthesia-resistant memory (ARM), an anesthesia-sensitive memory (ASM) of 3d and 30d 4xBRP flies compared to age-matched control (2xBRP) flies (n = 7 independent experiments; Kruskal- Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (n) Aversive olfactory memory performance 3 min after training (STM) higher in appl-gal4 > histone deacetylase-6 (HDAC6) RNAi in comparison to age-matched controls (n = 13–21; Kruskal-Wallis test with Spermidine Protects from Age Dependent Memory Decline by Suppressing Presynaptic Change Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory impairment. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes declined negligibly with age (Fig 5M). These experiments indicate that a genetically provoked “up-scaling” of the average AZ scaffold size is sufficient to induce an “early” decline in mem- ory, similar to AMI, which physiologically occurs over a time course of 20–30 d. A reduction in BRP levels, per se, might be expected to slow down the onset of AMI. To address this possibility, we removed a single gene copy of brp, and found BRP heterozygotes (brp69/+ or 1xBRP) to exhibit a considerable reduction in the levels of both BRP and RBP (S14A–S14F Fig), indicating that our antibody stainings can detect subtle changes and reaf- firming that BRP levels can directly steer the local amounts of other AZ components in the Drosophila brain. We found that 3d flies expressing only one BRP copy (brp69/+) displayed memory scores comparable to 3d control flies (2xBRP); however, these brp69/+ flies still exhib- ited a normally-occurringAMI (30d; S14G Fig). AZ scaffold-dependent control of neuronal plasticity is undoubtedly a complex process [24,41], and other mechanisms, operating in paral- lel to modulations in the amounts of scaffold proteins, might well contribute to the pace and extent of AMI. Lysine-acetylation of BRP was recently identified as a major node to control the SV release at larval AZs [42,43]. In particular, the loss of histone deacetylase-6 (HDAC6) was found to cause hyperacetylation of BRP and provoke a reduction in evoked SV release at AZs [43]. Interestingly, using immunoprecipitation followed by mass spectroscopic analysis, we found at least 13 lysine sites within BRP to be target for (de)acetylation, (S15 Fig). Next, we asked whether loss of HDAC6 might affect memory. While the odor avoidance and shock reac- tivity were mainly unaffected by knockdown of hdac6 (S1 Table), memory scores of both young and aged flies with pan-neuronal knockdown of hdac6 were higher than those of age- matched driver controls (Fig 5N). These findings are consistent with the idea that driving down the AZs towards the lower limit of their operational range might facilitate memory for- mation in aged animals. Though any implications of acetylation of BRP or potentially other AZ scaffold proteins with respect to aging process still require extensive analysis, this result shows that BRP-directed modifications, reported to reduce SV release, can in fact increase the efficacyof memory formation in aged animals. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 (a–d) Adult b Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory impairment. (a–d) Adult brains of 3d and 30d flies expressing 4xBRP together with age-matched controls brp (2xBRP), immunostained for BRP (using Nc82 and BRP N-terminal antibody) and RBP. Scale bar: 50 μm. (e, f) Quantification of BRP (using N-terminal antibody) as well as RBP intensity within the central brain region normalized to 3d flies (n = 12–13 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g–j) STED images of BRP label within the calyx region of 3d and 30d flies expressing 4xBRP as well as 2xBRP. Ring-shaped structures are indicated (arrowheads). Scale bar: 500 nm. (k) Quantification of BRP ring diameter in 3d and 30d 4xBRP flies along with age-matched 2xBRP flies (total of 47–68 BRP rings across eight independent animals, with at least six BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (l) Aversive associative memory performance 3 min after training (short-term memory; STM) markedly reduced in 3d 4xBRP flies in comparison to 3d wild-type 2xBRP flies (n = 10–16; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (m) Aversive associative memory performance at 3 h after training (intermediate-term memory; ITM), anesthesia-resistant memory (ARM), and anesthesia-sensitive memory (ASM) of 3d and 30d 4xBRP flies compared to age-matched control (2xBRP) flies (n = 7 independent experiments; Kruskal- Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (n) Aversive olfactory memory performance 3 min after training (STM) higher in appl-gal4 > histone deacetylase-6 (HDAC6) RNAi in comparison to age-matched controls (n = 13–21; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g005 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 12 / 34 Homeostasis of Odor-Driven Neuronal Ca2+ Signals in Aged Flies Finally, we asked how the postsynaptic compartment might respond to these age-associated pre- synaptic structuraland functional changes. To address this question, we used GCaMP3.0 fused to the postsynaptic protein Homer [15] and found the basal expression of Homer-GCamp3.0 to be largely unaffectedwith age (S2C Fig). Moreover, the sensor was found to be effectively targeted to the postsynaptic density of the PN::KC synapses, as manifested by its specific enrichment within the postsynaptic specializations formed by claw-like dendritic endings of multiple KCs surrounding a single PN bouton (Fig 6A). However, postsynaptic Ca2+ signals did not increase with age. Rather, a slight tendency towards a decrease of postsynaptic Ca2+ signals was observed in normally aged animals when compared to young controls (Fig 6A–6H). At the same time, aged flies treated with spermidine (30dSpd) produced signals more similar to untreated 3d- Homer-GCaMP3.0 flies than to untreated aged animals (Fig 6A–6H). In order to be certain that Homer-GCamp3.0 signals were not saturated, we used high-molar KCl treatment to determine the maximal postsynaptic Ca2+ response. Unlike the odor-evoked maximum change in Homer- GCamp3.0 fluorescence of about 55%, KCl stimulation resulted in a substantially higher ΔF/F0 value of more than 300% (S16 Fig), suggesting that sensor sensitivity was not a limiting factor for the postsynaptic Ca2+ signals. Meanwhile, when the cumulative postsynaptic Ca2+ activity was critically analyzed during the odor stimulation, we found that the Ca2+ responses reduced signifi- cantly in aged (30d) flies relative to young flies, while the Ca2+ signals were comparable between young flies and spermidine-fedaged animals (30dSpd; S17 Fig). PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 13 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. (b–c) False color-coded image of Homer- GCamp3.0 activity within the postsynaptic terminals of KCs in response to 3-Oct and MCH shown in (a). Warm colors indicate high activity and cold colors indicate low or no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). (d) Odor-evoked postsynaptic Ca2+ activity, measured by changes in fluorescence of Homer-Gamp3.0, of an individual fly over time, shown as false colors in dendritic claws of KCs in the calyx region. Homeostasis of Odor-Driven Neuronal Ca2+ Signals in Aged Flies The left panel is in response to the odorant 3-Oct, and the right panel is in response to MCH (n = 10 flies). (e) Time course of Ca2+ activity induced by 3-Oct in the dendritic terminals of KCs within the calyx region of 3d, 30d, and 30dSpd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (f) Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to 3-Oct within dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of Ca2+ activity induced by MCH in the dendritic terminals of KCs of 3d, 30d, and 30dSpd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (h) Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to MCH within dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. (b–c) False color-coded image of Homer- GCamp3.0 activity within the postsynaptic terminals of KCs in response to 3-Oct and MCH shown in (a). Warm colors indicate high activity and cold colors indicate low or no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). (d) Odor-evoked postsynaptic Ca2+ activity, measured by h i fl f H G 3 0 f i di id l fl ti h f l l i d d iti l f KC i th l i Th l ft l Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). The grey bars indicate the duration of the odor stimuli. * p < 0.05, ** p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g006 doi:10.1371/journal.pbio.1002563.g006 PNs provide cholinergic input to the KCs within the calyx [44]. We used a fusion of mush- room body-specificenhancer mb247 to the Dα7 subunit of the acetylcholine receptor (mb247:: Dα7GFP) to explicitly visualize postsynaptic acetylcholine receptors. We showed previously that expression of Dα7-GFP from KCs localized specificallyto the KC postsynaptic densities, where it closely matched the AZs of the PNs [45]. While we observedan age-related increase in BRP in 30d mb247::Dα7GFP flies in comparison to 3d mb247::Dα7GFP flies, the levels of Dα7 subunit (quantified using an antibody against GFP fused to the α7 subunit of acetylcholine receptors) did not change with age, and spermidine feeding had no effect on the level of the α7 subunit of acetylcholine receptors (Fig 7A–7E). Similarly, when we stained for endogenous Drep2, a postsynaptic scaffold protein that is known to express strongly within the postsynap- tic densities of PN::KC synapses [46], we also found Drep2 to remain unchanged with age (S18 Fig). g At first glance, the increase in release of SVs might be expected to translate into increased postsynaptic responses; however, ample evidence from various studies in different model organisms, including Drosophila, support the existence of homeostatic controls, allowing neu- rons to remain within a certain range of excitation [47,48] in order to avoid epileptic states and Ca2+-induced degeneration. In an attempt to directly examine the existence of such homeo- static controls, we wanted to determine whether an increase in the amount of depolarization required to trigger an action potential might influence the architecture of the apposed AZ scaf- fold. To achieve this, we used dORK1ΔC, a constitutively open K+ selective pore that causes hyperpolarization of neurons and subsequent inactivation of neuronal function [45,49]. dORK1ΔC was specificallyexpressed in the KCs, and presynaptic terminals of PNs within the calyx were analyzed for BRP levels (Fig 7F–7I). Indeed, we found a substantial increase in the levels of BRP in the calyces of both 3d as well as 10d mb247>dORK1ΔC flies, when compared to age-matched controls (Fig 7J). Thus, a drop in postsynaptic excitability can drive a homeo- static increase in presynaptic AZ scaffolds, leading to a potential increase in SV release at olfac- tory synapses—a finding similar to the one we found at aging synapses. Though the exact mechanisms allowing for homeostatic compensation of the elevated presynaptic release remain to be further worked out, it is tempting to speculate that homeostatic mechanisms coupling postsynaptic excitability to presynaptic release function might drive aging synapses towards the upper limit of their operational range and be critically involved in AMI (see model in Fig 7K) Homeostasis of Odor-Driven Neuronal Ca2+ Signals in Aged Flies (b–c) False color-coded image of Homer- GC 3 0 ti it ithi th t ti t i l f KC i t 3 O t d MCH h i ( ) W l i di t hi h ti it d ld l n the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in Cs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in g 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a Fig 6. Imaging of Homer GCamp3.0 within the dendritic claws of KCs to measure odor evoked Ca activity. (a) Expression of Homer GCamp3.0 in the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. (b–c) False color-coded image of Homer- GCamp3.0 activity within the postsynaptic terminals of KCs in response to 3-Oct and MCH shown in (a). Warm colors indicate high activity and cold colors indicate low or no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). (d) Odor-evoked postsynaptic Ca2+ activity, measured by changes in fluorescence of Homer-Gamp3.0, of an individual fly over time, shown as false colors in dendritic claws of KCs in the calyx region. The left panel is in response to the odorant 3-Oct, and the right panel is in response to MCH (n = 10 flies). (e) Time course of Ca2+ activity induced by 3-Oct in the dendritic terminals of KCs within the calyx region of 3d, 30d, and 30dSpd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (f) Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to 3-Oct within dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of Ca2+ activity induced by MCH in the dendritic terminals of KCs of 3d, 30d, and 30dSpd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (h) Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to MCH within dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 14 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Homeostasis at PN::KC synapses of aged flies. (a–c) Mushroom body calyx of 3d and 30d mb247::Dα7GFP flies and 30dSpd mb247:: Dα7GFP flies immunostained for GFP-labeled Dα7 as well as BRP (corresponding single z-planes are shown). Scale bar: 10 μm. Arrows indicate the recurrent presynapses of KCs that remain unopposed to acetylcholine-receptor rings within calycal neuropil; these KCs presynapses are spatially separated from the sites of cholinergic input onto KCs. (d, e) Quantification of signal intensity of Dα7 (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to 3d flies (n = 8–10 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p  0.05. (f–i) BRP immunostained within mushroom body calyx from adult brains of 3d and 10d flies expressing UAS-dORK1 ΔC in the KCs compared to age-matched controls. (j) Quantification of signal intensity of BRP (using Nc82) in the calyx region normalized to 3d flies (n = 10–12 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (k) Model showing the age-induced synaptic changes (in red). In the aged brain, the lowering of postsynaptic response with age, due to decrease in membrane excitability or Ca2+ homeostasis, might steer retrograde changes in the architecture of AZs. As a result, the AZ characterized by T-bar in flies enlarges in size, leading to higher release of SVs and causing aged synapses to function near the top of their presynaptic plasticity range, leaving little room for additional synaptic strengthening, and possibly impeding further learning. Underlying data is shown in S1 Data. Fig 7. Homeostasis at PN::KC synapses of aged flies. (a–c) Mushroom body calyx of 3d and 30d mb247::D doi:10.1371/journal.pbio.1002563.g007 odors used for learning experiments. Importantly, spermidine feeding was able to “protect” from both the functional and structural changes at aged AZs, arguing in favor of specific synap- tic changes to be causally relevant for AMI. Indeed, installing 4xBRP not only increased the size of BRP rings in young flies, similar to those found in aged animals, but also provoked memory impairment in young flies. Notably, a reduction of BRP levels has previously been reported to affect ARM but not ASM [50]. Here, we report that an increase in BRP levels (by changing the gene copy number of BRP from two to four copies) severely affected ASM. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 7. Homeostasis at PN::KC synapses of aged flies. (a–c) Mushroom body calyx of 3d and 30d mb247::Dα7GFP flies and 30dSpd mb247:: Dα7GFP flies immunostained for GFP-labeled Dα7 as well as BRP (corresponding single z-planes are shown). Scale bar: 10 μm. Arrows indicate the recurrent presynapses of KCs that remain unopposed to acetylcholine-receptor rings within calycal neuropil; these KCs presynapses are spatially separated from the sites of cholinergic input onto KCs. (d, e) Quantification of signal intensity of Dα7 (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to 3d flies (n = 8–10 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p  0.05. (f–i) BRP immunostained within mushroom body calyx from adult brains of 3d and 10d flies expressing UAS-dORK1 ΔC in the KCs compared to age-matched controls. (j) Quantification of signal intensity of BRP (using Nc82) in the calyx region normalized to 3d flies (n = 10–12 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (k) Model showing the age-induced synaptic changes (in red). In the aged brain, the lowering of postsynaptic response with age, due to decrease in membrane excitability or Ca2+ homeostasis, might steer retrograde changes in the architecture of AZs. As a result, the AZ characterized by T-bar in flies enlarges in size, leading to higher release of SVs and causing aged synapses to function near the top of their presynaptic plasticity range, leaving little room for additional synaptic strengthening, and possibly impeding further learning. Underlying data is shown in S1 Data. m body calyx of 3d and 30d mb247::Dα7GFP flies and 30dSpd mb247:: Dα7GFP flies gle z-planes are shown). Scale bar: 10 μm. Arrows indicate the recurrent gs within calycal neuropil; these KCs presynapses are spatially separated from the of Dα7 (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to multiple comparison test, p-values were subject to Bonferroni correction). * ned within mushroom body calyx from adult brains of 3d and 10d flies expressing tification of signal intensity of BRP (using Nc82) in the calyx region normalized to multiple comparison test, p-values were subject to Bonferroni correction). (k) ain, the lowering of postsynaptic response with age, due to decrease in Fig 7. Discussion The aging process, causing progressive deterioration of an organism, is subject to a complex interplay of regulatory mechanisms. One of the primary aims of aging research is to use the understanding of this process to delay or prevent age-related pathologies, including AMI. We previously showed that restoration of polyamine levels by dietary supplementation with sper- midine suppressed AMI in fruit flies [3], providing us with a protective paradigm to identify candidate processes that might be functionally associated with AMI. As an insight towards the synaptic basis of AMI, we describe an age-induced increase in the levels of core AZ proteins, BRP, and RBP and of the functionally critical release factor Unc13, together with a shift towards an enlargement of AZ scaffolds within the olfactory system. In addition, based on SynpH experiments, we observeda substantial increase in the release of SVs at aged synapses (PN-to-KC and KC-to-mushroom body output neuron [MBON] synapses) in response to PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 15 / 34 These findings suggest that the two PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 16 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes complementary forms of memory (ARM and ASM) might rely on the recruitment of distinct presynaptic “functional modules.” The loss of brp has been shown to severely reduce release function in response to single low frequency, but not in response to high-frequencystimulation [27], indicating that SV release at low-frequency stimulation might be particularly relevant for forming ARM, a memory component that develops gradually after training. On the other hand, mobilization of the SVs during high frequency stimulation has been suggested to be criti- cal for formation of ASM [50], a memory component that predominates early memory and decays with age. Thus, the increase in the size of the AZ scaffolds might potentially contribute directly to AMI by interfering with mechanisms facilitating SV availability in the course of forming ASM. Though the exact mechanisms underlying age-induced synaptic changes remain to be fully worked out, a reduction in autophagy-mediated protein degradation might well be involved [51–53]. Autophagy is a cellular digestion pathway that involves the sequestration of cyto- plasmic components within a double-membrane vesicle called autophagosome, which fuses with lysosomes (autolysosomes) to degrade autophagic cargo by acidic hydrolases [52]. Inter- estingly, spermidine was shown to induce autophagy in several model systems, including rodent tissues and cultured human cells [51,54,55]. Moreover, amelioration of a-synuclein neu- rotoxicity due to spermidine administration was accompanied by autophagy induction [56]. Of note, we also found that spermidine feeding prevented accumulation of poly-ubiquitinated proteins by plausibly halting normally occurringage-induced decline of autophagic clearance [3,57]. The gene atg7 encodes an E1-like enzyme required for activation of both Atg8 and Atg12, a step critical for the completion of the autophagic pathway [53]. We found that atg7- mutant flies (atg7-/-) exhibit reduced memory scores at a young age (3d), which declined fur- ther with age (20-d of age or 20d) [3]. Concurrently, spermidine-mediatedprotection from memory impairment was eliminated in atg7−/−flies (for both 3d- and 20d-flies) [3,57]. There- fore, we wondered whether the decrease in the autophagic pathway might, per se, provoke increase of AZ scaffold components. When staining for BRP in atg7-mutant brains (atg7-/-), we found a brain-wide increase in levels of BRP (for both BRPNc82 and BRPN-term antibodies), and spermidine feeding was unable to prevent this age-related increase (S19 Fig). PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes gyrus as well as the CA1 area of the brain [8,65,66]. Additionally, the “unitary” intracellular- evoked amplitude elicited by minimal stimulation protocols has been found to be greater in old than in young rodents [67], suggesting that the “surviving synapses” are stronger [68]. It is of note that the induction threshold for long-term potentiation, considered to be a synaptic corre- late of learning, has been reported to increase in aged rodents [10]. Similarly, an age-related increase in the amplitude of endplate potentials evoked has been reported at mouse neuromus- cular synapses [69,70]. By contrast, a study at neuromuscular junctions of C. elegans revealed that aged motor neurons undergo a progressive reduction in synaptic transmission [71]. In flies, however, an age-related increase in the amplitude of the excitatory postsynaptic potential at adult neuromuscular junctions has been reported recently; this increase was suggested to tune the response of the homeostatic signaling system and establish a new homeostatic set point [72]. Collectively, these findings suggest that the dynamic range of synaptic plasticity may change with advancing age and, thus, contribute to AMI. y g g g Why would an increase in the odor-evoked SV release and ultrastructural size of AZ scaf- folds impair the efficacy of forming new memories? Synapses appear to display a “finite ceil- ing and floor” that define a synaptic operating range [73]. In rodents, the formation of new memories seems to drive synaptic strength to the upper limit of a fixed operating range, thereby creating an imbalance [73]. As a result, if the synapses are not returned to the mid- point of the synaptic modification range, then additional strengthening required for new memory formation might be blocked, and the system is driven to employ homeostatic com- pensatory mechanisms to balance the change [74]. In our experiments, we found dendritic Ca2+ signals and postsynaptic receptor levels to remain largely unchanged with age, suggest- ing the existence of homeostatic mechanisms that might allow the up-scaling of presynaptic release to be compensated by lowering the postsynaptic response to a given amount of neuro- transmitter released. On the other hand, this upscaling of presynaptic structure and function might also be a homeostatic response to a reduction in postsynaptic excitability or Ca2+ homeostasis, steering retrograde enlargement of AZ scaffold and higher release of SVs (Fig 7K). PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 The finding that spermidine feeding in atg7−/−flies neither blocked the increase in BRP levels (S19 Fig) nor sup- pressed memory impairment [3] suggests that the integrity of the autophagic system is crucial for the spermidine-mediatedprotection from age-associated increase in AZ scaffold compo- nents. Spermidine effects were recently shown to involve widespread changes of both nuclear and cytosolic protein acetylation [58,59]. In primary neurons, autophagosomes have previously been observedto form at the distal end of the axon, indicating compartmentalization and spatial regulation of autophagosome biogenesis [60,61]. More recently, autophagosomes were demonstrated to form directly near synapses and were found to be required for presynaptic assembly at developing synaptic termi- nals of Caenorhabditis elegans [62]. Moreover, the crucial release factor Unc13 was found to accumulate under conditions of defective endosomal microautophagy (a specializedform of autophagy) at developing neuromuscular synapses of Drosophila, suggesting Unc13 to be a substrate of this form of autophagy [63]. Interestingly, we have shown recently that the synap- tic levels of Unc13-A isoform scale tightly with the levels of the BRP/RBP scaffold [64]. Thus, it is conceivable that some of the AZ proteins, whose levels increase with age (BRP/RBP/Unc13), might be direct substrates of “pre-synaptic autophagy,” and that spermidine feeding might aug- ment effective autophagic degradation of these proteins at aging synapses. We also observeda moderate decrease in synapse numbers in aging Drosophila brains, a phenotype that was unaffected by spermidine feeding. Our data compare favorably with studies in mammals. For example, loss of synapses in aged rodents has been reported in the dentate PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 17 / 34 Behavioral Assays Behavioral experiments were performedin dim red light at 25°C and 80% relative humidity with 3-Oct (1:150 dilution in mineral oil presented in a 14 mm cup) and MCH (1:100 dilution in min- eral oil presented in a 14 mm cup) servingas olfactorycues, and 120V AC current servingas a behavioral reinforcer. Standard single-cycle olfactoryassociative memory was performedas previ- ously described[3,4,46,84,85], with minor modifications.Briefly, about 60–80 flies received one training session, during which they were exposedsequentially to one odor (conditioned stimulus, CS+; 3-Oct or MCH) paired with electricshock (unconditionedstimulus, US) and then to a second odor (CS−; MCH or 3-Oct) without US for 60 s with 30 s rest interval betweeneach odor presenta- tion. During testing, the flies were exposedsimultaneously to the CS+ and CS−in a T-maze for 30 s. The conditioned odor avoidance was tested immediately after training for STM (memory tested immediately after odor conditioning). Subsequently, flies were trapped in either T-maze arm, anesthetized, and counted. From this distribution, a performance index was calculated as the number of flies avoiding the shocked odor minus the number avoiding the nonshocked odor divided by the total number of flies and, finally, timed by 100. A 50:50 distribution (no learning) yielded a PI of zero, and a 0:100 distribution away from the CS+ yielded a PI of 100. A final per- formance index was calculated by the average of both reciprocal indices for the two odors. For ITM, flies were trained as describedabove, but tested 3 h after training. As a component of ITM, ARM was separated from ASM by cold-amnestic treatment, during which the trained flies were anesthetized 90 s on ice at 30 min before testing. In the end, ASM was calculated by subtracting the performance index of ARM from that of ITM for each training session on the same day, respectively. Animal Rearing and Fly Strains All fly strains were reared under standard laboratory conditions [79] at 25°C and 70% humidity, with constant 12:12 h light/dark cycle. Flies from an isogenizedw1118 strain were used as the wild-type control for all experiments. Flies carrying P(acman) cacGFP, P(acman) brp83GFP and P(acman) brp83 [28] and mb247::Dα7GFP [45] were describedpreviously. The generation of UAS-homer-GCaMP3.0 flies are describedelsewhere [15]. Briefly, cDNA of dho- mer was amplified from w 1118 flies and inserted with a C-terminal linked GCaMP336 into pUAST. Both UAS-GCamp3.0 (on the 3rd chromosome) [80] and UAS-SynpH [81] were kindly provided by Gero Miesenböck.Atg7d14 and Atg7d77 flies were kind gifts from Thomas Neufeld [53]. In addition, mb247-Gal4 [82] and gh146-Gal4 [83] were used. As previously described[3], the fly food was prepared according to Bloomington media rec- ipe (www.flystocks.bio.indiana.edu/Fly_Work/media-recipes/media-recipes.htm) with minor modification,which was called Spd−or normal food. Spermidine (Sigma Aldrich) was prepared as a 2 M stock solution in sterile distilled water, aliquoted in single-use portions and stored at −20°C. After food had cooled down to 40°C, Spermidine was added to normal food to a final concentration of 1 mM or 5 mM Spd, and called Spd1mM+ or Spd5mM+, respectively. Parental flies mated on either Spd−or Spd5mM+ food for all experiments, and their progeny were allowed to develop on the respective food. Flies used in all experiments were F1 progeny. The flies were collected once a day for aging, as a results-specific age indicated is day ± 24 h. In fact, the influx of postsynaptic Ca2+ through glutamate receptors at the peripheral glutamatergic synapses of Drosophila has been reported to control presynaptic assembly by retrograde signalling [47,48,75]. While the exact nature of homeostatic controls connecting pre- with postsynaptic neurons in the olfactory system remains to be resolved, changes in plasma membrane excitability, a change in postsynaptic neurotransmitter sensitivity, or an increase in inhibitory GABAergic drive are obvious candidate processes. Taken together, we propose these synaptic changes steer the presynaptic AZs to function towards the upper limit of their operational range, making these synapses unable to react adequately to conditioning stimuli and provoke potentiation or depression of synapses in order to encode memory for- mation [11,12,76]. Sleep is widely believed to be critical for formation and consolidation of memories [77]. In sleep-deprived animals, neuronal circuits would exceed available space and/or saturate, thereby affecting an individual’s ability to learn [77]. Importantly, sleep deprivation has also been asso- ciated with widespread increases of BRP levels in the Drosophila brain [78]. Notably, we also observeda brain-wide increase in BRP levels in aged brains. It is tempting to speculate that both sleep deprivation and aging change the operational range over several synaptic relays and thereby affect memory formation—a topic that deserves further investigation in future. Taken together, our data show that upscaling of presynaptic structure and function contrib- ute to an AMI in Drosophila. Furthermore, and restoration of polyamine levels prevents these age-associated alterations as well as AMI. Thus, spermidine feeding provides a unique opportu- nity to further the molecular and functional dissection of the mechanisms underlying AMI with the ultimate goal of restoring memory function in older humans. 18 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Staining Protocol Whole-mount adult brains. Adult brains were dissected in HL3 (which contains 70 mM NaCl, 5 mM KCl, 20 mM MgCl2, 10 mM NaHCO3, 5 mM Trehalose, 115 mM Sucrose, 5 mM 19 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Hepes, added to 500 ml H2O; pH adjusted to 7.2) on ice and immediately fixed in cold 4% Paraformaldehyde (v/v) for 20 min at 20–30°C. After fixation, the brains were incubated in 1% PBT (phosphate-buffered saline (PBS) containing 1% Triton X-100; v/v) for 20 min and then preincubated in 0.3% PBT (PBS containing 0.3% Triton X-100) with 10% normal goat serum (NGS; v/v) at 20–30°C. For primary antibody treatment, samples were incubated in 0.3% PBT containing 5% NGS and the primary antibodies for 48 h at 20–30°C. After primary antibody incubation, brains were washed in 0.3% PBT, four times for 30 min at 20–30°C, and then over- night at 4°C. All samples were then incubated in 0.3% PBT with 5% NGS containing the sec- ondary antibodies for 24 h at 20–30°C. Brains were washed again four times for 30 min at 20– 30°C, then overnight at 4°C. Brains were finally mounted in Vectashield overnight before con- focal scanning (Vector Laboratories). Image acquisition, processing, and analysis. The images were acquired, processed, and analyzed as previously described[46,85], with minor modifications. Briefly, conventional con- focal images were acquired at room temperature with a Leica Microsystems TCS SP8 confocal microscope using a 63x 1.4 NA oil objective for detailed scans and a 20x 0.7 NA oil objective for overview scans. All images were acquired using Leica LCS AF software. Lateral pixel size was approximately 300 nm for overview scans, approximately 100 nm for detailed scans. Typi- cally, 1,024 x 1,024 images were scanned at 400 Hz using 4x line averaging. Images of calyces for cryostat sections were acquired at room temperature with a Leica Microsystems TCS SP5 CW STED microscope in confocal mode using a 100x 1.4 NA oil objective. STED images were acquired on a Leica TCS STED CW. Images were deconvolved using the built-in deconvolution algorithms of the Leica LAS-AF software. The PSF was generated by using a 2-D Lorentz func- tion with the full-width half-maximum set to 60 nm (as calculated on the image using the Wie- ner filter algorithm; regulation parameter: 0.05). Staining Protocol In order to analyze the brain scans, the signal intensity within a neuropil of interest (whole central brain, or CB; antennal lobes, or AL; and calyx) was determined using Amira software (Amira 5.3.3, FEI Visualization Sciences Group, Oregon, US). The region of interest within the 3-D image stack was masked using the tool Segmentation Editor by interpolating manual selec- tions between slices. Average intensity values were calculated for all pixels within each mask for each channel separately. For STED analysis, deconvolved BRP spots (stained with monoclonal Nc82 antiboby) were processed in ImageJ. The diameters of planar oriented BRP rings were measured using the line tool of ImageJ. The distance from intensity maximum to intensity maximum was acquired in the plot window of individual hand-drawn lines and transferred to Microsoft Excel. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Following embedding,sections of 60 nm, each, were cut using a Leica Ultracut E ultramicro- tome equipped with a 2 mm diamond knife. Sections were collected on 100 mesh copper grids (Plano GmbH, Germany) coated with 0.1% Pioloform resin. Contrast was enhanced by placing the grids in 2% uranyl acetate for 30 min, followed by washing with water three times and, then, incubation in lead citrate for 2 min. The grids were washed three times with water and dried. Images were acquired fully automatically on a FEI Tecnai Spirit transmission electron microscope operated at 120 kV equipped with a FEI 2K Eagle CCD camera using Leginon [86]. Regions of interest were first selected at 560x nominal magnification and then successively imaged at 4,400x and 26,000x nominal magnification, respectively. Series of more than 1,000 TEM images were then stitched to a single montage covering nearly the full calyx region using the TrakEM2 software [87] implemented in Fiji [88]. Immuno-EM Brains were dissected in HL3 solution and fixed for 20 min at room temperature with 4% paraformaldehyde and 0.2% Glutaraldehyde in a buffer containing 50 mM Sodium Cacody- late and 50 mM NaCl at pH 7.5. Afterwards, brains were washed twice in the buffer and dehydrated through a series of increasing alcohol concentrations. Samples were embedded in London-Resin (LR)-Gold resin by incubating them in Ethanol/LR-Gold 1:1 solution over- night at 4°C, followed by Ethanol/LR-Gold 1:5 solution for 4 h at room temperature. Thereaf- ter, the samples were washed first with LR-Gold/0.2% Benzil overnight, a second time for 4 h, and then again overnight. Finally, the brains were placed in BEEM capsules covered with LR-Gold/0.2% Benzil resin and placed under a UV lamp at 4°C for 5 d to allow for polymeri- zation of the resin. Following embedding, sections 70–80 nm, each, were cut using a Leica Ultracut E ultrami- crotome equipped with a 2 mm diamond knife. Sections were collected on 100 mesh nickel grids (Plano GmbH, Germany) coated with 0.1% Pioloform resin and transferred to a buffer solution (20 mM Tris-HCl, 0.9% NaCl, pH 8.0). Prior to staining, sections were blocked for 10 min with 0.04% BSA in buffer. Sections were incubated with the primary antibody (guinea pig- anti RBPSH3II+III and rabbit-anti BRPlast200, 1:500 dilution) in blocking solution overnight at 4°C. After washing four times in buffer, the sections were incubated in buffer containing the secondaryantibody (goat anti-guinea pig 10 nm colloidal gold, goat anti-rabbit 5 nm colloidal gold British Biocell, 1:100) for 2–3 h at room temperature. Finally, the sections were washed four times in buffer and three times in distilled water. Contrast was enhanced by placing the grids in 2% uranyl acetate for 30 min, followed by washing three times with water and, after- wards, incubation in lead citrate for 2 min. Afterwards, the grids were washed three times with water and dried. Images were acquired on a FEI Tecnai Spirit, 120 kV transmission electron microscope equipped with a FEI 2K Eagle CCD camera. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 EM Conventional EM. Brains were dissected in HL3 solution and fixed for 20 min at room temperature with 4% paraformaldehyde and 0.5% glutaraldehyde in a PBS. Subsequently, the brains were incubated overnight at 4°C with 2% glutaraldehyde in buffer containing 0.1 M sodium cacodylate at pH 7.2. Brains were then washed three times in cacodylate buffer for 10 min at 20–30°C. Afterwards, the brains were incubated with 1% Osmium tetroxide and 0.8% KFeCn (in 0.1 M cacodylate buffer) for 90 min on ice. Brains were then washed with cacodylate buffer for 10 min on ice and then three quick washes with distilled water. The brain were stained with 1% Uranylacetate (w/v) for 90 min on ice and dehydrated through a series of increasing alcohol concentrations. Samples were embedded in EPON resin by incubation sequentially in ethanol/EPON 1:1 solution for 45 min and 90 min at 20–30°C, then in pure EPON overnight at 15–20°C. Thereafter, the resin was changed once and brains were embed- ded in a single block at 60°C to allow for polymerization of the resin. 20 / 34 Optophysiological Imaging of GCaMP3.0, Homer-GCamp3.0 and SynaptopHlourin (SynpH) Female 3d or 30d flies were briefly anesthetized on ice and immobilized in a small chamber under thin sticky tape. A small window was cut through the sticky tape and the cuticle of the head capsule using a splint of a razor blade. Trachea were carefully removed and the brain was covered with Ringer’s solution (5 mM HEPES, 130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, pH = 7.3). Imaging was performed using an LSM 7 MP two-photon microscope (Carl Zeiss) equipped with a mode-lockedTi-sapphire Chameleon Vision II laser (Coherent), a 500– 550 nm bandpass filter, and a Plan-Apochromat 20×1.0 NA water-immersion objective (Carl Zeiss). A custom-built device to supply odorous air with a constant flow rate of 1 ml/s directly to the fly’s antennae was attached to the microscope. Odor stimulation (MCH or 3-Oct, diluted 1:100 or 1:150, respectively, in mineral oil or pure mineral oil) was controlled using a custom- written LABVIEW program (National instruments). GCamp3.0, homer-GCaMP, and SynpH were excited at 920 nm and fluorescence monitored at an image acquisition rate of 5 Hz. The odorants were presented with a 20 s break between stimulation, and each fly was exposed to five to six repetitive experiments. The images were aligned to reduce small shifts in the X–Y direction using a custom written ImageJ plugin. The mean intensity within the region of interest of five images before stimulus onset was used as baseline fluorescence (F0). The difference in intensity (ΔF) was calculated by subtracting F0 from the fluorescence intensity value within the ROI of each image (Fi) and, sub- sequently, divided by the baseline fluorescence. ΔF/F0 values of three or more repetitions were averaged for each fly. Odor-induced fluorescence changes of SynpH were considered in calycal PN boutons show- ing ΔF/F0 values more than twice the standard deviation of the baseline fluorescence. The bou- tons with the five highest odor-induced ΔF/F0 amplitudes were considered for further analysis. We found SynpH to exhibit rapid photo-bleaching, therefore, bleaching correction was per- formed on its ΔF/F0 values. For this, first, ΔF/F0 values from the onset of the stimulus until the decay of the signal were removed and then the best least square fit was obtained using the remaining ΔF/F0 values (second order polynomial decay function). Subsequently, this decay function was subtracted from the entire original ΔF/F0 curve, and the new modifieddata are the bleaching corrected data. Antibodies Used The following primary antibodies were used: MαBRPNc82 (ref. 9, 10; 1:100), GPαBRPN-term (1:800) [25,27], RbαRBPC-term (1:800) [29], MαSynapsin (1:20) [89], RatαSyb (1:100) [90], RbαSynaptotagmin-1C-term (1:500) [91], RbαGFP (Molecular Probes; 1:500), RbαDrep2C-term (1:500) [46], RbαUnc13C-term (1:500) [63], RbαBRPlast200 (1:500), and GPαRBPSH3II+III (1:500). The following secondary antibodies were used: GαM Alexa 488 (Molecular Probes; 1:400), GαR Alexa 488 (Molecular Probes; 1:500), GαGP Alexa 555 (Invitrogen; 1:800), GαM Cy3 (Dianova; 1:500), and GαR Cy5 (Invitrogen; 1:400). The following primary antibodies were used: MαBRPNc82 (ref. 9, 10; 1:100), GPαBRPN-term (1:800) [25,27], RbαRBPC-term (1:800) [29], MαSynapsin (1:20) [89], RatαSyb (1:100) [90], RbαSynaptotagmin-1C-term (1:500) [91], RbαGFP (Molecular Probes; 1:500), RbαDrep2C-term (1:500) [46], RbαUnc13C-term (1:500) [63], RbαBRPlast200 (1:500), and GPαRBPSH3II+III (1:500). The following secondary antibodies were used: GαM Alexa 488 (Molecular Probes; 1:400) The following secondary antibodies were used: GαM Alexa 488 (Molecular Probes; 1:400), GαR Alexa 488 (Molecular Probes; 1:500), GαGP Alexa 555 (Invitrogen; 1:800), GαM Cy3 (Dianova; 1:500), and GαR Cy5 (Invitrogen; 1:400). For Immunoprecipitation, BRPlast200 and IgG were used at final amount of 50 ug per 500 ul. For western blots, secondary antibody was used at a dilution 1:1,000. 21 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 D. Melanogaster Head Extract and Immunoprecipitation For the identification of (de)acetylated residues of BRP, we did “conventional” protein extrac- tions from Drosophila heads combined with BRP immunoprecipitations. The protocol could be divided into four main sections. 1) Precoupling of antibodies to matrix (50 ug antibody per reaction): 3 LoBind cups (2 ml; Eppendorf)containing Affiprep Protein A matrix were pre- pared: 1 X 30ul for specific antibody, 1 X 30ul for IgG control, 1 X 60 ul for head extract pre- clearing. The cups were washed 3 X with 500 ul H-buffer (25 mM HEPES pH 8.3 (NaOH), 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 10% Glycerol) by inverting several times, followed by centrifugation 1,000 gmax (3,000 rpm) for 1 min. 500 ul H-buffer (+ BRPlast200 or IgG) per cou- pling was prepared. 500 ul antibody solution (= 50 ug IgG) was added per 30 ul washed Protein A-beads. Beads were incubated with antibody solution for 2 h on the wheel at 4°C. The Affi- prep beads-antibody were collected by centrifugation for 3 min at 1,000 gmax. Affiprep beads- Antibody were washed 3 X by inverting tubes and 3 X for 10 min on wheel with IP buffer. 2) Homogenizing fly heads from stored fly heads [–80°C]. Fly heads were transferred with a clean spatula into 1 ml glass homogenizer. For 300 ul frozen fly heads, 300 ul Homogenization buffer (without detergent) was added, and heads were sheared at 900 rpm using an electronic over- head stirrer. Samples were collected in LoBind cups (2 ml; Eppendorf).2 X 300 ul was added to rinse pestle and homogenizer (Total volume in cups ~1,100–1,200 ul). Sodium-deoxycholic Acid (DOC) was added to a final concentration of 0.4% (28 ul of 10% stock spiked into homog- enate (1:25 v/v)). Triton X-100 was added to a final concentration of 1% (35 ul of 20% stock spiked into homogenate (1:20 v/v)). The samples (Homogenate) were incubated for 60 min at 4°C at level 8 (slow) on wheel. 20 ul of homogenate was stored for SDS-PAGE analysis for monitoring antigen during extraction/pull-downprocedure. Homogenate (H) was centrifuged for 15 min at 17,000 gmax. Supernatant (yellow in color) was transferred to a fresh LoBind cup. Centrifugation of S1 was repeated 4X to get rid of fat and remaining head debris. After final centrifugation step, remaining supernatant was diluted 1:1 with H-buffer (without detergent). Optophysiological Imaging of GCaMP3.0, Homer-GCamp3.0 and SynaptopHlourin (SynpH) Fluorescence emission of cytosolic GCamp was determined within specific boutons in the calyx that respond to the odor stimulus, and only the boutons showing ΔF/F0 values of more than 100% in four to five stimulations were averaged for each fly and considered for final anal- ysis. Fluorescence changes of mb247-Gal4; UAS-homer-GCamp flies were averaged over the five most responsive microglomerularstructures, as anatomically defined by basal fluorescence. False color-coded images were obtained by subtracting the image just before stimulus onset from the image at the maximum of the intensity difference (i.e., at 2 s after odor onset) and divided by the baseline fluorescence. The KCl experiments were performed using a fluorescence microscope (Zeiss) equipped with a xenon lamp (Lambda DG-4, Sutter Instrument), a 14-bit CCD camera (Coolsnap HQ, Photometrics) and a 20 × NA = 1 water-immersion objective. Images were acquired at 5 Hz using Metafluor (Visitron Systems). After recording some initial frames, KCl was added to the Ringer’s solution covering the fly brain (final concentration 0.05 M). Fluorescence changes were determined in a circular region covering the calyx (d = 20 μm), and background fluores- cence determined outside the calyx was subtracted. 22 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes D. Melanogaster Head Extract and Immunoprecipitation Total volume of Input was ~1,400 ul and of following composition: 25 mM Hepes pH 8.05 (NaOH), 150 mM NaCl, 0.5 mM MgCl2, 0.5 mM EGTA, 5% Glycerol, 0.2% DOC, 0,55% Tri- ton X-100. 3) Preclearing of fly head extract on Protein A-IgG beads: Diluted fly head extract was applied to preclearing beads and incubated for 60 min at 4°C while rotating on wheel. Pre- cleared extract was separated by centrifugation for 3 min at 1,000 gmax. Supernatant (IP input) was recovered. 4) Precipitation: Precleared extract (IP input) was applied to antibody-bead matrix (600 ul to specific Antibody-beads,600 ul to control IgGs) and antibody–antigen bind- ing was performed overnight at 4°C. Immunoprecipitates were collected by centrifugation at 1,000 gmax for 4 min at 4°C. Affiprep Beads-Antibody-Antigen were washed 3 X with a quick rinse followed by 2 X 20 min with 1 mL IP Buffer (H-buffer + 0.5% Triton-X 100 + 0.2% Na- DOC).Affiprep Beads-Antibody-Antigenwere resuspended in 1,000 ul IP buffer and trans- ferred to a clean LoBind cup (2 ml; Eppendorf).Affiprep Beads-Antibody-Antigenwere centri- fuged, and most of the supernatant was removed (without removing beads). 4.) Elution: For elution, 100 ul of 2X Laemmeli Buffer was added to Affiprep Beads-Antibody-Antigenand heated for 10 min at 95°C, 600 rpm, followed by centrifugation for 5 min at 1,000 gmax. Super- natant (IP eluate) was transferred into a fresh LoBind Cup (2 ml; Eppendorf).Immunoprecipi- tation was verified with western blot. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes separated on 4%–12% gradient SDS-PAGE (NuPAGE, Invitrogen). The gel lanes were cut into ten equal slices, the proteins were in-gel digested with trypsin (Promega) [92], and the resulting peptide mixtures were processed on STAGE tips [93] and analyzed by LC-MS/MS. Mass spectrometric(MS) measurements were performed on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to an Agilent 1200 nanoflow–HPLC (Agilent Technologies GmbH, Waldbronn, Germany) [94]. HPLC–column tips (fused silica) with 75 μm inner diameter (New Objective, Woburn, MA, USA) were self-packed with Reprosil– Pur 120 ODS–3 (Dr. Maisch, Ammerbuch, Germany) to a length of 20 cm. Samples were applied directly onto the column without a precolumn. A gradient of A (0.5% acetic acid (high purity, LGC Promochem, Wesel, Germany) in water and B (0.5% acetic acid in 80% acetonitrile (LC–MS grade, Wako, Germany) in water) with increasing organic proportion was used for peptide separation (loading of sample with 2% B; separation ramp: from 10%–30% B within 80 min). The flow rate was 250 nl/min and for sample application 500 nl/min. The mass spec- trometer was operated in the data-dependent mode and switched automatically between MS (maximum of 1 x 106 ions) and MS/MS. Each MS scan was followed by a maximum of five MS/MS scans in the linear ion trap using normalized collision energy of 35% and a target value of 5,000. Parent ions with a charge state from z = 1 and unassigned charge states were excluded for fragmentation. The mass range for MS was m/z = 370–2,000. The resolution was set to 60,000. MS parameters were as follows: spray voltage 2.3 kV; no sheath and auxiliary gas flow; ion transfer tube temperature 125°C. Identification of proteins and protein ratio assignment using MaxQuant. The MS raw data files were uploaded into the MaxQuant software version 1.4.1.2 [95] for peak detection, generation of peak lists of mass error corrected peptides, and for database searches. A full- length UniProt D. melanogaster database additionally containing common contaminants such as keratins and enzymes used for in-gel digestion (based on UniProt Drosophila FASTA ver- sion December 2013) was used as reference. Carbamidomethylcysteinewas set as fixed modifi- cation, methionine oxidation, protein amino-terminal acetylation, and lysine acetylation were set as variable modifications, and label-free was chosen as quantitation mode. Three missed cleavages were allowed, enzyme specificity was trypsin/P, and the MS/MS tolerance was set to 0.5 Da. The average mass precision of identified peptides was in general less than 1 ppm after recalibration. Peptide lists were further used by MaxQuant to identify and relatively quantify proteins using the following parameters: peptide and protein false discovery rates, based on a forward-reverse database, were set to 0.01, minimum peptide length was set to seven, mini- mum number of peptides for identification and quantitation of proteins was set to two, of which one must be unique, minimum ratio count was set to two, and identified proteins were requantified. The “match-between-run” option (2 min) was used. To analyze acetylation status of BRP, the data was processed using the freely available Per- seus software (Cox et al, 2011). For each IP, average acetylation intensity was calculated out of intensities of all sites identified in each replicate normalized to the respective protein intensity of BRP. Sample Preparation and Mass Spectrometry For identification of (de)acetylated lysine residues in BRP, IP eluate was heated in SDS-PAGE loading buffer, reduced with 1 mM DTT (Sigma-Aldrich) for 5 min at 95°C and alkylated using 5.5 mM iodoacetamide(Sigma-Aldrich) for 30 min at 20°C. The protein mixtures were 23 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. STM index plotted against shock number as experiencedduring training sessions with 120 V DC in 3d (light blue bars) and 30d (dark blue bars) wild-type w1118 flies (n = 6–8; Kruskal-Wal- lis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction).  p < 0.05,  p < 0.01,  p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. S2 Fig. Basal fluorescenceof different sensors (within the calyx neuropil) used for optoge- netic analysis. (a) Quantification of levels of GCamp3.0 in the PN terminals within the calyx region normalized to 3d flies (n = 6–7 independent calyces; Kruskal-Wallis test). (b) Quantifi- cation of levels of SynaptopHlourin (SynpH) in the PN terminals within the calyx region nor- malized to 3d flies (n = 7–12 independent calyces; Kruskal-Wallis test). (c) Quantification of levels of Homer-GCamp3.0 in the dendritic claws of KCs within the calyx region normalized to 3d flies (n = 10–12 independent calyces; Kruskal-Wallis test). ns = not significant, p  0.05. Underlying data is shown in S1 Data. (T ) S3 Fig. Ca2+-imaging in the PNs within the calyx region in response to odors in aged flies. (a) Expression of GCaMP3.0 in the PNs and imaged within the calyx neuropil. (b, c) False color-coded image of Ca2+ activity within the presynaptic terminals of PNs in response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, while cold colors low levels or no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). Scale bar: 10 μm. (d) Odor-evoked Ca2+ activity, measured by changes in fluorescence of Gamp3.0, of an individ- ual fly over time, shown as false colors in the presynaptic terminal of PNs in calyx region, in response to the odorants 3-Oct and MCH. (GCamp3.0 response averaged across three odor exposures from 6–7 animals). (e) Time course of Ca2+ activity induced by 3-Oct (averaged across three odor exposure) in the presynaptic terminals of PNs within calyx neuropil of 3d and 30d, together with 30dSpd flies (GCamp3.0 response averaged across three odor exposures from 6–7 animals). (f)Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to 3-Oct in PN boutons of 3d and 30d as well as 30dSpd flies (GCamp3.0 response averaged across three odor exposures from 6–7 animals; Kruskal-Wallis test). Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes order to avoid being biased by outliers, which are represented by solid circles. All p-values that are reported have been subject to Bonferroni correction for the number of comparisons. Addi- tional relevant information is indicated in the figure legends. The data for the behavioral stud- ies were collected with the investigator blind to the genotypes, treatment, and age of genotypes. There was no blinding in the other experiments. The data were collected and processed side by side in randomized order for all experiments. In order to analyze the difference in Homer- GCaMP3.0 responses (Fig 6 and S17 Fig), two-sided Kolmogorov-Smirnov tests were con- ducted in R, and the GCaMP3 responses only during odor stimulation and were compared. Supporting Information S1 Data. Excel spreadsheetcontaining, in separate sheets,numericaldata underlying panels 1e–1h, 2d, 2h, 2l–2n, 3e–3g, 4d, 4h, 4m–4n, 5e–5f, 5k, 5m–5n, 6e–6h, 7d–7e, 7j, S1, S2a– S2c, S3e–S3h, S4a–S4f, S5b–S5c, S6a–S6d, S7d, S8e–S8g, S9d, S10d–S10e, S11d–S11f, S14e–S14g, S16b–S16c, S17, S18d–S18f, S19e–S19f, S1 Table. (XLSX) PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Statistics Data were analyzed in R v3.1.2 using the additional CRAN package dunn.test v1.2.2. Asterisks are used in the figures to denote significance:  p < 0.05,  p < 0.01,  p < 0.001, ns = not sig- nificant. Nonparametric methods were used because of the small sample sizes and because of failure of tests for normality for parts of the data (Shapiro-Wilk test). Unless indicated other- wise, the different groups in each figure were first compared using the Kruskal-Wallis test, fol- lowed by Dunn’s test for posthoc multiple comparisons. Nonparametric tests were used in 24 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. (g) Time course of Ca2+ activity induced by MCH (averaged across three odor exposure) in the presynaptic terminals of PNs within calyx neuropil of 3d and 30d flies, together with 30dSpd flies (GCamp3.0 response PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 25 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes averaged across three odor exposures from 6–7 animals). (h) Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to MCH in PN boutons of 3d, 30d, and 30dSpd flies (GCamp3.0 response averaged across three odor exposures from 6–7 animals; Kruskal-Wallis test). The grey bars indicate the duration of the odor stimuli. ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S4 Fig. Decay time constant (τ) of odor-evoked SynpH response. (a) Overall decay time con- stant (τ) of SynpH signal in response to 3-Oct (3-Octonal). (b) Fast component of decay time constant (τ) of SynpH signal in response to 3-Oct. (c) Slow component of decay time constant (τ) of SynpH signal in response to 3-Oct. (d) Overall decay time constant (τ) of SynpH signal in response to MCH. (e) Fast component of decay time constant (τ) of SynpH signal in response to MCH. (f)Slow component of decay time constant (τ) of SynpH signal in response to MCH. (n = 6–7 flies; Kruskal-Wallis test). ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S5 Fig. KCl-induced changes in fluorescenceof SynpH within the calyx neuropil. (a) SynpH expressed in the PNs and imaged within the calyx region. The two rings indicate the region of interest (calyx neuropil) and background region used for analysis. Scale bar: 50 μm. (b) KCl- induced release of SVs, measured by changes in fluorescence (ΔF/F in %) of SynpH of a single fly over time. (c) Maximum change in fluorescence (ΔF/F in %) of SynpH response to KCl in 3d, 30d, and 30dSpd flies (n = 5–6 flies; Kruskal-Wallis test). ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S6 Fig. Imaging of SynpH at KC-to-MBON synapses to measure odor-evoked SV release. (a) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of KCs within the horizontal lobe of mushroom body of 3d, 30d, and 30dSpd animals (SynpH response aver- aged across three odor exposures from 6–7 flies). S8 Fig. BRP and RBP increase progressively with age. (a–d) Adult brains of 3d, 10d, 20d, and 30d w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. Analysis of the endogenous expression of BRPGFP in adult brains. (a–c) Adult brains of 3d- and 30d-BRP(83-ex13)GFP flies, and 30dSpd- BRP(83-ex13)GFP flies (BRPGFP). Brains were fixed in 5% PFA and scanned for GFP signal. Scale bar: 50 μm. (d) Quantification of GFP signal within the central brain region normalized to 3d flies (n = 9–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction).  p < 0.05,  p < 0.01,  p < 0.001. Underlying data is shown in S1 Data. (TIF) S10 Fig. Analysis of Ca2+ channel and BRP in wild-typebrains. (a–c) Mushroom body calyx of 3d, 30d, and 30dSpd flies expressing GFP-labeled genomic construct of α1 subunit Cacoph- ony (CacGFP) and immunostained for GFP as well as BRP (corresponding single z-planes are shown). Scale bar: 10 μm. (d,e) Quantification of signal intensity of CacGFP (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to 3d flies (n = 7–9 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction).  p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S11 Fig. Analysis of for Unc13 and BRP in wild-typebrains. (a–c) Adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies, immunostained for BRP (using Nc82 and N- terminal antibody) and Unc13. Scale bar: 50 μm. (d–f)Quantification of signal intensity of the proteins in the central brain region normalized to 3d flies (n = 10–15 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction).  p < 0.05,  p < 0.01,  p < 0.001. Underlying data is shown in S1 Data. (TIF) S12 Fig. EM of PN-to-KC synapses.Electron micrographs revealed that the alignment of the plasma membrane, with evident increase in extracellular spacing between cellular elements, to be affected in 30d w1118 flies, when compared to 3d or 30dSpd w1118 flies. Scale bar: 500 nm. The arrowheads point to the alignment of the plasma membrane between subcellular entities. (TIF) S13 Fig. STED analysis of BRP ring diameter at PN-to-KC synapses.Examples of confocal and STED images of BRP spots within the calyx region of 3d and 30d w1118 flies, together with 30dSpd w1118 flies. Scale bar: 500 nm. S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. (b) Maximum change in SynpH fluorescence (ΔF/F in %) in response to 3-Oct within the presynaptic terminals of KCs of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal- Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correc- tion). (c) Time course of SynpH activity induced by MCH in the presynaptic terminals KCs within the horizontal lobe of mushroom body of 3d, 30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies) (d) Maximum change in SynpH fluores- cence (ΔF/F in %) in response to MCH within the presynaptic terminals of KCs of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal- Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correc- tion).  p < 0.05,  p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. ( ) S7 Fig. Analysis of Syb in wild-typebrains. (a–c) Adult brains of 3d, 30d and 30dSpd w1118 flies immunostained for Syb. Scale bar: 50 μm. (d) Quantification of Syb intensity within the central brain region normalized to 3d flies (n = 6–9 independent brains; Kruskal-Wallis test). ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S7 Fig. Analysis of Syb in wild-typebrains. (a–c) Adult brains of 3d, 30d and 30dSpd w1118 flies immunostained for Syb. Scale bar: 50 μm. (d) Quantification of Syb intensity within the central brain region normalized to 3d flies (n = 6–9 independent brains; Kruskal-Wallis test). ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S8 Fig. BRP and RBP increase progressively with age. (a–d) Adult brains of 3d, 10d, 20d, and 30d w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. 26 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Scale bar: 50 μm (e–g) Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d flies (n = 10–12 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction).  p < 0.01,  p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. S9 Fig. PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes type (2xBRP) flies (n = 7–12; Kruskal-Wallis test with Dunn’s multiple comparison test, p-val- ues were subject to Bonferroni correction).  p < 0.05,  p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S15 Fig. Acetylation and mass spectroscopy. (a) BRP sequence with acetylated peptide frag- ments (yellow) and lysine sites positive for acetylation (red) identified through mass spectros- copy. (b) Position of possible lysine residues that undergo (de)acetylation within BRP. (TIF) type (2xBRP) flies (n = 7–12; Kruskal-Wallis test with Dunn’s multiple comparison test, p-val- ues were subject to Bonferroni correction).  p < 0.05,  p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) type (2xBRP) flies (n = 7–12; Kruskal-Wallis test with Dunn’s multiple comparison test, p-val- ues were subject to Bonferroni correction).  p < 0.05,  p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S15 Fig. Acetylation and mass spectroscopy. (a) BRP sequence with acetylated peptide frag- ments (yellow) and lysine sites positive for acetylation (red) identified through mass spectros- copy. (b) Position of possible lysine residues that undergo (de)acetylation within BRP. (TIF) S15 Fig. Acetylation and mass spectroscopy. (a) BRP sequence with acetylated peptide frag- ments (yellow) and lysine sites positive for acetylation (red) identified through mass spectros- copy. (b) Position of possible lysine residues that undergo (de)acetylation within BRP. (TIF) S16 Fig. KCl-inducedchanges in fluorescenceof Homer GCamp3.0 within the calyx neuropil. (a) Homer GCamp3.0 expressed in the dendritic claws of KCs and imaged within the calyx region. The two rings indicate the region of interest (calyx neuropil) and background region used for analy- sis. Scalebar:50 μm. (b) KCl-induced influx of postsynaptic Ca2+ ion, measured by changes in fluo- rescence (ΔF/F in %) of Homer GCamp3.0 of a single fly over time. (c) Maximum change in fluorescence(ΔF/F in %) of Homer GCamp3.0 response to KCl in 3d, 30d, and 30dSpd flies (n = 8–9 flies;Kruskal-Wallis test). ns = not significant,p  0.05. Underlying data is shown in S1 Data. (TIF) S17 Fig. Empirical cumulative distribution functions for postsynaptic Homer-GCaMP3.0 response during odor stimulation. Empirical cumulative distribution functions for 3-Oct and MCH, as used in the Kolmogorov-Smirnov test. Only the GCaMP3 response during odor pre- sentation (seconds 1–3, grey bars in Fig 3K and 3M) was used. Two-sided Kolmogorov-Smir- nov tests were conducted for the analysis of difference. The differences for 3-Oct were not significant. The differences for MCH between 3d and 30d (), as well as between 30d and 30dSpd () were significant after Bonferroni correction for three groups.  p < 0.05,  p < 0.01. Underlying data is shown in S1 Data. (TIF) S18 Fig. Calyx neuropil from wild-typebrains immunostained for Drep2 and BRP. (a–c) Mushroom body calyx from adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Drep2 and BRP (using Nc82 as well as N-terminal antibodies) (corre- sponding single z-planes are shown). Scale bar: 10 μm. (d–f)Quantification of signal intensity of these proteins in the calyx region normalized to 3d flies (n = 10 independent brains; Krus- kal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni cor- rection).  p < 0.01,  p < 0.001, ns = not significant, p  0.05. Underlying data is shown in S1 Data. S19 Fig. Autophagy required for spermidine-mediatedsuppression of age-associated increase in BRP levels. (a–d) Comparison of BRP signal intensity (using Nc82 and N-terminal antibodies) in brains of 3d w1118 control animals, 3d and 20-d old (20d) atg7-/- flies, raised either on normal or spermidine-supplemented food. Scale bar: 50 μm. (e–f)Quantification of BRP within the central brain region of young (3d) and old (20d) atg7-/- mutants normalized to 3d w1118 flies (n = 9–12 independent brains; Kruskal-Wallis test with Dunn’s multiple compari- son test, p-values were subject to Bonferroni correction).  p < 0.01, ns = not significant, p  0.05. Underlying data is shown in S1 Data. (TIF) S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. These calyces were also stained for Drep2, a protein found highly enriched in dendritic claws of KCs, allowing the quantification of the diameter of BRP spots that mark the synapse between PNs and KCs. (TIF) S14 Fig. Effect of removing one-copy of BRP on memory formation. (a–d) Adult brains of 3d and 30d brp69/+ (1xBRP) flies together with age-matched controls (2xBRP), immunos- tained for BRP (using Nc82 and N-terminal antibody), and RBP. Scale bar: 50 μm. (e, f)Quan- tification of BRP (using N-terminal antibody) and RBP intensity within the central brain region normalized to 3d flies (9–10 independent brains; Kruskal-Wallis test with Dunn’s multi- ple comparison test, p-values were subject to Bonferroni correction). (g) Aversive associative memory performance 3 min after training (STM) of brp69/+ (1xBRP) flies compared to wild- PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 27 / 34 S1 Table. Aversive odor avoidance and shock reactivity in different genotypes. Underlying data is shown in S1 Data. (TIF) 28 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563 September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Writing – original draft: VKG FM SJS. 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STAT5BN642H is a driver mutation for T cell neoplasia
˜The œJournal of clinical investigation/˜The œjournal of clinical investigation
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Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 R E S E A R C H A R T I C L E Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: April 12, 2017; Accepted: October 5, 2017. License: This work is licensed under the Creative Commons Attribution 4.0 Inter- national License. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Reference information: J Clin Invest. 2018;128(1):387–401. https://doi.org/10.1172/JCI94509. Introduction due to differences in the level of STAT5 proteins and possible dis- tinct chromatin-remodeling capabilities as a result of interactions with other transcriptional regulatory proteins (10–12). Recently, the hSTAT5BN642H mutation was described as a gain-of-function (GOF) mutation in leukemic patients that causes enhanced and prolonged tyrosine phosphorylation (13–27). This mutation is associated with a poor prognosis and an increased risk of relapse, despite the use of combination chemotherapy (13). The N642H mutation is located in the center of the STAT5B SH2 domain, the phosphotyrosine-binding domain that is essential for the forma- tion of parallel STAT5 dimers and efficient nuclear transloca- tion (1). STAT5BN642H has been found in more than 90 patients with 7 types of aggressive leukemia or lymphoma including γδ T cell–derived lymphoma, hepatosplenic T cell lymphomas, large granular lymphocytic (LGL) leukemia, T cell acute lymphoblas- tic leukemia (T-ALL), T cell prolymphocytic leukemia, NK/T cell lymphoma, and enteropathy-associated T cell lymphoma (13–25). The 2 signal transducer and activator of transcription 5 proteins, STAT5A and STAT5B, encoded by 2 different genes with 89% DNA sequence homology, are downstream targets of cytokines and growth factors (1, 2). STATs are highly expressed and/or hyperactivated by tyrosine and serine phosphorylation in numer- ous hematopoietic cancers (3–6). The 2 STAT5 proteins have been reported to have redundant functions largely due to overlapping genome binding sites (7–9). There are different phenotypes upon genetic loss or somatic point mutation resulting in hyperactivation of STAT5A or STAT5B. STAT5B has a dominant role in immune cells, as suggested by its higher expression levels compared with STAT5A in NK or T cell subsets (7–9). Interestingly, mutations in cancer patients have predominantly been found in the Src homology 2 (SH2) domain of human STAT5B (hSTAT5B). This suggests that differences in hematopoietic transformation are Studies in mice have implicated STAT5 signaling in the expan- sion of CD8+ T cells as well as the development of lymphoblastic lymphoma (28). Nevertheless, there is no evidence of whether the hSTAT5BN642H mutation is capable of driving the development and progression of leukemia. Drug-sensitivity tests on hSTAT5BN642H- expressing leukemic blasts from T-ALL patients indicated that the mutation provides a survival advantage in leukemic cells (17). STAT5BN642H is a driver mutation for T cell neoplasia Ha Thi Thanh Pham,1,2 Barbara Maurer,1,2 Michaela Prchal-Murphy,3 Reinhard Grausenburger,3 Eva Grundschober,3 Tahereh Javaheri,1,2 Harini Nivarthi,4 Auke Boersma,5 Thomas Kolbe,6,7 Mohamed Elabd,1 Florian Halbritter,4 Jan Pencik,1 Zahra Kazemi,8,9 Florian Grebien,1 Markus Hengstschläger,10 Lukas Kenner,1,11,12 Stefan Kubicek,4 Matthias Farlik,4 Christoph Bock,4,8,13 Peter Valent,14,15 Mathias Müller,2 Thomas Rülicke,5 Veronika Sexl,3 and Richard Moriggl1,2,8 1Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria. 2Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria. 3Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria. 4CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria. 5Institute of Laboratory Animal Science, and 6Biomodels Austria (Biat), University of Veterinary Medicine Vienna, Vienna, Austria. 7IFA-Tulln, University of Natural Resources and Life Sciences, Tulln, Austria. 8Medical University of Vienna, Vienna, Austria. 9Center of Physiology and Pharmacology, Vienna, Austria. 10Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria. 11Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria. 12Unit of Pathology of Laboratory Animals, University of Veterinary Medicine Vienna, Vienna, Austria. 13Max Planck Institute for Informatics, Saarbrücken, Germany. 14Department of Internal Medicine I, Division of Hematology and Hemostaseology, and 15Ludwig Boltzmann-Cluster Oncology, Medical University of Vienna, Vienna, Austria. STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B- expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy. jci.org      Volume 128      Number 1      January 2018 Results (TET2) and DNA methyltransferase 3α (DNMT3A) affecting DNA methylation are frequently found in T cell lymphoma (29). TET1/2 was also shown to interact with STAT5 in Tregs that are strictly dependent on STAT5 because of direct regulation of the STAT5 target genes FOXP3 and CD25 (30). Interestingly, the DNMT3A gene was shown to be controlled by STAT5 in AML cells (31). Drugs interfering with epigenetic changes are powerful tools in cancer drug development and have found entry into therapeu- tic strategies (29). A key role of STAT5 is to support the process of histone acetylation and methylation in T cells, which was shown for the TCR locus (32, 33). Furthermore, the histone methyltrans- ferase EZH2 and histone deacetylase 1 (HDAC1) were shown to be recruited via STAT5 binding (34, 35). hSTAT5BN642H is an activating mutation in hematopoietic cells. Somatic mutations of STAT5B, many of which are located in the SH2 domain, have been found in patients with lymphoid neoplasia (Figure 1A) (13–26, 36). To investigate the impact of these somatic mutations on hSTAT5B activity, we analyzed different potential GOF mutations in 293T cells using tyrosine phosphorylation of STAT5 (pY-STAT5) as a correlation for activity. We observed high pY-STAT5 levels under steady-state conditions in cells expressing the N642H muta- tion, the most frequent STAT5 mutation in patients with leukemia or lymphoma. The 2 SH2 domain variants Y665H and Y665F also showed enhanced activity in the absence of cytokine stimulation (Figure 1B). We observed a similar pattern of pY-STAT5B upon expression of the N642H mutant in the murine pro–B cell line Ba/ F3 and the murine myeloid cell line 32D (Figure 1C). In contrast, expression of hSTAT5B at comparable levels failed to induce signif- icant pY-STAT5 in the absence of IL-3 stimulation (Figure 1C and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI94509DS1). Impor- tantly, hSTAT5BN642H rendered Ba/F3 and 32D cells cytokine inde- pendent, supporting the finding of a proto-oncogenic potential of hSTAT5BN642H (15) (Supplemental Figure 1B). Here, we investigated the oncogenic potential of the hSTAT5BN642H mutation compared with the nonmutated hSTAT5B using Vav1-driven transgenic mouse models. In contrast to WT hSTAT5B, moderate hSTAT5BN642H expression levels triggered leu- kemia or lymphoma development, which manifested as a transplant- able CD8+ T cell disease. Related Commentary: p. 113 Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: April 12, 2017; Accepted: October 5, 2017. License: This work is licensed under the Creative Commons Attribution 4.0 Inter- national License. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Reference information: J Clin Invest. 2018;128(1):387–401. https://doi.org/10.1172/JCI94509. Epigenetic abnormalities are major drivers of hematopoiet- ic malignancies. Mutations in Tet methylcytosine dioxygenase 2 jci.org      Volume 128      Number 1      January 2018 3 8 7 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 1. hSTAT5BN642H is an activating mutation. (A) Schematic of STAT5B mutations identified in leukemia and lymphoma patients. Each dot represents 1 patient. (B) WB analysis of pY-STAT5, total STAT5 protein, and HSC70 in 293T cells that were transiently transfected with different hSTAT5B (hS5B) variants using a pMSCV-IRES-GFP vector, with or without growth hormone (GH) stimulation. (C) WB analysis of pY-STAT5, STAT5, FLAG, and HSC70 in hSTAT5B- or hSTAT5BN642H-expressing (N642H) Ba/F3 cells with or without IL-3 stimulation. (B and C) Nontransfected and pMSCV-transfected cells are shown as controls. Data presented in B and C are representative of 3 independent experiments. Samples were run on parallel gels for B and C, and a load- ing control is provided for each gel. Figure 1. hSTAT5BN642H is an activating mutation. (A) Schematic of STAT5B mutations identified in leukemia and lymphoma patients. Each dot represents 1 patient. (B) WB analysis of pY-STAT5, total STAT5 protein, and HSC70 in 293T cells that were transiently transfected with different hSTAT5B (hS5B) variants using a pMSCV-IRES-GFP vector, with or without growth hormone (GH) stimulation. (C) WB analysis of pY-STAT5, STAT5, FLAG, and HSC70 in hSTAT5B- or hSTAT5BN642H-expressing (N642H) Ba/F3 cells with or without IL-3 stimulation. (B and C) Nontransfected and pMSCV-transfected cells are shown as controls. Data presented in B and C are representative of 3 independent experiments. Samples were run on parallel gels for B and C, and a load- ing control is provided for each gel. Results (A) Survival curve shows Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs (CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs (CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Analysis of WBC counts in hSTAT5BN642H mice revealed an increase of approximately 20-fold compared with that detected in hSTAT5B and WT mice (Figure 3C). The WBC count in hSTAT5B mice only increased slightly with age but remained within a phys- iological range (Supplemental Figure 3B). The drastic increase in the WBC count in STAT5BN642H mice was correlated with an expan- sion of CD8+ T cells (Figure 3C). Results Transcriptome and DNA methylome anal- yses illustrated profound changes in gene expression and reduced DNA methylation of potential EZH2-binding sites, with Aurora kinas- es being one of the most altered genes in hSTAT5BN642H-transgenic animals. In line with this, we found that Aurora kinase and JAK inhib- itors were effective in blocking neoplastic T cell expansion and organ infiltration driven by hSTAT5BN642H. This suggested that inhibitors of Aurora kinases and JAK have potential as a treatment for patients suf- fering from hSTAT5N642H-driven lymphoma or leukemia. Vav1-driven expression of hSTAT5BN642H induces cancer. Given that hSTAT5BN642H was found in different hematopoietic cancers, we expressed hSTAT5B or hSTAT5BN642H in mice during early hematopoiesis using the Vav1 oncogene promoter. This led to transgene expression primarily in cells of the hematopoietic sys- 3 8 8 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E The Journal of Clinical Investigation      R E S E A R C H A R T I C L E Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs (CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. Results (D) CD8/CD4 T cell ratios in LNs were determined using flow cytometry. Analyses included 7-week-old WT (n = 5), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. (E) Quantification of the absolute number of CD4+ and CD8+ T cells, myeloid cells (CD11b+Gr1+), and B cells (CD19+) in spleens from hSTAT5BN642H- and hSTAT5B-transgenic mice and WT mice. Analyses included 7-week-old WT (n = 13), hSTAT5B (n = 6), and hSTAT5BN642H (n = 6 and 11) mice. (F) CD3+CD8+ splenic cells were analyzed by flow cytometry for their expression of CD25. Analyses included 8-week-old WT (n = 8), hSTAT5B (n = 9), and (n = 6) hSTAT5BN642H mice. (G) CD3+CD8+ splenic cells were further analyzed for CD62L and CD44 expression. Analyses included WT (n = 8), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice at 8 weeks of age. Data represent the mean ± SD. n ≥ 6. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. cells expressed surface markers indicative of mature T cells with an activated phenotype and high expression of IL-2Rα (CD25), a direct target gene of STAT5 (41) (Figure 3F). Fifty percent of the diseased CD8+ T cells also expressed markers reminiscent of cen- tral memory T cells. Moreover, we found that the percentage of cells expressing markers for effector memory T cells was elevated in the diseased mice compared with that observed in WT controls (Figure 3G). High numbers of proliferating T cells were associated with splenomegaly and lymphoma formation, and proliferating T cells were found to heavily infiltrate peripheral organs, leading to fatal pulmonary obstruction (Figure 4 and Supplemental Figure 4). tion of CD8+ T cells (Figure 5, C and D). Of note, the i.v. injection of CD8+ T cells from diseased mice into nonirradiated Ly5.1+CD45.1+ recipient mice was sufficient to phenotypically recapitulate the pri- mary disease, identifying the CD8+ T cells as the malignant cell pool (Figure 5, E–G, and Supplemental Figure 5, C and D). JAK inhibitors suppress disease progression in the hSTAT5BN642H- driven disease model. A number of treatment regimens have been suggested for leukemia and lymphoma patients carrying the hSTAT5BN642H mutation. However, there is limited knowledge about the effectiveness of these treatments, partially because of the lack of a suitable preclinical model (17, 18). Results https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hSTAT5BN642H and hSTAT5B mouse spleens and LNs with those from WT mice. Scale bars: 1 cm. (B) Modified Wright staining of blood smears from hSTAT5BN642H (N642H), hSTAT5B (hS5B), and WT mice (original magnification, ×100). (C) WBC count using an animal blood counter (scil Vet ABC). CD8/CD4 ratios in the peripheral blood were determined using flow cytometry. Analysis included 7- to 10-week-old WT (n = 20), hSTAT5B (n = 15), and hSTAT5BN642H (n = 20) mice. (D) CD8/CD4 T cell ratios in LNs were determined using flow cytometry. Analyses included 7-week-old WT (n = 5), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. (E) Quantification of the absolute number of CD4+ and CD8+ T cells, myeloid cells (CD11b+Gr1+), and B cells (CD19+) in spleens from hSTAT5BN642H- and hSTAT5B-transgenic mice and WT mice. Analyses included 7-week-old WT (n = 13), hSTAT5B (n = 6), and hSTAT5BN642H (n = 6 and 11) mice. (F) CD3+CD8+ splenic cells were analyzed by flow cytometry for their expression of CD25. Analyses included 8-week-old WT (n = 8), hSTAT5B (n = 9), and (n = 6) hSTAT5BN642H mice. (G) CD3+CD8+ splenic cells were further analyzed for CD62L and CD44 expression. Analyses included WT (n = 8), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice at 8 weeks of age. Data represent the mean ± SD. n ≥ 6. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hS Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hSTAT5BN642H and hSTAT5B mouse spleens and LNs with those from WT mice. Scale bars: 1 cm. (B) Modified Wright staining of blood smears from hSTAT5BN642H (N642H), hSTAT5B (hS5B), and WT mice (original magnification, ×100). (C) WBC count using an animal blood counter (scil Vet ABC). CD8/CD4 ratios in the peripheral blood were determined using flow cytometry. Analysis included 7- to 10-week-old WT (n = 20), hSTAT5B (n = 15), and hSTAT5BN642H (n = 20) mice. jci.org      Volume 128      Number 1      January 2018 Results Similarly, CD8+ T cells increased by 3-fold in the lymph nodes (LNs) of hSTAT5BN642H mice (Fig- ure 3D), which was confirmed by immunohistochemical staining (Supplemental Figure 3C). The numbers of CD4+ T cells were also moderately increased, whereas the percentage, but not the total number, of CD19+ B cells was reduced in the LNs of hSTAT5BN642H mice compared with controls (Figure 3E and Supplemental Fig- ure 3D). Hematocrit levels were comparable in all mouse models (Supplemental Figure 3E). We also observed a mild expansion of other hematopoietic cell types such as CD19+ B cells, CD4+ T cells, and CD11b+Gr1+ myeloid cells in the spleen (Figure 3E and Supple- mental Figure 3F). tem, including hematopoietic stem cells (HSCs) (37) (Supplemen- tal Figure 2, A and B). Transgenic mice expressing hSTAT5BN642H rapidly developed malignant disease leading to death between 40 and 100 days of age. hSTAT5B-transgenic mice showed no signs of disease when sacrificed at the age of 12 months or older (Figure 2A). Despite expressing comparable levels of total STAT5, only hSTAT5BN642H-transgenic mice showed elevated pY-STAT5 signals, indicating strong and persistent tyrosine phosphorylation (Figure 2B). In line with this observation, Vav1-driven expression of hSTAT5BN642H, but not hSTAT5B, led to increased numbers of progenitor cells throughout all early hematopoietic compart- ments, including lineage–Sca1+c-Kit+ cells (LSKs), long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progen- itors (MPPs) (CD150+CD48–, CD150+CD48+, CD150–CD48+ frac- tions) (Figure 2C). Likewise, the numbers of common lymphoid progenitors (CLPs) and myeloid progenitor cells (MPCs) were sig- nificantly elevated in mice expressing hSTAT5BN642H (Figure 2D). hSTAT5BN642H mice had 3 times more CLPs than did WT mice, which translated into expansion of CD3+ T cells, but not CD19+, B cells in their BM (Figure 2E and Supplemental Figure 2C). The elevated number of MPCs was also accompanied by a signifi- cant increase in the percentage of CD11b+Gr1+ cells in the BM of hSTAT5BN642H mice (Supplemental Figure 2C). We used flow cytometry to analyze markers for T cell acti- vation (CD25) and surface markers for T cell subpopulations, including naive CD8+ T cells (CD62LhiCD44lo), central memory CD8+ T cells (CD62LhiCD44hi), and effector memory CD8+ T cells (CD62LloCD44hi) (38–40). This analysis showed that the leukemic jci.org      Volume 128      Number 1      January 2018 3 8 9 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). lation of 371 genes in T cells derived from hSTAT5BN642H compared with that observed in WT T cells (FDR-adjusted P < 0.05) (Sup- plemental Figure 7A). As expected, known STAT5 targets such as Ccl5, Ccr5, Pim1, Bcl2, and Il2r were among the top upregulated genes, confirming hSTAT5BN642H transgene specificity (Supple- mental Figure 7, B and C and Supplemental Tables 2 and 3) (7–9). Importantly, gene set enrichment analysis (GSEA) confirmed that genes upregulated in CD8+ T cell lymphoma patients were high- ly enriched, which emphasized the validity of our model (Figure 7B) (43, 44). Additional pathway analysis showed that E2F targets, the G2M checkpoint, and MYC targets were the most upregulated pathways, underlining the high proliferation rate of leukemic cells and indicating hSTAT5BN642H as a driver for cell-cycle progres- sion (Figure 7C and Supplemental Figure 7, D and E). In contrast, inflammatory gene pathways or developmental core cancer path- ways were significantly downregulated (P < 0.05), as analyzed by pathway analysis (Supplemental Figure 7E). whether cells carrying the hSTAT5BN642H mutation are sensitive to JAK inhibition. As expected, the FDA-approved JAK inhibitors rux- olitinib and tofacitinib reduced the activation of STAT5 and cell viability, with an IC50 of 0.11 μM (ruxolitinib) and 0.12 μM (tofaci- tinib) and comparable IC50 values for all genotypes (Figure 6, B and C, and Supplemental Figure 6A). R E S E A R C H A R T I C L E Moreover, other FDA-approved drugs such as HDAC inhibitors for the treatment of T cell lympho- ma were tested (42). Entinostat and several other drugs were also found to be effective in inducing apoptosis in T cells, with an IC50 in the nanomolar range, but did not exert differential effects between hSTAT5B- and hSTAT5N642H-expressing cells (Supplemental Figure 6A and Supplemental Table 1). Following the in vitro data, we investigated the effect of rux- olitinib in vivo by treating hSTAT5BN642H CD8+ T cell recipient Ly5.1+CD45.1 mice, 60 days after transplantation, with ruxolitinib (45 mg/kg) for a period of 30 days. The treatment significantly reduced the size of LNs and spleens (Figure 6, D and E). The WBC count as well as CD25 expression on donor hSTAT5BN642H CD8+ T cells were also reduced upon ruxolitinib treatment (Figure 6F and Supplemental Figure 6B). Furthermore, ruxolitinib decreased the degree of T cell infiltration into the lungs, skin, BM, LNs, and spleens of treated mice, leading to a substantial reduction in dis- ease burden (Figure 6G and Supplemental Figure 6C). The treat- ment did not significantly affect the myeloid cell population in the hematopoietic organs (Supplemental Figure 6D). Besides its function as a transcription factor, STAT5 can shape chromatin by interacting with other chromatin-remodeling enzymes such as EZH2 (35, 45). As changes in DNA methylation patterns have recently been associated with malignant disease and particularly with leukemia (46, 47), we questioned wheth- er the dramatic changes observed in the expression profiles of hSTAT5BN642H CD8+ T cells would also be reflected by alterations in the DNA methylome. Using reduced representation bisul- fite sequencing (RRBS), we found that overall DNA methylation across CpG islands (CGIs) among hSTAT5BN642H and WT T cells was highly consistent (Pearson’s r = 0.98), with only 1,380 CGIs being substantially different (absolute difference ≥5 percentage points) (Figure 7D) (48). When comparing WT and hSTAT5B CD8+ T cells, we found weaker differences and overlaps (Supplemental Figure 8A). Combining DNA methylation analysis with mRNA expression data, we identified a small set of genes with substan- tial and concordant changes in DNA methylation and expression of genes within the proximity of differentially methylated CGIs (Supplemental Figure 8, B and C). Interestingly, the genes with higher expression in hSTAT5BN642H T cells and concordant loss hSTAT5BN642H CD8+ T cells exhibit substantial changes in gene expression profile, accompanied by specific changes in DNA methyla- tion. Results Typically, STAT5 is activated in response to cytokine signaling, and cells harboring the hSTAT5BN642H mutant show prolonged pY-STAT5 levels upon stimulation rather than being constitutively active (18). When we analyzed the level of pY-STAT5 in primary T cells derived from the LNs of WT and hSTAT5B- and hSTAT5BN642H-transgenic mice, we detected dras- tically reduced levels of pY-STAT5 one hour after IL-2 deprivation in WT and hSTAT5B-expressing T cells. In contrast, low levels of pY-STAT5 remained detectable up to 4 hours after IL-2 removal in hSTAT5BN642H-expressing T cells (Figure 6A). The finding that cytokines efficiently activated hSTAT5BN642H prompted us to test To test whether the T cell disease in hSTAT5BN642H-transgenic mice was transplantable, we transferred BM cells from mutant or WT control mice i.v. into nonirradiated, immunocompromised NSG recipient mice. The recipients of mutant cells became termi- nally sick approximately 3 months after injection (Figure 5A). Bone marrow transplantations (BMTs) induced disease, with a phenotype comparable to that of hSTAT5BN642H-transgenic mice. The disease was characterized by enlarged spleens and lymphoma formation, with T cell infiltration into peripheral organs (Figure 5B and Supple- mental Figure 5, A and B) caused by excessive expansion and infiltra- 3 9 0 jci.org      Volume 128      Number 1      January 2018 The Journal of Clinical Investigation      R E S E A R C H A R T I C L E Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E (A) Percentage of disease-free survival following hSTAT5BN642H whole BMT into 8-week- old NSG recipient mice compared with WT BMT. (B) Macroscopic view of LNs and spleen from a hSTAT5BN642H BMT recipient mouse compared with those from a WT BMT recipient mouse. Scale bar: 1 cm. (C) Flow cytometric analysis shows the quantity of CD3+ cells and CD8/CD4 T cell ratio in the spleens of BMT recipient mice. (D) Histological analysis of CD3+ cells from the lungs of NSG recipient mice after hSTAT5BN642H or WT BMT. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). (E) Percentage of disease-free survival after hSTAT5BN642H or WT CD8+ T cell transplantation into nonirradiated 8-week-old Ly5.1/CD45.1 recipient mice. (F) Flow cytometric analysis shows the quantity of splenic CD3+CD8+ cells in CD8+ T cell–transplanted mice. (G) Spleen versus BW ratios of WT and hSTAT5BN642H CD8+ T cell–transplanted Ly5.1/CD45.1 mice. (A–C) n = 4 WT mice and n = 5 hSTAT5BN642H mice; (E–G) n = 6 WT mice and n = 5 hSTAT5BN642H mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test. pared with that detected in WT murine STAT5B and hSTAT5B. The mutated STAT5 increased its binding to the Cis promoter and was also found at the promoter regions of the EZH2 known targets Cdkn2a and Ccnd2. In addition, it bound to the less methylated CGI in association with Aurkb (Figure 8C). Although EZH2 bind- ing was found to be reduced in hSTAT5BN642H CD8+ T cells, EZH2 retained its binding at the same CGI (Figure 8D and Supplemental Figure 9C). However, hSTAT5BN642H was not shown to have direct interactions with EZH2 (Figure 8E). of DNA methylation at nearby CGIs included the mitotic check- point protein KNTC1 (49) and the oncogene topoisomerase type IIα (TOP2A) (36, 50), which is known to regulate DNA topologi- cal structure and cell-cycle progression (Supplemental Figure 8B, right, sector II). None of these genes was substantially affected in hSTAT5B CD8+ T cells (Supplemental Figure 8B). Specific DNA methylation changes in hSTAT5BN642H reveal targets for therapy. Location overlap analysis (LOLA) (51) of regions that lost methylation in T cells expressing hSTAT5BN642H compared with WT cells revealed significant enrichment for sites known to bind EZH2 and/or SUZ12 proteins. R E S E A R C H A R T I C L E Given the leukemogenic effect of hSTAT5BN642H, which is not shared by WT hSTAT5B, we next investigated alterations in gene expression and epigenetic modifications in T cells derived from both mouse models. CD8+ T cells were isolated from the LNs of 13-week-old WT and hSTAT5B and hSTAT5BN642H diseased mice, and mRNA sequencing analysis was performed. While the glob- al expression patterns of WT and hSTAT5B CD8+ T cells were comparable, the gene expression signature of cells expressing hSTAT5BN642H showed a distinct pattern (Figure 7A). We found a significant upregulation of 564 genes and a significant downregu- 3 9 1 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation   S E A R C H A R T I C L E wnloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 5. hSTAT5BN642H CD8+ T cells are the cancer-initiating cells. (A) Percentage of disease-free survival following hSTAT5BN642H whole BMT into 8-week- old NSG recipient mice compared with WT BMT. (B) Macroscopic view of LNs and spleen from a hSTAT5BN642H BMT recipient mouse compared with those from a WT BMT recipient mouse. Scale bar: 1 cm. (C) Flow cytometric analysis shows the quantity of CD3+ cells and CD8/CD4 T cell ratio in the spleens of BMT recipient mice. (D) Histological analysis of CD3+ cells from the lungs of NSG recipient mice after hSTAT5BN642H or WT BMT. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). (E) Percentage of disease-free survival after hSTAT5BN642H or WT CD8+ T cell transplantation into nonirradiated 8-week-old Ly5.1/CD45.1 recipient mice. (F) Flow cytometric analysis shows the quantity of splenic CD3+CD8+ cells in CD8+ T cell–transplanted mice. (G) Spleen versus BW ratios of WT and hSTAT5BN642H CD8+ T cell–transplanted Ly5.1/CD45.1 mice. (A–C) n = 4 WT mice and n = 5 hSTAT5BN642H mice; (E–G) n = 6 WT mice and n = 5 hSTAT5BN642H mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test. Figure 5. hSTAT5BN642H CD8+ T cells are the cancer-initiating cells. jci.org      Volume 128      Number 1      January 2018 R E S E A R C H A R T I C L E These are components of poly- comb repressor complex 2 (PCR2), which promotes methylation of histone 3 at lysine 27 (FDR-adjusted P ≤ 0.05, Fisher’s exact test) (Figure 8A, top, Supplemental Figure 9A, and Supplemental Table 4). STAT5 has recently been reported to oppose a network of transcription factors such as NF-κB and IKAROS in B cell acute lymphoblastic leukemia (52) and to interact with EZH2 (35). Fur- thermore, TOP2A expression has been previously linked to EZH2 expression in aggressive prostate cancer (53). Consistently, target genes of EZH2 and SUZ12 were found to be enriched in CD8+ T cells derived from hSTAT5BN642H mice (Figure 8B, Supplemental Figure 9B, and Supplemental Table 5). STAT5BN642H-expressing T cells are sensitive to AURKB inhibition. Among EZH2 target genes, the genes encoding Aurora kinase B (Aurkb) and DNA topoisomerase 2α (Top2a) were significantly upreg- ulated, and AURKB targets were highly enriched in hSTAT5BN642H- expressing CD8+ T cells (Figure 8B and Supplemental Figure 10A). Western blot analysis showed that hSTAT5BN642H mice had higher AURKB activity, and quantitative PCR (qPCR) analysis validat- ed the hSTAT5BN642H-dependent upregulation of Aurkb levels in CD8+ T cells (Figure 9A and Supplemental Figure 10B). This led us to test the dual-specific JAK and Aurora kinase inhibitor AT9283 as a potential therapeutic in hSTAT5BN642H-expressing cells. We found that hSTAT5BN642H-expressing T cells were exquisitely more sensitive to AT9382, with a 10-fold lower IC50 compared with that of hSTAT5B-expressing T cells (Figure 9B), but not to etoposide, a TOP2A inhibitor (Supplemental Figure 10C). AT9283 was not effective in reducing STAT5 activation compared with ruxolitinib but efficiently reduced AURKB activity (Figure 9C). The high sen- To investigate whether STAT5BN642H has a role in the upregula- tion of these genes, we performed ChIP with isolated CD8+ T cells from WT, hSTAT5B, and hSTAT5BN642H mice. Given its hyperac- tivation status, binding of hSTAT5BN642H to DNA increased com- 3 9 2 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation      R E S E A R C H A R T I C L Figure 6. hSTAT5BN642H-driven diseased T cells can be treated with JAK inhibitors. (A) WB analysis of pY-STAT5 levels in isolated and cultivated LN T cells from hSTAT5BN642H, hSTAT5B, and WT mice after IL-2 removal. R E S E A R C H A R T I C L E (B) Dose-response curve of WT, hSTAT5BN642H, and hSTAT5B T cells 72 hours after ruxolitinib treatment, analyzed using CellTiter-Glo (CTG) assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. (C) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with ruxolitinib or tofacitinib, analyzed for pY-STAT5. (D) Macroscopic view of LNs and spleens from CD8+ T cell–transplanted mice treated with ruxolitinib compared with vehicle controls. CD8+ T cell–recipient mice were treated with ruxolitinib at the dosage of 45 mg/kg twice a day for 30 days. (E) Quantification of spleen versus BW ratio of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice. (F) WBC counts of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice, measured using a scil Vet ABC animal blood counter. Flow cytometric analysis of CD25 expression in peripheral blood CD8+ T cells. MFI, mean fluorescence intensity. (G) Histological analysis of CD3+ cells in the lungs of recipient mice after treatment with ruxolitinib compared with the vehicle-treated group. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). n = 5 vehicle-treated mice and n = 4 ruxolitinib-treated mice. Data represent the mean ± SD. n ≥ 6. *P < 0.05, by unpaired, 2-tailed Student’s t test. Data presented in A–C are representative of 3 independent experiments. Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 6. hSTAT5BN642H-driven diseased T cells can be treated with JAK inhibitors. (A) WB analysis of pY-STAT5 levels in isolated and cultivated LN T cells from hSTAT5BN642H, hSTAT5B, and WT mice after IL-2 removal. (B) Dose-response curve of WT, hSTAT5BN642H, and hSTAT5B T cells 72 hours after ruxolitinib treatment, analyzed using CellTiter-Glo (CTG) assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. (C) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with ruxolitinib or tofacitinib, analyzed for pY-STAT5. (D) Macroscopic view of LNs and spleens from CD8+ T cell–transplanted mice treated with ruxolitinib compared with vehicle controls. R E S E A R C H A R T I C L E CD8+ T cell–recipient mice were treated with ruxolitinib at the dosage of 45 mg/kg twice a day for 30 days. (E) Quantification of spleen versus BW ratio of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice. (F) WBC counts of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice, measured using a scil Vet ABC animal blood counter. Flow cytometric analysis of CD25 expression in peripheral blood CD8+ T cells. MFI, mean fluorescence intensity. (G) Histological analysis of CD3+ cells in the lungs of recipient mice after treatment with ruxolitinib compared with the vehicle-treated group. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). n = 5 vehicle-treated mice and n = 4 ruxolitinib-treated mice. Data represent the mean ± SD. n ≥ 6. *P < 0.05, by unpaired, 2-tailed Student’s t test. Data presented in A–C are representative of 3 independent experiments. Discussion sitivity of AT9283 could be an attribute of Aurora serine/threonine and JAK tyrosine/serine kinase combinatorial inhibition, as IC50 values of ruxolitinib and tofacitinib were similar in all genotypes (Figure 6 and Supplemental Figure 6). Combinatory treatment with ruxolitinib and AZD1152, an AURKB-specific inhibitor, showed an additive effect, which further supported our hypothesis (Supple- mental Figure 10D). Although AZD1152 treatment did not affect STAT5 phosphorylation in all genotypes, it efficiently inhibited AURKB activity in hSTAT5BN642H-expressing T cells (Figure 9D). Here, we provide evidence that the STAT5BN642H mutation is a direct driver and not a bystander mutation for lymphoid malig- nancy. Expression of hSTAT5BN642H triggers the development of leukemia or lymphoma characterized by highly proliferative and invasive CD8+ T cells. hSTAT5BN642H activation remains large- ly cytokine dependent, which renders the diseased cells sensi- tive to JAK inhibition. When comparing T cells from transgenic hSTAT5BN642H mice with those from their hSTAT5B counterparts, jci.org      Volume 128      Number 1      January 2018 3 9 3 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+ T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values in B and C were determined by Kolmogorov-Smirnov test. Discussion (A) Heatmap stantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA me Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+ T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values in B and C were determined by Kolmogorov-Smirnov test. (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. 59, 60). Transgenic mouse models expressing high levels of murine Stat5a or Stat5b developed lymphoblastic lymphoma at low pene- trance (5%–25%) and with a late onset (up to 456 days) (28, 61). we found reduced DNA methylation of EZH2-binding sites. This correlated with an increase in the transcription of STAT5B and EZH2 target genes including the cell-cycle regulators Top2A and Aurkb, for which AURKB represents a potential therapeutic target. Discussion (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+ T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values in B and C were determined by Kolmogorov-Smirnov test. (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T c Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. 3 9 4 jci.org      Volume 128      Number 1      January 2018 Discussion We now show that moderate expression of hSTAT5BN642H, but not hSTAT5B, is sufficient to trigger an aggressive disease that causes rapid lethality at a young age, with full penetrance irrespective of gender, demonstrating the potent oncogenic role of the hSTAT5BN642H mutation. Despite the Vav1 promoter– dependent expression of hSTAT5BN642H throughout the entire hematopoietic system, malignancy evolved in CD8+ T cells. This T cells express considerably more STAT5B than do other cell types of the hematopoietic system (54–57), suggesting a privileged role for STAT5B in the T cell compartment (58). Moreover, STAT5B is the dominant STAT5 protein in effector and regulatory T cells, and the differences in STAT5A and STAT5B governing T cell func- tion are largely associated with paralog expression differences (7, 3 9 4 jci.org      Volume 128      Number 1      January 2018 3 9 4 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E ure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 result in the induction of Aurora ase B. (A) Region set enrichment analysis testing CGIs with lower DNA methylation in hSTAT5BN642H cells than in WT cells (top) or lower DNA methyla- n in WT cells than in hSTAT5BN642H cells (bottom). Enrichment was determined using LOLA (51). Each dot represents 1 ChIP-seq experiment for a given nscription factor from the CODEX database. The vertical dashed line represents the significance threshold (FDR-adjusted P ≤ 0.05). (B) Enrichment t of EZH2 target genes in HSCs, together with their methylation states of EZH2-bound and EZH2-unbound CGIs 100 kb up- and downstream of the nscriptional start sites (TSSs). Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and gative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from the MSigDB (72). Black circles indicate CGIs overlapping h EZH2-binding sites. p.p., percentage points. n = 2 per genotype. ChIP with anti-STAT5 (C) or anti-EZH2 (D) in CD8+ T cells isolated from WT (n = 7), TAT5B (n = 7), or hSTAT5BN642H (n = 4) mice. Discussion Binding of STAT5 to the Cis and Ccnd2 promoters or binding of EZH2 to the promoter regions of Cdkn2A and nd2 served as positive controls. Horizontal dotted line indicates the threshold for nonspecific binding. (E) ChIP with anti-STAT5, anti-EZH2, or IgG in AT5BN642H-expressing CD8+ T cells, followed by WB analysis. IB, immunoblot. Data presented in C–E are representative of 2 independent experiments. or bars indicate the mean ± SD. Figure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 re Figure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 result in the induction of Aurora kinase B. (A) Region set enrichment analysis testing CGIs with lower DNA methylation in hSTAT5BN642H cells than in WT cells (top) or lower DNA methyla- tion in WT cells than in hSTAT5BN642H cells (bottom). Enrichment was determined using LOLA (51). Each dot represents 1 ChIP-seq experiment for a given transcription factor from the CODEX database. The vertical dashed line represents the significance threshold (FDR-adjusted P ≤ 0.05). (B) Enrichment blot of EZH2 target genes in HSCs, together with their methylation states of EZH2-bound and EZH2-unbound CGIs 100 kb up- and downstream of the transcriptional start sites (TSSs). Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from the MSigDB (72). Black circles indicate CGIs overlapping with EZH2-binding sites. p.p., percentage points. n = 2 per genotype. ChIP with anti-STAT5 (C) or anti-EZH2 (D) in CD8+ T cells isolated from WT (n = 7), hSTAT5B (n = 7), or hSTAT5BN642H (n = 4) mice. Binding of STAT5 to the Cis and Ccnd2 promoters or binding of EZH2 to the promoter regions of Cdkn2A and Ccnd2 served as positive controls. Horizontal dotted line indicates the threshold for nonspecific binding. (E) ChIP with anti-STAT5, anti-EZH2, or IgG in STAT5BN642H-expressing CD8+ T cells, followed by WB analysis. IB, immunoblot. Data presented in C–E are representative of 2 independent experiments. Error bars indicate the mean ± SD. STAT5BN642H has been shown previously to render Ba/F3 cells cytokine independent and to be constitutively active in HeLa cells (13, 15, 17). jci.org      Volume 128      Number 1      January 2018 Discussion IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. n = 6 per genotype. (C and D) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with AT9283 or AZD1152, for determination of pY-STAT5, total STAT5, p-AURKB, total AURKB, p-H3 (Ser10), and total H3 levels. Data presented in A–D are representative of 3 independent experiments. Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 9. hSTAT5BN642H-driven diseased T cells are sensitive to Aurora kinase B inhibition. (A) WB analysis of p-AURKB, total AURKB, and HSC70 in LNs from WT and hSTAT5BN642H- and hSTAT5B-transgenic mice. WB quantification (bar graph) was performed using ImageJ. (B) Dose-response curves of WT, hSTAT5BN642H, or hSTAT5B T cells in response to AT9283 after 72 hours of treatment, analyzed using a CTG assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. n = 6 per genotype. (C and D) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with AT9283 or AZD1152, for determination of pY-STAT5, total STAT5, p-AURKB, total AURKB, p-H3 (Ser10), and total H3 levels. Data presented in A–D are representative of 3 independent experiments. gested that STAT5 and EZH2 compete for binding to regulatory sites, as shown in B cells and mammary epithelial cells (52, 75). We observed that, as a consequence of STAT5B hyperactivation, STAT5BN642H bound more to DNA and subsequently upregulated many cell-cycle–regulating genes including Top2a and Aurkb. The fact that the cells were particularly sensitive to Aurora kinase inhi- bition underlines this observation. tantly, the most upregulated genes were E2F and MYC targets, which highlights the proliferative nature of the diseased T cells and explains the upregulation of numerous genes (65). STAT5BN642H is hyperphosphorylated, and it would be interesting to study its potential different interactions with CD8+ T cell–specific activators or repressors compared with the less active WT STAT5B. Discussion Ba/F3 cells have been used to determine the oncogenic potential of many leukemogenic drivers, however, the expression level of the oncogene is often very high, and the cells might have acquired additional mutations as a result of long-term cultivation. In cytokine-independent cell lines such as HeLa or HEK293T, STAT5 might be activated by other available growth stimuli. Cells expressing low levels of STAT5BN642H, however, remain dependent on cytokine stimulation, as shown in our diseased T cell model. This was also observed in NK cells by Küçük and colleagues (18). development could be a result of the CD8+ T cell sensitivity to the Stat5a/b gene dosage that was described previously in mice (62). Moreover, it has been reported that CD8+ T cells are more susceptible to oncogenic drivers, especially when these drivers are activated by cytokines or triggered via T cell receptors (62, 63). Similarly, hSTAT5BN642H activation remains cytokine depen- dent, and the upregulation of IL-2Rα, a direct target of STAT5, resulted in CD8+ T cells becoming more sensitive to low doses of cytokine stimulation. So far, hSTAT5BN642H mutations have primarily been found in patients with T cell or NK cell malignan- cies, pointing toward the sensitivity of these patients to aberrant STAT5 activation. When STAT5BN642H was identified in CD8+ T cells in patients, such as those with T cell LGL (T-LGL) or epi- theliotropic intestinal T cell lymphoma (13, 23, 64), it gave rise to more aggressive disease (26). The malignant transformation and expansion of CD8+ T cells in transgenic mice correlated with the upregulation of direct STAT5 target genes such as D-type cyclins, Bcl2 family members, and Pim kinases, which promote cell-cycle progression and survival. Impor- 3 9 5 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation   R E S E A R C H A R T I C L E Figure 9. hSTAT5BN642H-driven diseased T cells are sensitive to Aurora kinase B inhibition. (A) WB analysis of p-AURKB, total AURKB, and HSC70 in LNs from WT and hSTAT5BN642H- and hSTAT5B-transgenic mice. WB quantification (bar graph) was performed using ImageJ. (B) Dose-response curves of WT, hSTAT5BN642H, or hSTAT5B T cells in response to AT9283 after 72 hours of treatment, analyzed using a CTG assay. jci.org      Volume 128      Number 1      January 2018 Methods Plasmid construction/mutagenesis and transfection. hSTAT5B variants were generated using site-directed mutagenesis (80). Mutagenic PCR was performed using KOD Polymerase (Novagen). PCR products were subsequently digested with DpnI enzyme (New England BioLabs) to remove the methylated template according to the manufacturer’s pro- tocol. E. coli was transformed with the digested reaction, and positive clones were selected by Sanger sequencing (81). Plasmid transfection was performed using Lipofectamine 2000 Reagent (Invitrogen, Ther- mo Fisher Scientific). Cytokine stimulation of T cells was performed with human IL-2 (100 U/ml; ProleukinÒ; Novartis), murine IL-4 (100 ng/ml; R&D Sys- tems), or murine IL-7 (10 ng/ml; R&D Systems). The 293T and 32D cell lines were gifts of M. Hengstschläger (Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria) and F. Grebien (Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria), respectively. The Ba/F3 cell line was provided by A. D’An- drea (Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA). The cases of patients harboring the STAT5BN642H mutation were assembled from previously published work (13–26, 36). Animals and generation of transgenic mice. Transgenic mice were generated and bred on a C57BL/6NCrl background and maintained in a specific pathogen–free environment in the experimental mouse facility at the University of Veterinary Medicine (Vienna, Austria). We used the Vav1-hematopoietic vector Vav1-hCD4 (HS21/45) (37) to generate several transgenic mouse lines expressing hSTAT5B and hSTAT5BN642H in the hematopoietic system and selected the lines B6N-Tg(STAT5B)731Biat and B6N-Tg(STAT5BN/H)726Biat, respec- tively, for further experiments. The hSTAT5BN642H construct was gen- erated using overlapping PCR technology as previously described (80) (forward primer: GAAAGAATGTTTTGGCATCTGATGCCTTTTAC; reverse primer: GTAAAAGGCATCAGATGCCAAAACATTCTTTC). The construct was digested with the HindIII restriction enzyme and gel purified for pronuclear injection (82). The transgenic mice were identified by genotyping PCR (forward primer: ACGCAGGACACA- GAGAATGAG; reverse primer: GTGATGGTGGCGTTGACCTC). WT (C57BL/6NCrl) mice and B6-Ly5.1/Cr (B6.SJL-PtprcaPepcb/BoyCrCrl) mice were purchased from Charles River Laboratories, and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from The Jack- son Laboratory. Given the rapid development and strong phenotype of the hSTAT5BN642H-transgenic mice, the colony was propagated via in vitro fertilization with archived sperm cells (83). Transplantation experiments. BM cells (1 × 106) from hSTAT5BN642H or WT mice were transplanted by lateral tail vein injection into nonirra- diated NSG mice. Mice were monitored daily and evaluated at the first sign of disease onset. Methods CD8+ T cells were isolated using a CD8+ Magni­ Sort Mouse T Cell Enrichment Kit (eBioscience), and sorted cells were checked with flow cytometry for their purity. Cells (1 × 106) were inject- ed i.v. into nonirradiated Ly5.1/CD45.1 mice. IHC. Mouse organs were incubated overnight in 4% phos- phate-buffered formaldehyde solution (Roti-Histofix; Carl Roth), dehydrated, embedded, and cut (4-μm-thick sections). For immu- nohistochemical staining, heat-mediated antigen retrieval was per- formed in citrate buffer at pH 6.0 (Dako) and stained with antibod- ies against CD3 (Thermo Fisher Scientific; RM-9107-S0; dilution 1:300); Ki67 (Novocastra, Leica Biosystem; NCL-Ki67p; dilution 1:1,000); and STAT5B (Santa Cruz Biotechnology; sc-1656; dilution 1:200) using standard protocols. Images were taken using a Zeiss Imager Z.1 microscope. Western blot analysis. Western blotting (WB) was performed using standard protocols. The antibodies used were: monoclonal rabbit anti- mouse phosphorylated STAT5 (p-STAT5) (Invitrogen, Thermo Fisher Scientific; 716900; dilution 1:1,000); purified mouse anti-STAT5 (BD; 610191; dilution 1:2,000); monoclonal mouse anti-mouse HSC70 (Santa Cruz Biotechnology; sc-7298; dilution 1:10,000); monoclonal mouse anti-Flag M2 ( MilliporeSigma; F3156; dilution 1:1,000); mono- clonal rabbit anti–p–Aurora A (Thr288), p–Aurora B (Thr232), and p–Aurora C (Thr198) (Cell Signaling Technology; 2914; 1:1,000); monoclonal rabbit anti-Aurora B/AIM1 (Cell Signaling Technology; 3094; 1:1,000); monoclonal rabbit anti–histone H3 (anti-H3) (Cell Signaling Technology; 4499; 1:1,000); monoclonal rabbit anti–p-H3 (Ser10) (Cell Signaling Technology; 53348; 1:1,000); ECL anti-mouse IgG; HRP-linked whole antibody from sheep (GE Healthcare; NA931V; dilution 1:10,000); ECL anti-rabbit IgG; and HRP-linked whole anti- body from sheep (GE Healthcare; NA934V; dilution 1:10,000). WB quantification was performed using ImageJ software (NIH). (See the complete unedited blots in the supplemental material.) Hematocytometry and flow cytometry. Mouse blood was collect- ed into EDTA tubes (Greiner Bio-One Mini-Collect K3EDTA Tubes; Thermo Fisher Scientific) from the facial vein or from euthanized mice via cardiac puncture, and blood smears were stained using mod- ified Wright staining. WBC counts were measured using an animal blood counter (scil Vet ABC). For flow cytometry, erythrocytes were lysed using Gay’s solution (10 mM KHCO3 and 75 mM NH4Cl, pH 7.4). Single-cell suspensions were prepared by mincing organs through a 70-μm cell strainer (BD Biosciences). HSC staining was performed as previously described (84). All antibodies used for flow cytometry were purchased from eBioscience and BD (see Supplemental Table 6 for the list of the anti- bodies). All analyses were performed on the BD FACSCanto II using FACSDiva software (BD). Further analysis was performed using FlowJo software. Discussion Recent work suggested that altered DNA methylation patterns in T cells are indicative or even causative for T cell transformation and that methylation of gene bodies was correlated with active transcription contributing to carcinogenesis (66, 67). Epigenetic regulators such as EZH2, TET1/2, and HDAC play important roles in leukemogenesis (68–71) and have been shown to interact with STAT5 (30, 31, 34, 35). EZH2 has been linked to the long-term repopulating capability, proliferation, and inhibition of apoptosis of HSCs (72, 73), all of which are important for transformed cells as well as for governing peripheral T cell fates (74). We demonstrate here that the expression of hSTAT5BN642H not only led to transcrip- tional changes but also changed DNA methylation. Decreased methylation at EZH2- and SUZ12-binding sites in hSTAT5BN642H T cells resulted in the upregulation of EZH2 target genes. There are conflicting reports regarding the interaction between EZH2 and STAT5. In 2011, Mandal and colleagues reported that STAT5 plays an essential part in the recruitment of EZH2 to repress Ig κ-chain (Igk) transcription in progenitor B cells (35). Others sug- Work by many groups identified STAT5 as an important tar- get for therapy, since it is essential for JAK2V617F-, Flt3-ITD-, and BCR/ABL-driven diseases (76–78). Currently, intensive efforts are being made to inhibit STAT5 by blocking its SH2 domain (79). However, effective targeting of STAT5 remains challenging. Sev- eral different strategies have been suggested for the treatment of hSTAT5BN642H-expressing cells including the use of BCL2, MEK1/2, and JAK inhibitors (17, 18). Although some patients respond to JAK inhibitors, the lack of sensitivity in other patients requires broader therapeutic targets (16, 17). We believe that the hSTAT5BN642H-transgenic mouse model will serve as a valuable preclinical model. Using this model, we showed that the com- bined use of Aurora kinase and JAK inhibitors is a potential thera- peutic strategy to treat lymphoma and leukemia patients with the STAT5BN642H mutation. We show here that hSTAT5BN642H acts as a driver mutation in the development of leukemia and lymphoma and propose that 3 9 6 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 R E S E A R C H A R T I C L E The Journal of Clinical Investigation upstream inhibition of JAK activation or the chromatin-remodel- ing partners of STAT5 could be an alternative targeting strategy for enhanced STAT5 activation. mycin) (all from Gibco, Thermo Fisher Scientific) supplemented with IL-3 (1 ng/ml; ImmunoTools). IL-3 stimulation was performed with 10 ng/ml IL-3 for 20 minutes. The hSTAT5BN642H, hSTAT5B, and B6N WT T cells were isolated from LNs and spleens from 8- to 12-week-old mice. Following T cell activation by anti-CD3 (BD), T cells were grown in complete RPMI 1640 medium containing 10 mM HEPES, 1× MEM nonessential ami- no acids, 50 μM β-mercaptoethanol (all from Gibco, Thermo Fisher Scientific), 1 mM sodium pyruvate (MilliporeSigma), and 100 U/ml human IL-2 (ProleukinÒ; Novartis). R E S E A R C H A R T I C L E After alignment of reads against contam- inating sequences (mitochondrial and ribosomal DNA), the remain- ing reads were aligned against GRCm37 using transcriptome-guided alignment with TopHat, version 1.4.1 (http://ccb.jhu.edu/software/ tophat/index.shtml). Next, the htseq-count (http://htseq.readthedocs. io/en/master/count.html) with mode union was used to obtain gene counts for union gene models. Then, differentially expressed genes (log2 fold change >2 and FDR-adjusted q < 0.1) were determined using DESeq2, version 1.12.4 (Bioconductor). For heatmaps, centered and scaled rlog-transformed library size– normalized counts were visualized using the heatmap.2 function of R package gplots, version 3.0.1 (https://www.rdocumentation.org/ packages/gplots/versions/3.0.1). Viability assay. Murine T cells (5 × 104) from hSTAT5BN642H and WT mice were seeded in triplicate in 96-well plates. JQ1, 5-azacyti- dine, entinostat, etoposide, AT9283, tofacitinib, and ruxolitinib (all from Selleckchem) were added and incubated for 72 hours. All com- pounds were solubilized in DMSO (MilliporeSigma). DMSO and bor- tezomib (Selleckchem) were used as a negative and positive control, respectively. CellTiter-Glo reagent (Promega) was used to determine viability, measured on an EnSpire plate reader (PerkinElmer). IC50 val- ues were determined by nonlinear regression using GraphPad Prism 6 (GraphPad Software). Gene lists from differential expression analyses were ranked for the log­2 fold changes between hSTAT5BN642H and WT or hSTAT5BN642H and hSTAT5B CD8+ T cells. Ranking lists were subsequently used for GSEA via the Broad Institute’s GSEAPreranked tool at the standard setting. Gene sets were obtained from current publications or from the Broad Institute’s Molecular Signatures Database (MSigDB). RNA-seq data and a description of the experimental design are available in the NCBI’s Gene Expression Omnibus (GEO) database (GEO GSE104557). RRBS and analysis. Genomic DNA from purified CD8+ T cells was isolated using an AllPrep DNA/RNA Mini Kit (QIAGEN) and subsequently subjected to RRBS and analysis. RRBS was carried out as described earlier (85). In brief, 100 ng genomic DNA was digest- ed for 12 hours at 37°C with 20 units of MspI (New England Bio- Labs; R0106L) in 30 μl of 1× NEB Buffer 2. Fill-in and A-tailing were performed by the addition of Klenow Fragment 3′→ 5′ exo- (New England BioLabs; M0212L) and dNTP mix (10 mM dATP, 1 mM dCTP, 1 mM dGTP). After ligation to methylated Illumina TruSeq LT v2 adaptors using Quick Ligase (New England BioLabs; M2200L), the libraries were size selected by performing a 0.75× clean-up with AMPure XP beads (Beckman Coulter; A63881). R E S E A R C H A R T I C L E Up to 12 libraries were pooled in equal amounts on the basis of qPCR data and bisulfite converted using the EZ DNA Methylation Direct Kit (Zymo Research; D5020) with the following changes to the manufacturer’s protocol: the conversion reagent was used at ×0.9 concentration; incubation was performed for 20 cycles of 1 minute each at 95°C, followed by 10 minutes at 60°C; and the desulfonation time was extended to 30 minutes. Bisulfite-converted libraries were enriched for up to 17 cycles using PfuTurbo Cx Hotstart DNA Polymerase (Agilent Tech- nologies; 600412). After a 2× AMPure XP clean-up, quality control was performed using a Qubit dsDNA HS Assay Kit (Life Technolo- gies, Thermo Fisher Scientific; Q32854) and an Experion DNA 1K Analysis Kit (Bio-Rad; 700-7107). Sequencing was performed on an Illumina HiSeq 3000/4000 System using the 5-bp single-end mode. Initial data processing was carried out at the Biomedical Sequencing Facility of the Medical University of Vienna (http://www.biomed- ical-sequencing.at) using an in-house pipeline based on Pypiper (http://databio.org/pypiper) and Looper (http://databio.org/loop- In vivo ruxolitinib treatment. hSTAT5BN642H CD8+ T cell transplant recipients were treated with ruxolitinib (Chemietek) twice a day by oral gavage at a dosage of 45 mg/kg. Ruxolitinib was dissolved in DMSO (MilliporeSigma) and subsequently diluted in 0.5% methylcel- lulose (w/v) (MilliporeSigma). ChIP. CD8+ T cells (107 cells) from WT, hSTAT5B, and hSTAT5BN642H mice were isolated using a CD8+ MagniSort Enrichment Kit. Isolat- ed cells were washed twice with ice-cold PBS supplemented with inhibitors (1 mM Na3VO4, 1 mM NaF, 1× cOmplete Protease Inhibitor Cocktail [PIC], Roche) and fixed with DSG (2 mM, 30 min; Thermo Fisher Scientific). Cells were washed twice with cold PBS supple- mented with inhibitor and fixed with formaldehyde (1%, 10 min; MilliporeSigma). Fixation was quenched by incubation with glycine (125 mM, 5 min; MilliporeSigma). T cells were subsequently harvest- ed by centrifugation (350 g, 5 min). Cell lysis was performed with 1% SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris [pH 8.1], 1 mM Na3VO4, 1 mM phenylmethylsulphonyl) at 4°C for 30 minutes and sonicated using a Diagenode Bioruptor (20 cycles with 30 sec- onds on, 30 seconds off, high magnitude). Sonication was followed by chromatin dilution (1:10) in dilution buffer (167 mM NaCl, 16.7 mM Tris [pH 8.1], 1.2 mM EDTA, 1.1% Triton-X, 0.01% SDS). Clear chromatin was harvested by centrifugation (10,000 g, 10 min, 4°C). R E S E A R C H A R T I C L E er). Sequences were trimmed for adapters using Trimmomatic (86) with the ILLUMINACLIP settings “:2:40:7 SLIDINGWINDOW:4:15 MAXINFO:20:0.50 MINLEN:18.” All reads were aligned to the GRCm38 (mm10) assembly of the mouse genome using BSMAP in its RRBS mapping mode (87, 88). DNA methylation levels for individual CpGs were calculated using custom Python scripts and loaded into RnBeads (89) for exploratory analysis and to aggregate DNA meth- ylation estimates per CGI. The aggregated values were loaded into R for further analysis. Differentially methylated regions (absolute dif- ference ≥5 percentage points) were compared with ChIP-seq peaks from the CODEX database (90) using LOLA (51) to find significant overlaps (FDR-adjusted P ≤ 0.05) with potential regulators and effec- tors of DNA methylation differences. To compare DNA methylation at CGIs with genes, each CGI was associated with all genes within a 10-kb window after conversion of the gene coordinates to the mm10 reference genome using the UCSC LiftOver tool (https://genome. ucsc.edu/cgi-bin/hgLiftOver). RRBS sequencing data were deposited in the NCBI’s GEO database (GEO GSE104557). er). Sequences were trimmed for adapters using Trimmomatic (86) with the ILLUMINACLIP settings “:2:40:7 SLIDINGWINDOW:4:15 MAXINFO:20:0.50 MINLEN:18.” All reads were aligned to the GRCm38 (mm10) assembly of the mouse genome using BSMAP in its RRBS mapping mode (87, 88). DNA methylation levels for individual CpGs were calculated using custom Python scripts and loaded into RnBeads (89) for exploratory analysis and to aggregate DNA meth- ylation estimates per CGI. The aggregated values were loaded into R for further analysis. Differentially methylated regions (absolute dif- ference ≥5 percentage points) were compared with ChIP-seq peaks from the CODEX database (90) using LOLA (51) to find significant overlaps (FDR-adjusted P ≤ 0.05) with potential regulators and effec- tors of DNA methylation differences. To compare DNA methylation at CGIs with genes, each CGI was associated with all genes within a 10-kb window after conversion of the gene coordinates to the mm10 reference genome using the UCSC LiftOver tool (https://genome. ucsc.edu/cgi-bin/hgLiftOver). RRBS sequencing data were deposited in the NCBI’s GEO database (GEO GSE104557). were enriched using a CD8+ MagniSort Enrichment Kit, and mRNA was isolated using TRIzol (MilliporeSigma) in combination with an RNeasy Mini Kit (QIAGEN). mRNA library preparation (SENSE mRNA-Seq Library preparation) and RNA sequencing (RNA-seq) was performed with an Illumina HiSeq 2500 at the Vienna Biocenter Core Facility (VBCF) Next-Generation Sequencing (NGS) Unit (www. vbcf.ac.at). Adapter trimming and removal of low-quality bases were performed using cutadapt. Methods Cell culture. 293T cells were cultivated with complete DMEM medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-streptomy- cin). Ba/F3 and 32D cells were cultivated with complete RPMI 1640 medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-strepto- RNA sequencing and analysis. mRNA was isolated from CD8+ T cells harvested from LNs from mice of all 3 genotypes. CD8+ T cells 3 9 7 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 2015;208(4):115–128. 10. Bamford S, et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and web- site. Br J Cancer. 2004;91(2):355–358. 20. Ma X, et al. Rare occurrence of a STAT5B N642H mutation in adult T-cell acute lymphoblastic leu- kemia. Cancer Genet. 2015;208(1–2):52–53. 2. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1–2):203–214. 11. Imada K, et al. Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity. J Exp Med. 1998;188(11):2067–2074. 21. Jiang L, et al. Exome sequencing identifies somat- ic mutations of DDX3X in natural killer/T-cell lymphoma. Nat Genet. 2015;47(9):1061–1066. 3. Nivarthi H, Friedbichler K, Moriggl R. Stat5 as a Hematopoietic Master Regulator for Differenti- ation and Neoplasia Development. In: Decker T, Müller M, eds. Jak-Stat Signaling: From Basics to Disease. Vienna: Springer;2012:153–167. 12. Lin JX, Leonard WJ. The role of Stat5a and Stat5b in signaling by IL-2 family cytokines. Oncogene. 2000;19(21):2566–2576. 22. López C, et al. Genes encoding members of the JAK-STAT pathway or epigenetic regulators are recurrently mutated in T-cell prolymphocytic leukaemia. Br J Haematol. 2016;173(2):265–273. 13. Rajala HL, et al. Discovery of somatic STAT5b mutations in large granular lymphocytic leuke- mia. Blood. 2013;121(22):4541–4550. 4. Bunting KD. STAT5 signaling in normal and pathologic hematopoiesis. Front Biosci. 2007;12:2807–2820. 23. Nairismägi ML, et al. JAK-STAT and G-protein- coupled receptor signaling pathways are fre- quently altered in epitheliotropic intestinal T-cell lymphoma. Leukemia. 2016;30(6):1311–1319. 14. Nicolae A, et al. Frequent STAT5B mutations in γδ hepatosplenic T-cell lymphomas. Leukemia. 2014;28(11):2244–2248. 5. Heltemes-Harris LM, Farrar MA. The role of STAT5 in lymphocyte development and transfor- mation. Curr Opin Immunol. 2012;24(2):146–152. 15. Bandapalli OR, et al. The activating STAT5B N642H mutation is a common abnormality in pediatric T-cell acute lymphoblastic leukemia and confers a higher risk of relapse. Haematologi- ca. 2014;99(10):e188–e192. 24. Lavallée VP, et al. Chemo-genomic interrogation of CEBPA mutated AML reveals recurrent CSF3R mutations and subgroup sensitivity to JAK inhibi- tors. Blood. 2016;127(24):3054–3061. 6. Ferbeyre G, Moriggl R. The role of Stat5 transcrip- tion factors as tumor suppressors or oncogenes. Biochim Biophys Acta. 2011;1815(1):104–114. 7. Villarino A, et al. Signal transducer and activator of transcription 5 (STAT5) paralog dose governs T cell effector and regulatory functions. Elife. 2016;5:e08384. 25. Simpson HM, et al. Concurrent mutations in ATM and genes associated with common γ chain signaling in peripheral T cell lymphoma. PLoS One. 2015;10(11):e0141906. 16. Kiel MJ, et al. Author contributions ium chloride wash buffer (0.5 M LiCl2, 50 mM HEPES, 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40) and then once in Tris-EDTA (TE) buffer containing 50 mM NaCl. Chromatin was eluted in 2× 100 μl elution buffer (1% SDS, 50 mM Tris, 10 mM EDTA). Eluted chro- matin (20 μl) was used for WB analysis. Samples and inputs were incubated with 8 μl of 5 M NaCl at 65°C overnight and subsequently incubated with 0.5 M EDTA, 1 M Tris (pH 6.5), and proteinase K (10 mg/ml) for 2 hours at 55°C. RNA was lysed for 1 hour at 37°C using 0.2 mg/ml RNase-A (MilliporeSigma). Chromatin clean-up was per- formed using a PCR purification kit (QIAGEN). DNA was subjected to qPCR using GoTaq Real-Time qPCR (Promega), and the amount of amplification was quantified using standard curves. Primers are listed in Supplemental Table 7. RM designed and supervised the study. HTTP, BM, MPM, EG, TJ, HN, ZK, TK, AB, SK, MF, MM, TR, VS, and RM designed and/or performed experiments. HTTP, RG, FH, and MPM analyzed data. JP and FG contributed to the interpretation of the data. LK inter- preted IHC results. ME designed and performed experiments. PV, MH, and CB revised the manuscript with regard to critical intellec- tual content. HTTP, BM, VS, and RM wrote the manuscript. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Cleared chromatin was incubated with rolling at 4°C with 5 μg STAT5 (C-17) (Santa Cruz Biotechnology; sc-835 X), EZH2 (Diagenode; pAb-039-050), or IgG (Santa Cruz Biotechnology; sc-2027 X) over- night at 4°C. Diluted chromatin (1%) was kept as the input. Blocked Dynal Magnetic Beads (65 μl; Life Technologies, Thermo Fisher Sci- entific) were added per IP the following day and incubated for an additional 4 hours at 4°C. IP samples were washed 5 times with lith- 3 9 8 jci.org      Volume 128      Number 1      January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 2. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1–2):203–214. 1. Stark GR, Darnell JE. The JAK-STAT pathway at twenty. Immunity. 2012;36(4):503–514. Acknowledgments We would like to thank Gregor Hörmann, Safia Zahma, Graham Tebb, Michaela Schlederer, Katrin Meissl, Johannes Schmöllerl, Patricia Stiedl, Helmut Dolznig, Margit Rosner, Thomas Weich- hart, Claus Vogl, and all members of Ludwig Boltzmann Institute for Cancer Research (LBI-CR) for their help and support. This work and RM, HTTP, BM, MM, VS, SK, CB, MF, and PV are sup- ported by the Austrian Science Fund (FWF) SFB grant F47 and F61 subprojects F4701-B20, F4704-B20, F4706-B20, F4707-B20, F6101, F6102, F6105, F6106, and F6107. FG is supported by the European Research Council (ERC) Starting Grant ONCOME- CHAML. FH is supported by a postdoctoral fellowship from the German Research Council (DFG) (HA 7723/1-1). Statistics. Flow cytometric data are reported as the mean ± SD and were analyzed using GraphPad Prism 6 (GraphPad Software). Differ- ences were assessed for statistical significance by an unpaired, 2-tailed Student’s t test and 1-way ANOVA with Bonferroni’s correction. Kaplan-Meier plots were analyzed using a log-rank (Mantel-Cox) test. P values for GSEA were determined using the Kolmogorov-Smirnov test. A P value of less than 0.05 was accepted as statistically significant. Study approval. All animal experiments were approved by the institutional ethics committee and the Austrian Ministry BMWFW authorities under the animal license protocols BMWFW-68.205/ 0166-WF/V/3b/2015), BMWFW-68.205/0117-WF/V/3b/2016, and BMWFW-68.205/0103-WF/V/3b/2015. 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https://openalex.org/W4388265398
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English
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Modelling public attitude towards drone delivery in Germany
European transport research review
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10,573
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Abstract Background  Last-mile delivery by drone is expected to be a promising innovation for future urban logistics. How- ever, in addition to adoption of services by customers, leveraging this delivery method will depend essentially on a positive public perception of such services in urban airspace. Objective  This article provides novel and comprehensive insights into factors driving or impeding citizens’ attitudes towards drone delivery. Methodology  The article develops a structural equation model that  derives from a sequential exploratory mixed methods design. In the first step, factors affecting attitudes towards drone delivery were identified within the scope of five focus groups and converted into the development of a questionnaire. In the second step, a German pop- ulation-representative survey was conducted through telephone interviews, which provided reliable data to test the model (n = 819). Results  Expected risks (particularly stress due to traffic in lower airspace, noise, and visual disturbances), as well as expected benefits (particularly fast and time-flexible delivery), significantly affect attitudes towards drone-based delivery, while the individual level of technological openness (technophilia) does not have a significant association. Moreover, the model reveals that the expected risks of drone deliveries are stronger associated with public attitude than with expected benefits. Conclusions  The provided framework suggests fashioning policies and drone delivery applications that focus on mitigating social, spatial, and visual risks while achieving maximum utility for customers. Keywords  Drone delivery, Urban air mobility, Public attitude, Acceptance, SEM, Modelling, Expected benefits, Expected risks, Technophilia Keywords  Drone delivery, Urban air mobility, Public attitude, Acceptance, SEM, Modelling, Expected benefits, Expected risks, Technophilia referred to in this paper, can lift weights of 2–3 kg and fulfil flight missions within a diameter of about 15 km [3]. Thereby, delivery drones generate plenty of applications ranging from medical use [4, 5], intralogistical applica- tions [6] to commercial last mile delivery, e.g. for online retailing or food delivery [7, 8]. Though still in its infancy, drone delivery may form a serious option for innovating future urban and rural transport networks. 1.1  Background Driven by economic motivations of making processes faster, more flexible, reliable, and paired with the attri- bution of a “green” transport option, drone delivery has climbed high on industrial and political agendas world- wide [1, 2]. Technically, standard delivery drones, as From a customer perspective, drone delivery is said to address the demand for ever-faster delivery as well as to open the opportunity for customers to receive their delivery within a narrow or even well-defined time- frame [9]. However, while economic and political actors *Correspondence: Robin Kellermann robin.kellermann@tu-berlin.de 1 Mobility Research Cluster, Department of Work, Technology and Participation, Technische Universität Berlin, 10587 Berlin, Germany Kellermann et al. European Transport Research Review (2023) 15:38 https://doi.org/10.1186/s12544-023-00606-0 Kellermann et al. European Transport Research Review (2023) 15:38 https://doi.org/10.1186/s12544-023-00606-0 European Transport Research Review Open Access Open Access Modelling public attitude towards drone delivery in Germany Robin Kellermann1*   , Tobias Biehle1    and Hamid Mostofi1 Kellermann et al. European Transport Research Review (2023) 15:38 Page 2 of 14 Page 2 of 14 Nevertheless, acceptance research on consumer adop- tion of drone delivery and the willingness to pay for drone delivery services has grown significantly over the past decade. Drawing on a bibliometric analysis of respective empirical studies [14], results show that the variables’ intention to use, ‘attitude’, ‘risk perception’, and ‘personal innovativeness’ are being used most frequently to analyse the acceptance of drone delivery. Moreover, the devel- opment of research hypotheses most often derives from modifications of the Technology Acceptance Model [15], the Theory of Reasoned Action [16], and the Diffusion of Innovation model [17]. For example, Yoo et  al. [18] presented an acceptance model with a specific focus on delivery drones, which applied the Diffusion of Innova- tion model [17] and the Technology Acceptance Model to develop their hypotheses. By testing their model, they showed that the delivery speed, the ease of use, as well as individual characteristics such as the personal level of innovativeness, are central in determining the attitude towards the adoption of drone delivery. The latter is also confirmed by Hwang and colleagues, who did extensive research on the adoption of drone-based food delivery in South Korea [19].i increasingly pursue ambitions to enable deliveries in low- level airspace, the creation of a ‘third dimension’ for cargo transport may not only become a disruptive moment for customers but may also mark a turning point in people’s perception of space and traditional notions of land use. Given the potential of wider diffusion of drone delivery in retail business and the respective possible sizes of drone fleets [10], it remains a relevant question of how citizens position themselves regarding the collective exposure to traffic in low-level airspace as a consequence of using the technology [11]. Thus, the vision of drone delivery not only involves an individual service choice for customers but also entails an inherently public dimension. In cur- rent acceptance research, however, this comprehensive understanding appears rather underrepresented, posing a risk to private sector ambitions and the jurisdiction of policymakers alike. Against this background, this paper presents and tests a structural equation model (SEM) aiming to explain the public’s attitude towards the deployment of delivery drones for consumer goods in public space. As citizens are both users and bystanders, the model incorporates relevant aspects of consumer preferences as well as novel societal and spatial dimensions, which are also expected to influence attitudes. The selected factors are subdivided into three latent variables (expected benefits, expected risks, technophilia) and derive from a sequential explora- tory mixed methods design. Therefore, in the first step, a set of focus group discussions enabled to qualitatively explore and extract genuine acceptance factors. In a sec- ond step, these factors were investigated in a population- representative survey conducted in Germany in early 2020. In the third step, a large sample (n = 819) was tested in our model. Regarding the potential benefits of drone delivery from a customer point of view, the notion of (food) delivery services being “green” have shown a relevant positive association towards the attitude of respondents [20, 21]. Interestingly, lower delivery fees have not been found to significantly influence the attractiveness of drone deliv- ery in general [22] but are suggested to be moderated by the specific context and circumstance, such as the parcel value or the availability of drop-off locations [23]. From a consumer risk perception, the functionality of the service is in question, including risks of technical delivery fail- ures [24], low service reliability [18], unattended deliver- ies, and theft [25]. 1.2  Literature review Public support for commercial drone delivery currently appears low. For example, results of a representative pop- ulation survey in Germany conducted in early 2020 show that only 25% of the respondents support or strongly sup- port the idea of using the technology for the delivery of consumer goods, while 55% of the respondents disagree or strongly disagree [12]. Those results correspond to a cross-European study by EASA [13], in which partici- pants were asked to rank their three most useful drone applications out of fourteen possible. Drone delivery of goods ordered through online shopping was a priority for about 20% of the respondents. Moreover, only 18% of respondents considered drone-based food delivery in urban environments to be one of the three most useful applications [13]. In sharp contrast to medical and emer- gency applications, the delivery of consumer goods thus doesn’t appear to be perceived as a key necessity. However, Zhu et al. [26] in particular, have shown that such delivery-related risks are not adequately reflecting the overall risk belief systems of the public towards drone delivery. Lateral and public risk dimensions, such as the risks of drones making the sky less pleasant to look at or causing property damage, can be strongly intervened to those believes [26]. Regarding the public dimension of drone delivery, especially safety and security concerns are broadly anticipated issues [27, 28]. Researchers also show that respondents perceive privacy risks as a sig- nificant concern in relation to the public application of drone delivery [21, 29]. In addition to that, noise has pre- dominantly been surveyed as a central barrier to public acceptance of drones for transportation [30]. In addition, Kähler et al. [31] conducted an experiment exploring the salience of drones to the observer in various environ- ments and applications, suggesting that the perception Kellermann et al. European Transport Research Review (2023) 15:38 Page 3 of 14 Fig. 1  Sequential mixed-methods approach Fig. 1  Sequential mixed-methods approach SEM includes the structural model and measure- ment model. The measurement model (Factor analysis) indicates how observable variables measure the latent variables. The latent variables represent intangible or psychological concepts such as attitudes, behaviors, and emotions, which cannot be directly measured through single items [34]. The structural model (path analysis) indicates the association between the latent variables and examines the hypothesized associations. Regression models test the strength and direction of relationships between predictors and a dependent variable. 1.2  Literature review However, SEM includes regression relationships simultaneously among latent variables and between observed and latent variables. In contrast to most statistical methods, SEM can analyze linear associations among variables at the same time by counting the measurement error. and aesthetic evaluation of drones is a relevant accept- ance factor. Consequently, concerns regarding visual pol- lution from drones should be considered more strongly in future research [31]. From an empathetic social per- spective, also the potential replacement of post officers generated by automated drone delivery might be a rel- evant factor to consider, as both Aydin [32] as well as Al Haddad et  al. [33] demonstrate a linkage between con- cerns about possible job losses and lower acceptance of drones. Reviewing the extensive body of research, we can state that studies currently either focus on consumer adoption or the influence of contextual aspects of drone delivery (environment, aesthetics, and noise). However, accept- ance research on drone delivery strongly demands an integrated and applicable approach that incorporates aspects of both individual dimensions of consumer preferences and the various contextual aspects of drone delivery that refer to its public and societal dimen- sions. Against this background, the article is motivated to achieve an integrated perspective through a model- oriented expansion of current attitudinal research, com- bining aspects of drone deliveries’ consumer and public dimensions. By this, we will provide more comprehensive as well as statistically robust insights on relevant fac- tors driving or impeding attitudes towards drone deliv- ery. Beyond the modeling approach creating conceptual relevance for attitudinal and social science technology acceptance in the field, the results can be of specific rel- evance for market research, the private sector, and poli- cymakers with respect to future jurisdiction and the development of drone-related transport policies. Factor analysis tests hypotheses on how well sets of observed variables in an existing dataset measure latent construct (i.e., factors). For this reason, in the litera- ture, factor analysis is also known as a measurement model. The constructs’ reliability was tested by using the value of Cronbach’s alpha for the constructs. Com- posite reliability (sometimes called construct reliabil- ity) is a measure of internal consistency in scale items, much like Cronbach’s alpha [35]. α = N · c v + (N −1) · c where, N = the number of items, c = average covariance between item pairs, v = average variance. 2.1  Structural equation model (SEM) Regarding the research purpose of this paper, we applied the approach of SEM. SEM contains concurrent statisti- cal approaches, such as analysis of variance, covariance, regression, path analysis, and factor analysis [34]. SEM contains the comparisons between-group and within- group variance, generally related to the ANOVA analysis. These analyses are usually performed by applying data in means or correlations and covariances (i.e., unstandard- ized correlations). 1.2  Literature review A high level of alpha means that the observed vari- ables are highly correlated and can be representative of a latent variable. Many studies suggest that the value of Cronbach’s alpha should be above 0.70 to indicate the reliability of the constructs [35]. 2.2  Data set To develop and test the presented model, a sequential exploratory mixed methods design was applied [36, To develop and test the presented model, a sequential exploratory mixed methods design was applied [36, 37]. In this approach (Fig. 1), genuine qualitative data was collected in the first step using focus groups. To guide the development of a model structure, results Page 4 of 14 Kellermann et al. European Transport Research Review (2023) 15:38 Fig. 2  Structural model and hypotheses Fig. 2  Structural model and hypotheses Fig. 2  Structural model and hypotheses Participants were chosen according to a pre-screening questionnaire aiming to exclude participants who worked in the drone industry and those who had never heard about drones before. Given that technology acceptance is often age-related [38–40], four of the five groups were separated into a younger (18–44) and an older (45–65) age-group. Since some studies also demonstrated a rela- tionship between technology acceptance and gender [41, 42], gender balance was assured. In addition, different levels of education, income and household sizes were analyzed to avoid selection effects [43]. Following the methodological procedure proposed by Benighaus and Benighaus [44], eight to ten participants of each group were supplied with information on delivery drones and, in the following, were guided through a group discussion by a professional moderator. Each group discussion was initiated by the moderator providing a 10-min presen- tation, which was compiled beforehand by the research team. The presentation defined the concept of "drone" and showed different use cases in a neutral and objective were evaluated using content analysis and interpreted within the theoretical framework of technology accept- ance research. Building on this identification and clas- sification of relevant variables that possibly influence the perception and attitudes towards drone delivery, quantitative data was gathered in a second step by con- ducting a telephone survey. The relevant variables were queried in a standardized questionnaire, allowing to build and test the SEM (Fig. 2). 2.2.2  Model and hypothesesh manner. In addition, a short video clip was shown, which demonstrated what a parcel delivery by drone might look like in practice. Based on a pre-developed guide- line, the participants then discussed the subject of fully automated delivery drones as a possible element of future urban logistics. The proposed attitudinal model consists of 17 observed variables that are entirely based on the qualitative results of the five focus group discussions. Grounded on these results and informed by key concepts from technol- ogy acceptance theories, the four latent constructs of expected benefits, expected risks, technophilia, and atti- tudes were defined. The following sections present the four latent constructs in more detail, while Fig. 2 shows the structural model. The focus groups were analyzed within the theoreti- cal framework of technology acceptance theory [45, 46] and by applying a qualitative content analysis [47]. The different attitudinal dimensions and acceptance factors were generated inductively throughout the analysis of the transcripts [48]. Firstly, this assured the invariable identi- fication of all factors that were considered relevant by the discussants. Secondly, this allowed for the identification of factors that had not been identified in previous stud- ies. A comprehensive review of the focus group results is provided in Kellermann and Fischer [49]. More precisely, four key results can be depicted, which informed the sub- sequent development of a structural model and the for- mulation of hypotheses: 2.2.2.1  Attitude  Attitude represents a central construct in the original Technology Acceptance Model (TAM) presented by Davis [15] and describes a person’s posi- tive or negative perceptions regarding the performance of an action [50]. In drone-related acceptance research, attitude has already been proposed as a dependent vari- able, mostly to predict the intention to use drones or drone-related services [18, 51, 52]. In line with that, the construct of attitude was derived from the focus group discussions. Since the discussions were not only related to individual interests in using drone delivery services but primarily to the technology’s expected influence on urban societies, the term public attitude is used. Consequently, in this research, attitude represents the target variable, and hypotheses are tested to explain the formation of (public) attitudes towards drone delivery. 1. 2.2.2  Model and hypothesesh The focus groups unveiled contrasting attitudes regarding the participants’ assessments of whether drone delivery would contribute to a higher quality of life in cities, if these new services would be useful or if they are rather to be seen as a threat to public safety and the common vision of sustainable societies.h In the survey, public attitude was measured by asking respondents how much they would agree to the state- ments of drone delivery (1) bringing advantages in the respondents’ everyday life, (2) being generally safe, (3) having a positive effect on the quality of life in cities, and (4) being more environmentally friendly than current transport alternatives. 2. The focus groups strongly emphasized the adverse impacts of automated drone delivery. Anticipated were potential job losses of delivery personal and intensified trends of social isolation, especially of the elderly. The expected impact of drone delivery on urban sound and landscape was a key issue in all the focus groups. What is more, there were broad discus- sions on how the quality of life in cities would change with respect to this new form of urban air traffic. 2.2.2.2  Expected risks  The concept of risk was introduced to attitudinal and behavioral research to explain the con- sequences of an action or the circumstances surrounding it cause uncertainty, uneasiness, or anxiety [53]. In drone- related research, the variable of (perceived) risk has been con- ceptualized early onwards [52] and proved as a significant predictor of attitude formation in prior models on drone delivery [18]. In this research, expected risks are understood as concerns with respect to the future introduction of drone delivery in urban areas, directly affecting the attitude. fi 3. Participants positively assessed the outlook of drones to provide the possibility to determine a package drop-off by time and location. Other stated expec- tations of potential benefits were the high reliability of drone delivery and the option to receive packages faster. Furthermore, the eventuality of drone delivery being environmentally friendly was highlighted posi- tively, thus forming a central aspiration. 4. Finally, discussions about drone delivery tended to be strongly influenced by participants’ opinions about technology in general. On the one hand, some partici- pants expressed enthusiastic technological optimism, often coupled with a fascination for new technolo- gies. In stark contrast to that stood the technological skepticism or even technophobia expressed by other participants. 2.2.1  Focus groupsh The variables included in the presented model are derived from the analysis of five focus group discussions, which scrutinized delivery drones from a societal perspective. They were conducted in the German capital Berlin and in the state capitals, Stuttgart and Erfurt, in September and October 2019. The aim of these focus groups was to explore the public’s basic perception as well as the asso- ciated beneficial expectations and concerns towards automated drone delivery as a possible element of future urban logistics. Kellermann et al. European Transport Research Review (2023) 15:38 Page 5 of 14 2.3  Survey Based on the focus group findings, the survey questionnaire was designed and administered in a fully structured com- puter-assisted telephone interview (CATI) with 1000 inter- views in January 2020. In the beginning, respondents were informed that the survey thematizes the future of urban transport. All items were then asked with the agreement or disagreement of different statements on a five-point Likert scale. In addition, "don’t know/no answer" was included as a response option. To minimize distortion of the response behavior due to fixed item sequences, the corresponding item batteries were randomized [58]. Moreover, to mini- mize Acquiescence Response Bias [59], the questionnaire followed a query of factors by an alternation of positively and negatively formulated items. The composition of the sample corresponds to the structure of the total German- speaking population in Germany aged 18 and over. This means that the original dataset is representative. 2.2.2.3  Expected benefits  Following TAM, the perceived usefulness of a new technology has a direct correlation to respondents’ attitudes towards the object [15]. The same concept was already adopted in drone-related research [51, 54]. In this research, the variable of expected benefits describes a similar concept and refers to personal but also public benefits that a widespread introduction of drone delivery might contribute to. A direct correlation between expected benefits on attitude is proclaimed. H2  There is a significant association between the sub- ject’s expectation of benefits and its attitude to delivery drones. As potential predictors for the latent variable of expected benefits, in the survey, it was asked how impor- tant it would be for the respondent that parcel deliveries with drones would be (1) reliable, (2) fast, (3) environ- mentally friendly, (4) flexible in delivery time, and how important it would be (5) that drop-off locations can be determined by the customer. H3  There is a significant association between a subject’s technophilia and its attitude to delivery drones. H3  There is a significant association between a subject’s technophilia and its attitude to delivery drones. The latent variable of technophilia was measured by evaluating the respondents’ affinity to new technologies. The survey design drew on established questionnaires in the field, which already measured technophilia as a latent variable [57]. Accordingly, to measure technophilia, (1) the respondents’ subjective level of information about technologies, (2) their ability to get easily enthusiastic for new technology, and (3) their general interest in technol- ogy were surveyed. 2.2.2  Model and hypothesesh H1  There is a significant association between a subject’s expectation of risks and its attitude to delivery drones. Expected risks were measured by how problematic respondents considered (1) the noise from parcel deliv- eries with drones, (2) the potential decrease of personal Kellermann et al. European Transport Research Review (2023) 15:38 Page 6 of 14 Page 6 of 14 contacts between recipients and deliverers/drivers, (3) caused stress due to delivery drones flying around, (4) potential job losses of deliverers/drivers due to automa- tion of flights, (5) a potentially blocked view to the sky. 2.4  Sample After having removed entries with missing values from the initial dataset, a sample size of 819 respondents was employed for the statistical analysis of the model. The characteristics of the sample are indicated in Appendix 1. Around 29% were aged 18 to 39. The age group 40 to 59 was represented by about 40% of the respondents, and around 30% of persons were age 60+ years. About 39% of respondents had completed a technical or university degree. About 23% held a secondary school leaving cer- tificate or technical college entrance qualification. Only 37% of respondents were either retired or unemployed while most respondents reported a monthly net house- hold income of 4500 euros or more (about 27%). Spa- tially, about 21.1% of respondents lived in major cities of 500,000 or more inhabitants, while with 50%, most of the respondents originated from smaller to mid-size towns with a population between 5000 and 100,000 inhabitants. 2.2.2.4  Technophilia  While external variables, such as demographics, have already been part of TAM [15], lat- ter extensions to the model emphasized more strongly on individual dispositions [42]. In this research, a person having a technophile disposition is considered to evalu- ate and perceive novel technologies in a positive way to improve his or her own life as well as society as a whole [55]. Constructs such as personal innovativeness [18] or cognitively motivated consumer innovativeness [21] were already emphasized by acceptance research studies in the field [18],Lin [56]. Henceforth, a direct association of tech- nophilia with attitude is suggested. Despite the removal of entries with missing values hav- ing reduced the sample size, the sample of 819 respond- ents still closely corresponded to the socio-demographics of Germany (Statistisches [60–63]. Larger deviations from the socio-demographics of Germany exist, however, regarding gender distribution, as around 51% of the pop- ulation in Germany are women [64] while in the survey, they account for only 45.5%. 3  Data analysis and results Job loss …that parcel deliveries with drones would block your free view of the sky Blocked sky  Expected benefits How important would be for you… …that parcel deliveries with drones would be reliable Reliability 0.789 …that you would get your parcels delivered quickly Fast delivery …that parcel deliveries with drones would be environmentally friendly Environmentally friendly …that you could use drones to have parcels delivered to a place of your choice, e.g., balcony or garden Spatial flexibility …that your parcels would be delivered at an exact time of your choice Time flexibility  Attitude How much would you agree to the following statement: I think that parcel deliveries with drones are environmentally friendly than package deliveries with a delivery van Environment 0.834 I imagine parcel deliveries with drones to be safe Safety Parcel deliveries with drones would have a positive effect on the quality of life in cities Quality of life Parcel deliveries with drones would bring me advantages in my everyday life Utility Variable names Cronbach alpha The comparative fit index (CFI) analyses the model fit by evaluating the discrepancy between the data and the hypothesized model, and its value is in the range from 0 to 1, and the larger values indicate better fit. A CFI value of 0.90 or larger is considered to indicate an accept- able model fit. The calculated CFI in this model is 0.936. Tucker–Lewis index (TLI), also known as the non-nor- med fit index, is an incremental fit index. Bentler and Bonett [68] recommended that TLI > 0.90 indicates an acceptable fit. In this model TLI is 0.923. RMSEA (root mean square error of approximation) is one of the most applied measures to check structural equation models. RMSEA is the root mean square error of approxima- tion, indicating how well the model, with unknown but optimally chosen parameters, would fit the populations’ covariance matrix [69]. It is “one of the most informa- tive fit indicators because of its sensitivity to the number of estimators in the model” [70]: 85). The values of 0.01, 0.05 and 0.08 indicate excellent, good, and mediocre fit respectively. In this model, RMSEA is around 0.05 which indicates good fit. threshold (0.7), which confirmed the high reliability of the constructs. The result of the Cronbach test is indi- cated in Table 1. 3  Data analysis and results Before examining the structural model, we checked the multicollinearity assumption by using the value of the variance inflation factor (VIF). All the con- structs were considered predictors of one of the constructs and calculated the VIF scores. The VIF scores are less than 2.00, which is less than the recommended value of 10, sug- gesting minimal collinearity [65]. SEM was then utilized to estimate the hypothesized relationships. The analyses pro- vided acceptable fit indices for the structural model. 3  Data analysis and results For the statistical analysis of the model, we employed the above-mentioned 819 samples. All the values of Cron- bach’s alpha and CR were above the recommended Kellermann et al. European Transport Research Review (2023) 15:38 Page 7 of 14 Table 1  Reliability of constructs by Cronbach Alpha Test Latent variable Observed variable Variable names Cronbach alph  Technophilia How much would you agree to the following statement: In general, I am well informed about new technologies Well informed 0.798 I get easily enthusiastic about new technologies Enthusiastic I am always interested in new technologies Interested  Expected risks How bad it would be for you… …the noise from parcel deliveries with drones Noise 0.778 …the fact that parcel deliveries with drones would mean the personal contact between recipient and delivery driver would disappear? Loss of personal contact …the stress caused by drones flying around to deliver packages Stress …the fact that delivery drivers would lose their jobs as a result of parcel deliveries with drones? 3  Data analysis and results Job loss …that parcel deliveries with drones would block your free view of the sky Blocked sky  Expected benefits How important would be for you… …that parcel deliveries with drones would be reliable Reliability 0.789 …that you would get your parcels delivered quickly Fast delivery …that parcel deliveries with drones would be environmentally friendly Environmentally friendly …that you could use drones to have parcels delivered to a place of your choice, e.g., balcony or garden Spatial flexibility …that your parcels would be delivered at an exact time of your choice Time flexibility  Attitude How much would you agree to the following statement: I think that parcel deliveries with drones are environmentally friendly than package deliveries with a delivery van Environment 0.834 I imagine parcel deliveries with drones to be safe Safety Parcel deliveries with drones would have a positive effect on the quality of life in cities Quality of life Parcel deliveries with drones would bring me advantages in my everyday life Utility Table 1  Reliability of constructs by Cronbach Alpha Test Latent variable Observed variable Variable names Cronbach al  Technophilia How much would you agree to the following statement: In general, I am well informed about new technologies Well informed 0.798 I get easily enthusiastic about new technologies Enthusiastic I am always interested in new technologies Interested  Expected risks How bad it would be for you… …the noise from parcel deliveries with drones Noise 0.778 …the fact that parcel deliveries with drones would mean the personal contact between recipient and delivery driver would disappear? Loss of personal contact …the stress caused by drones flying around to deliver packages Stress …the fact that delivery drivers would lose their jobs as a result of parcel deliveries with drones? 3.2  Factor loadings Each of the four constructs in the model is measured by at least three observed variables. The load factors are indicated in Table 2 and visualized in Fig. 3. Results sug- gest which observed variables most reliably measure the respective construct. i Therefore, the first relevant result of our research is the robust composition of attitude as a target vari- able. In addition to individual aspects (personal utility of drone delivery), the composition includes a strong community-oriented character (positive environmen- tal effects, quality of urban life, public safety), which is why the here-used term "public" attitude seems appro- priate. The standardized estimates show that especially the impact of drone delivery on the urban quality of life (0.798) serves as a good predictor for public attitude. The standardized estimates between the latent variable attitude and its measurement indicate that the observed variable of the environment has the least standardized estimates (0.67), while the other three measurements have almost similar load factors, which are quality of life (0.8), Safety (0.74), utility (0.79). Testing of the research hypotheses showed mixed results. Among the measurements for the latent variable expected benefits, the observed variables of fast deliv- ery and flexibility in time show the highest standardized load factor by 0.76 and 0.74, respectively. The influence of delivery drones’ relative advantage of environmental friendliness (0.40) is considerably weaker, nevertheless significant. i. The model reveals a significant association of atti- tude with expected risks (H1). These results are generally consistent with previous risk studies [50, 71, 72], which illustrate a negative effect of poten- tial risk factors on attitude and technology adop- tion. The more the movement of delivery drones in urban space is expected to be stressful (0.838) and noisy (0.678), would generate the impression of blocked skies (0.718), the more respondents create a negative attitude towards the technology. While i. The model reveals a significant association of atti- tude with expected risks (H1). These results are generally consistent with previous risk studies [50, 71, 72], which illustrate a negative effect of poten- tial risk factors on attitude and technology adop- tion. The more the movement of delivery drones in urban space is expected to be stressful (0.838) and noisy (0.678), would generate the impression of blocked skies (0.718), the more respondents create a negative attitude towards the technology. 4  Discussionh The aim of this study was to identify significant factors and their direction of influence on the formation of atti- tudes towards drone delivery. For this purpose, a SEM was tested on a sample of 819 respondents and showed reliable results. The Comparative Fit Index (CFI), the Tucker-Lewis Index (TLI), and the Root Mean Square Error of Approximation (RMSEA) indicate an accept- able model fit of 0.936, 0.923 and 0.05, respectively. The composition of the four latent constructs of public atti- tude, expected risks, expected benefits, and technophilia from the total of 17 observed variables derived from qualitative research can be confirmed. 3.3  Structural model and hypothesis Table  3 shows the results of the relationships. The model reveals significant associations of attitude with expected benefits (H2) by (β = 0.439, p < 0.001) and expected risks (H1) by (β = −0.738, p < 0.001). Hence, H1 and H2 are confirmed. In contrast, the p-value of H3 is 0.111, which means that the model does not sug- gest a significant association between technophilia and attitude. 3.1  Fitness of modelhi The fitness of the model is checked through the compara- tive fit index (CFI), the Tucker–Lewis index (TLI), and the root mean square error of approximation (RMSEA). As the chi-square statistic is sensitive to the large sample size [66], it is recommended to use the normed chi-square, that the chi-square is divided by the degrees of freedom (χ2/df) as a measure of model fit, with the acceptance-value of 5 or less [67]. The chi-square of this model is 458.389 with df = 113, therefore the normed chi-square is 4.05. Kellermann et al. European Transport Research Review (2023) 15:38 Page 8 of 14 Table 2  Results of the measurements and constructs  (***P ≤ 0.001) Estimate S.E Standardized estimates P Attitude Environment .856 .045 .666 *** Quality of life .991 .042 .798 *** Safety .872 .041 .737 *** Utility 1.000 .789 Expected risks Noise 1.000 .678 Loss of personal contact .917 .072 .503 *** Stress 1.345 .069 .838 *** Job loss .722 .061 .465 *** Blocked sky 1.299 .074 .718 *** Expected benefits Time flexibility 1.000 .737 Spatial flexibility .993 .054 .708 *** Fast delivery .968 .050 .760 *** Reliability .853 .051 .646 *** Environmentally friendly .456 .043 .401 *** Technophilia Well informed .743 .045 .612 *** Enthusiasm 1.086 .056 .844 *** Interested 1.000 .811 Table 2  Results of the measurements and constructs  (***P ≤ 0.001) Finally, technophilia is measured most reliably by interest and enthusiasm in new technologies with standardized estimates of 0.81 and 0.844, respectively, while the level of being informed shows a weaker yet significant influence on technophilia (0.61). 3.2  Factor loadings While i In the construct of expected risks and its measure- ment, the observed variables of stress and blocked sky view show the highest standardized estimates, which are 0.84 and 0.72, respectively, while the variable of Job loss has the lowest impact factor (0.46) on the latent variable. Kellermann et al. European Transport Research Review (2023) 15:38 Page 9 of 14 Fig. 3  Structural equation model noise, in particular, has been addressed in accept- ance research and engineering sciences [73, 74], the above-mentioned aesthetic implications of drones and their salient for citizens have yet to receive little attention [31, 75]. Furthermore, our study unveiled the significance of losing personal contact with delivery personnel (0.503) in forming the atti- tude towards drone delivery. This aspect was pre- viously confirmed in the context of adopting self- service parcel lockers [76], but—apart from general considerations [23]—had not yet been explic- itly analyzed in the context of drone delivery. As another social factor, the risk perception of drone delivery generating job losses (0.465) was shown to have a significant negative effect on the public atti- tude. This outcome appears to be strongly consist- ent with related works, particularly Aydin [32]. ii. The SEM reveals a significant association of atti- tude with expected benefits (H2). These results are consistent with findings within technology accept- ance research which have repeatedly demonstrated the central relevance of beneficial factors for posi- tively affecting attitudes towards technology adop- tion in various contexts [15, 50, 72]. Specifically, our study provides evidence that people form a more positive attitude towards the technology who expect the delivery to be faster (0.760) and more flexible regarding delivery date (0.737) and location of parcel drop-off (0.708). Moreover, the expecta- tion of delivery drones providing a reliable service (0.646) and being environmentally friendly (0.401) were also shown to form a more positive attitude. Fig. 3  Structural equation model Table 3  Results of the structural mode (***P ≤ 0.001) Hypotheses Standardized Estimates P Results H1: Attitude < –- Expected risks − 0.520 *** Supported H2: Attitude < –- Expected benefits 0.424 *** Supported H3: Attitude < –- Technophilia 0.053 0.111 Not supported the central relevance of beneficial factors for posi- tively affecting attitudes towards technology adop- tion in various contexts [15, 50, 72]. 3.2  Factor loadings Specifically, our study provides evidence that people form a more positive attitude towards the technology who expect the delivery to be faster (0.760) and more flexible regarding delivery date (0.737) and location of parcel drop-off (0.708). Moreover, the expecta- tion of delivery drones providing a reliable service (0.646) and being environmentally friendly (0.401) were also shown to form a more positive attitude. noise, in particular, has been addressed in accept- ance research and engineering sciences [73, 74], the above-mentioned aesthetic implications of drones and their salient for citizens have yet to receive little attention [31, 75]. Furthermore, our study unveiled the significance of losing personal contact with delivery personnel (0.503) in forming the atti- tude towards drone delivery. This aspect was pre- viously confirmed in the context of adopting self- service parcel lockers [76], but—apart from general considerations [23]—had not yet been explic- itly analyzed in the context of drone delivery. As another social factor, the risk perception of drone delivery generating job losses (0.465) was shown to have a significant negative effect on the public atti- tude. This outcome appears to be strongly consist- ent with related works, particularly Aydin [32]. Table 3  Results of the structural mode (***P ≤ 0.001) Hypotheses Standardized Estimates P Results H1: Attitude < –- Expected risks − 0.520 *** Supported H2: Attitude < –- Expected benefits 0.424 *** Supported H3: Attitude < –- Technophilia 0.053 0.111 Not supported ble 3  Results of the structural mode (***P ≤ 0.001) ii. The SEM reveals a significant association of atti- tude with expected benefits (H2). These results are consistent with findings within technology accept- ance research which have repeatedly demonstrated Page 10 of 14 Kellermann et al. European Transport Research Review (2023) 15:38 Page 10 of 14 Kellermann et al. European Transport Research Review (2023) 15:38 These results are partly consistent with studies focusing on drone deliveries, which also found the relative advantages of speed and environmental friendliness [18], respectively a “green image” [21], to be significant predictors for a positive attitude towards delivery drone usage. In addition, the pre- ceding focus groups have qualitatively revealed the factors of reliability and flexibility (time, location as new and previously uncharted factors influenc- ing attitudes towards drone delivery. 3.2  Factor loadings In contrast to other studies that rather focused on examining the factor of reliability in the context of a poten- tial "performance risk" [18, 25], the focus groups unveiled that participants actually considered ser- vice  reliability as a beneficial factor. This concep- tual re-interpretation was confirmed in our model and may contribute to a more precise definition of acceptance factors in future studies. of drone delivery might yet appear abstract and hardly imaginable, such as the suggested flexible drop off time and locations for packages, or benefits might be per- ceived as not sufficiently compelling. This, in turn, may to some extent, be explained by the lack of familiarity and experience with potential beneficial features. Leading behavioral and technology adoption models have demon- strated the relevance of familiarity and experience with a technology [17]. This may particularly apply to drone ser- vices, which have just been rarely implemented. Conse- quently, experience and real-life applications with drone deliveries may become a relevant attitudinal aspect [79]. On the other hand, individuals without real-life experi- ence might more easily imagine obvious threats of drone delivery (physically falling, being misused for criminal purposes, being noisy, or violating privacy) compared to imagining potential benefits. i Beyond that, the comparably lower influence of expected benefits against expected risks strengthens the assumption that in the sample, drone delivery is not con- sidered a prior necessity for improving the delivery sys- tem. This may also be explained by respondents’ strongly varying environments. Most respondents live in rural areas or smaller towns or cities below 500,000 inhabit- ants (79% of respondents), where congestion problems affecting last-mile delivery traffic may not be experienced as critical as in bigger urban areas of more than 500,000 inhabitants (21% of respondents). Moreover, respondents may live in heterogeneous housing conditions (single house, multi-story apartment building etc.), thus possi- bly creating varying perceptions of drone deliveries being practically feasible in their individual environments. iii. i. The model does not suggest a significant associa- tion between a person’s technophilia and attitude (H3) towards drone delivery. The result seems trustworthy, as the latent variable was measured in accordance with an established questionnaire [57] by three separate items that show a good Cron- bach alpha (0.8). Appendix 1: Socio‑demographic distribution of the sample Appendix 1: Socio‑demographic distribution of the sample pp g p of the sample Attribute Value Count % Gender Male 445 54.3 Female 373 45.5 Divers 1 0.1 Age 18–29 years 84 10.3 30–39 years 152 18.6 40–49 years 157 19.2 50–59 years 173 21.1 60+ years 253 30.9 Education Without lower secondary/ vocational school leaving certificate 3 0.4 Lower secondary/voca- tional school leaving certificate 62 7.6 Secondary school leaving certificate, technical college entrance qualification 199 24.3 Completion of polytechnic secondary school (8th/10th grade) 20 2.4 Advanced technical college entrance qualification, completion of a specialized upper secondary school 63 7.7 High school diploma, general or subject-linked higher education entrance qualification 144 17.6 Technical/college studies 317 38.7 Other school-leaving qualification 9 1.1 No indication 2 0.2 Employment Employed 519 63.4 Unemployed 300 36.6 Household monthly income Below 500 EUR 4 0.5 3.2  Factor loadings This suggests that even consum- ers who have a general interest in learning about new technologies, who consider themselves to know a lot about new technologies and easily feel enthusiastic about using and trying them, don’t create significantly more positive attitudes towards drone delivery services. This finding is in contrast to previous studies that highlighted the relevance of concepts such as “personal innovativeness” [18] or “cognitively motivated consumer innovative- ness” [21] in significantly influencing attitudes and, thus technology adoption. Our finding of a person’s technophilia proving to be neglectable for forming attitudes towards drone delivery appears especially remarkably as technophilia was found to have a sig- nificant positive correlation with forming attitudes towards the adoption of electric vehicles [77] or on the attitude towards drone-enabled passenger transport in air taxis [78]. The later may indirectly hint at drone logistics being perceived as less spec- tacular and rather pragmatic compared to the indi- vidual thrill and risk of being transported as a pas- senger in an automated drone. Irrespective of the geographical context, prior stud- ies demonstrated that commercial drone delivery is not considered among the most relevant use cases of drone technology [13]. Furthermore, results from technology assessment show that added values of delivery drones often remain abstract and must be made more plausible to the public in order to sustain a publicly accepted usage of urban airspace as a new transport layer [80]. Strong added values can be created, however when the deploy- ment of the technology is more evidently targeted to serve the common good, e.g. in medical or humanitarian use cases [81]). This study faces several limitations. First, the transfer- ability of results may be restricted as data were collected in Germany only. However, a cross-national study on the acceptance of drone applications in Europe shows no serious divergences between the examined member countries [13]. Against the background of this finding, the model framework can be considered a relevant research reference for future studies in the European context or regions with comparable socio-demographic features as Concerning the relationship between expected benefits and risks, we found the latter to have a stronger asso- ciation with the attitude towards drone delivery (−0.52) than the expected benefits (0.424). On the one hand, this may be explained by the assumption that the benefits Kellermann et al. European Transport Research Review (2023) 15:38 Kellermann et al. 3.2  Factor loadings European Transport Research Review (2023) 15:38 Page 11 of 14 Page 11 of 14 used in this paper. Secondly, the survey data on drone delivery had the limitation of asking respondents about a technology that is not yet tangible in the daily life of citizens. Future applications of drone delivery should emphasize accompanying social science research to col- lect alternative data. Third, the database for this study consists of a survey that was conducted in January 2020, so right before the outbreak of the Covid-19 pandemic. As various studies have highlighted that the pandemic induced (long-term) changes in mobility and consump- tion behaviors [82, 83], our findings may not reflect these changed societal framework conditions. As people con- fronted with the pandemic might have perceived a big- ger value of contact-less delivery methods than before the pandemic, a share of respondents might have slightly reduced their expectation of risks and might have slightly increased their expectation of benefits related to drone delivery [84]. In addition, the results of this research show that techno- philia, as we framed the individual innovativeness towards technology, has no significant association with the attitude towards drone delivery. This may have general implications for the industry as drone service providers may not easily be able to count on the innovativeness of certain consumer subgroups, e.g., innovators or early adopters. Finally, the presented model may be of methodologi- cal utility as both the qualitative identification of novel acceptance factors and their quantitative transition into a survey and the test in a model highlights the appropriate- ness of following a mixed methods approach in technol- ogy acceptance research. Availability of data and materials The primary survey dataset (telephone survey) is available on request to the authors. 17. Rogers, E. M. (2003). Diffusion of innovations (5th ed.). Free Pre 18. Yoo, W., Eun, Yu., & Jung, J. (2018). Drone delivery: factors affecting the public’s attitude and intention to adopt. Telematics and Informatics, 35(6), 1687–1700. https://​doi.​org/​10.​1016/j.​tele.​2018.​04.​014 Funding Funding Open Access funding enabled and organized by Projekt DEAL. g Open Access funding enabled and organized by Projekt DEAL. 16. Fishbein, M., & Ajzen, I. (1975). Belief, attitude, intention, and behavior: An introduction to theory and research. Addison-Wesley series in social psychology. Reading, Mass: Addison-Wesley Pub. Co. Received: 20 May 2022 Accepted: 28 August 2023 21. Mathew, A. O., Jha, A. N., Lingappa, A. K., & Sinha, P. (2021). Attitude towards drone food delivery services—Role of innovativeness, perceived risk, and green image. Journal of Open Innovation: Technology, Market, and Complexity, 7(2), 144. https://​doi.​org/​10.​3390/​joitm​c7020​144 5  Conclusionsh Delivery drones from a technology assessment perspective. Institute for Technology Assessement Vienna (ITA). http://​epub.​oeaw.​ac.​at/​ita/​ita-​proje​ktber​ichte/​2018-​01.​pdf. 11. Nentwich, M., & Horváth, D. M. (2018). Delivery drones from a technology assessment perspective. Institute for Technology Assessement Vienna (ITA). http://​epub.​oeaw.​ac.​at/​ita/​ita-​proje​ktber​ichte/​2018-​01.​pdf. Acknowledgements h d b f h 12. Dannenberger, N., Schmid-Loertzer, V., Fischer, L., Schwarzbach, V., Kellermann, R., Biehle, T. (2020). Traffic solution or technical hype? Repre- sentative population survey on delivery drones and air taxis in Germany. https://​skyli​mits.​info/​wp-​conte​nt/​uploa​ds/​2020/​06/​Sky-​Limit​s-_​Resul​ts_​ Survey_​engli​sh.​pdf. The data base of this article (focus groups and telephone survey) originates from the research project “Sky Limits” that was conducted between Janu- ary 2019 and December 2020 (funded by the German Federal Ministry for Research and Education (BMBF), Grant Number: 16ITA216A). 13. EASA. (2021). Study on the societal acceptance of Urban Air Mobility in Europe. https://​www.​easa.​europa.​eu/​sites/​defau​lt/​files/​dfu/​uam-​full-​ report.​pdf. 13. EASA. (2021). Study on the societal acceptance of Urban Air Mobility in Europe. https://​www.​easa.​europa.​eu/​sites/​defau​lt/​files/​dfu/​uam-​full-​ report.​pdf. Author contributions Dr. Robin Kellermann: Conceptualization, Project Administration, Supervision, Writing—Original Draft. Tobias Biehle: Conceptualization, Formal analysis, Methodology, Visualization, Writing—Review & Editing. Dr. Hamid Mostofi: Data Curation, Formal analysis, Methodology, Visualization, Validation, Writing— Review & Editing. 14. Cesur, A., Yi̇Ğenoğlu, K., Aydin, İ, Çeli̇K, Z. (2022). A biblyometric analysis of empirical studies on drone delivery. Yüzüncü Yıl Üniversitesi Sosyal Bilimler Enstitüsü Dergisi. https://​doi.​org/​10.​53568/​yyusb​ed.​11720​19. 14. Cesur, A., Yi̇Ğenoğlu, K., Aydin, İ, Çeli̇K, Z. (2022). A biblyometric analysis of empirical studies on drone delivery. Yüzüncü Yıl Üniversitesi Sosyal Bilimler Enstitüsü Dergisi. https://​doi.​org/​10.​53568/​yyusb​ed.​11720​19. 15. Davis, F. D. (1989). Perceived usefulness, perceived ease of use, and user acceptance of information technology. MIS Quarterly, 13(3), 319. https://​ doi.​org/​10.​2307/​249008 Declarations 19. Hwang, J., Kim, J. J., Lee, K.-W. (2021). Investigating consumer innovative- ness in the context of drone food delivery services: Its impact on attitude and behavioral intentions. Technological Forecasting and Social Change 163(February). https://​doi.​org/​10.​1016/j.​techf​ore.​2020.​120433. 4. Haidari, L. A., Brown, S. T., Ferguson, M., Bancroft, E., Spiker, M., Wilcox, A., Ambikapathi, R., Sampath, V., Connor, D. L., Lee, B. Y. (2016). The economic 5  Conclusionsh The vision of commercial drone delivery in urban envi- ronments incorporates not only an individual service choice for potential customers but also entails an inher- ently public dimension. In current acceptance research, this comprehensive understanding appears rather under- represented. Therefore, this study aimed to build and test an integrated model to define and explain the public atti- tude towards drone delivery that includes both relevant aspects of consumer preferences as well as significant societal and public dimensions. Using a large sample (n = 819) from a representative survey in Germany from 2020, the results of the model reveal insights of practical utility. We confirm that par- ticularly the expectation of a fast and time-flexible delivery are highly valued attributes from a customer per- spective. On the other hand, especially the expectation of spatial and visual implications (noise, visual distur- bance, and rising stress levels through drone movements) are relevant factors that negatively impact respondents’ attitudes from a citizen’s perspective. Rather than solely focusing on individual aspects of adopting the use of drone delivery, we, therefore, suggest that the planning of business cases and drone-related policies should give stronger emphasis on the consideration of public dimen- sions of drone delivery. What is more, by highlighting the centrality of social risk factors that outweigh individual benefits, the results suggest that the public is currently not willing to accept the risks of large-scale drone delivery of consumer goods. From an acceptance perspective, rather than focusing on business-to-customer solutions, drone delivery service providers may generate more robust business cases by focusing on business to business applications. Kellermann et al. European Transport Research Review (2023) 15:38 Page 12 of 14 Attribute Value Count % 500 until below 1.000 EUR 21 2.6 1.000 until below 1.500 EUR 53 6.5 1.500 until below 2.000 EUR 58 7.1 2.000 until below 2.500 EUR 87 10.6 2.500 until below 3.000 EUR 74 9.0 3.000 until below 3.500 EUR 72 8.8 3.500 until below 4.000 EUR 76 9.3 4.000 until below 4.500 EUR 80 9.8 4.500 and more 222 27.1 No indication 72 8.8 City size Below 5.000 EW 119 14.5 Between 5.000 and 20.000 177 21.6 Between 20.000 and 100.000 227 27.7 Between 100.000 and 500.000 123 15 More than 500.000 173 21.1 10. Doole, M., Ellerbroek, J., Hoekstra, J. (2018). Drone delivery: Urban airspace traffic density estimation. https://​www.​sesar​ju.​eu/​sites/​defau​lt/​files/​ docum​ents/​sid/​2018/​papers/​SIDs_​2018_​paper_3.​pdf. 11. Nentwich, M., & Horváth, D. M. (2018). Competing interests Not applicable. Competing interests Not applicable. Not applicable. 20. Hwang, J., & Kim, H. (2019). Consequences of a green image of drone food delivery services: The moderating role of gender and age. Business Strategy and the Environment, 28(5), 872–884. https://​doi.​org/​10.​1002/​bse.​ 2289 Received: 20 May 2022 Accepted: 28 August 2023 Received: 20 May 2022 Accepted: 28 August 2023 References 1. ITF. (2021). Ready for take off? Integrating drones into the transport system. ITF Research Reports. Paris: OECD Publishing. 1. ITF. (2021). Ready for take off? Integrating drones into the transport system. ITF Research Reports. Paris: OECD Publishing. 22. Mittendorf, C., Franzmann, D., Ostermann, U. (2017). Why would custom- ers engage in drone deliveries? AIS Electronic Library. https://​core.​ac.​uk/​ reader/​30137​1848. 2. SESAR Joint Undertaking. (2018). 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16B8509 Multiple Case Studies
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BB 4312 ORGANIZATIONAL DEVELOPMENT AND CHANGE INDIVIDUAL REPORT “Multiple Case Studies” Prepared By: Nurul Azzimatul Adawiyah Hj Omar Student ID: 16B8509 Prepared For: Pg Dr Siti Rozaidah Pg Hj Idris Submission Date: 5 May 2020 (Tuesday) Table of Contents Abstract ................................................................................................................................................... 3 1.0 Introduction ....................................................................................................................................... 4 1.1 Problem Statement ........................................................................................................................ 4 1.1.1 BruTEL 's 'Going Paperless' Initiative ................................................................................... 4 1.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District .................... 4 1.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project .............. 5 2.0 Discussion ......................................................................................................................................... 6 2.1 Case Analysis ................................................................................................................................ 6 2.1.1 BruTEL's 'Going Paperless' Initiative .................................................................................... 6 2.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District .................... 7 2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project .............. 8 2.2 Alternative Solutions .................................................................................................................... 9 2.2.1 BruTEL's 'Going Paperless' Initiative .................................................................................... 9 2.2.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District .................... 9 2.2.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project ............ 10 3.0 Recommendation ............................................................................................................................ 11 References ............................................................................................................................................. 12 Table of Contents Abstract This is a multiple case study report of three local cases: 1) BruTEL 's 'Going Paperless' Initiative, 2) Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District, and 3) Society for Community Outreach and Training (SCOT)’s Green Xchange Project. The objective of this report is to identify and discuss the organizational development and change strategy or strategies employed in each of the cases. The writer also discusses the key raised issues based on some scholar’s planned change theories. A few recommended solutions are included in this report. It is found that communication is the most optimal strategy solution that befit into all three cases. This is because, this type of strategy is the most general and best fit to all types of interventions when planning a change within the organization. Keywords: ODC strategies, planned change theory, Lewin’s three step model, Kotter’s eight step model, positive model, communication 1.0 Introduction This multiple case study report consists of the analyses of three cases: 1) BruTEL 's 'Going Paperless' Initiative, 2) Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District, and 3) Society for Community Outreach and Training (SCOT)’s Green Xchange Project. The main objective of this multiple case study report is to identify the organizational development and change strategy or strategies employed in these three cases. The writer will first identify the key raised issues from each of these cases. These issues will later be discussed based on the organizational change theory in the Discussion section. A number of solutions will also be discussed. At the end of this report, the writer will recommend the most optimal solutions to be deployed in these cases, followed by the conclusion of this case studies. 1.1 Problem Statement In this section, it will consists of the overview of the raised issue(s) from each of the cases (assuming the reader has read the cases mentioned). 1.1.1 BruTEL 's 'Going Paperless' Initiative It is stated that BruTel is planning to ‘go paperless’ where they wished to introduce an absolute utilization of the technology in their business and omit the usage of papers. The main aim of this plan is to accomplish the third goal of BruTel i.e. to conserve the natural environment. However, some segments of its customers are giving the adverse response – especially the elder people. It is said that majority of the elderly experienced some difficulties on using the technology due to the computers incompetency which caused them to insist on the traditional system (using paper to make transactions). This therefore hinders BruTel to achieve its third goal. Hence, the main issue for BruTel is the reluctance or hesitance of these customer segments to the intended system change. It is also stated that if BruTel succeed on persuading this group of customers to switch to the new system, BruTel could smoothly develop and execute its strategies on the ‘go paperless’ system. 1.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District Similar to the BruTel case, this case is also facing the challenges on implementing the green activities. It is said that Sumbiling Eco Village (SEV) is having issue in obtaining regular sources of electricity and water. This is because the location of SEV is far from the city where the flow of these sources are quite limited. Besides having a scarce source of electricity and water, SEV is also scarce in skilled workers who are well versed in the green activities. Another issue is that SEV struggles to maintain the facilities such as the benches and chairs due to the inevitable circumstances such as fluctuations in climate i.e. Brunei humid weather and the salinity of the wind (this is because SEV is located next to the salt-water river bank of Sungai Temburong). These type of nature caused the fast erosion of SEV facilities and architectures. In this regard, SEV’s founder wanted help from the building schemes that utilized green practices to prolong its facilities ‘endurance. Not only that, SEV also felt threatened by the existence of other ecotourism spots in the neighbouring countries such as Sarawak and Miri; which they may have better offers in its tourism businesses. Additionally, the local communities and the indigenous cultures may also be ‘polluted’ by the foreign visitors and wealth. 2.1.1 BruTEL's 'Going Paperless' Initiative It is stated that BruTel is having challenges in implementing the new change initiative. It is also stated that the status quo are from some of its customer segments – particularly the elder people, where they are hesitant or resistant to the change initiative i.e. go paperless project; this project is also in line with BruTel’s third goal of corporate social responsibilities (to be conservative to the natural environment). It is understood that the main cause for these resistance are because the elder people are not techno-savvy and are not familiar to the usage of technology. This indicates that BruTel’s main issue is the difficulty in making the customer (elder people) to ‘digest’ and adapt to the new intended change (switching from traditional transaction to electronic billing). Therefore, it is assumed that BruTel is utilizing the Lewin’s three step model. Lewin’s three step model functions to evaluate the organizational change process; and to evaluate the challenges caused from the status quo on the implementation of the change. Beforehand, the following is the brief explanation of the three stages of Lewin’s model (Cummings & Worley, 2009): 1) Freezing – at this stage, the organization prepares the initial set of activities of the intended change. This is to gently introduce the organization members or other parties on the intended change. 2) Moving – this is a process when people starts to ‘digest’ the new change. At this stage, status quo may still exist. 3) Refreezing – this is when people are accustomed and stabilized to the new change. And when this occur, the organization is ready to consolidate its planned change initiatives. Referring to the above model, at the first stage, BruTel has successfully prepared the change initiative (‘go paperless’ initiative) as in line with its third corporate social responsibilities goal i.e. being conservative to the natural environment. At the second stage, it is where BruTel faces the status quo of its customers (elderly), where they resist to the change of going paperless due to not being a techno-savvy and unfamiliar with the usage of technology. According to Kotter & Schlesinger (2008), these resistance to change are due to the misunderstanding and the lack of trust to the change. It is misunderstood of the boon effects of the change. These people assumes that the change would give them more bane as compared to boon. 2.1 Case Analysis 2.1 Case Analysis 1.1.1 BruTEL 's 'Going Paperless' Initiative Nevertheless, SEV founder is persistent in the establishment of SEV and the founder wanted to change people’s mind sets– especially the local community, about ecotourism. It is also stated that an active tourism activities in the natural areas tend to lead to environmental degradation. Therefore, it is crucial for SEV to set a thorough plans and management in order to minimize the negative impact from its activities – especially the jungle camping activities, towards the natural environment. 1.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project 1.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project Last but not least, the SCOT case. It can be considered that SCOT has run its Non-Government Organization (NGO) quite well. However, there are several issues raised throughout its operation. The first issue is, SCOT is highly reliant to the donations and funding from the government and private organization. The main advantage from these donations and funding are to realize SCOT’s next projects (after one project ends). This means that if there is not enough money to conduct the next project, SCOT would have difficulties in continuing the NGO to help the underprivileged people in entrepreneurship. Hence, SCOT’s mission of reducing poverty could also be hindered because the underprivileged people is dependent on SCOT’s project e.g. building coops for chicken rearing to earn money. In this regard, SCOT hopes that their Green Xchange project is sufficient enough to generate their own financial source. The Green Xchange project is to collect recyclable materials from the public and exchange it with basic necessities such as rice. However, through this activity, another issue arise. The price for the basic necessities are uncertain and may fluctuate as they are highly dependent to some factors such as supplier countries’ climate and currency rate. This in result, will affect the price of exchange rate between recyclable materials and the basic necessities. 2.0 Discussion This section will include the discussion of the root causes of the key raised issues. The writer will discuss these issues based of the assumed theories that is implemented in each cases based on the scholar’s planned change theory. The second subsection consists of the possible solution(s) that can be employed in the respective cases. 2.1 Case Analysis 2.1.1 BruTEL's 'Going Paperless' Initiative Thus, resulting in the lack of trust to the organization who implemented the change. In this case, the elder people can be considered to misunderstand of the benefit of ‘go paperless’ initiative. At the final stage, as per stated in the case study, if these elder customers are successfully persuade to switch to the new paperless system, then BruTel can proceed to develop and implement its green strategies. This indicate that BruTel’s dilemma on whether to continue or stop the green initiative to accomplish its third goal. This dilemma is as a response to the non-techno-savvy customer. Hence, it is concluded that BruTel failed to proceed to the final stage of the Lewin’s three step model due to the challenges of status quo caused from its elderly customers. It indicates that the main issue of BruTel is to get the change implementation to be fully digested and accepted by its elderly customers. 2.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District In is assumed that SEV utilizes the Kotter’s eight step model. Kotter’s eight step model consists of the of eight stages: 1) Establishing a sense of urgency, 2) Creating the guiding coalition, 3) Developing a vision and strategy, 4) Communicating the change, 5) Empowering the broad-based action, 6) Generating short-term wins, 7) Consolidating gains and producing more change, and 8) Anchoring new approaches in the culture (Cummings & Worley, 2009). Below are the brief explanation of each stages (“The 8-Step Process”, n.d.), followed by the writer’s analysis based on these eight stages: 1) Establishing a sense of urgency – in this stage, the organization are to seek an urgent and affirmed attention from the others and telling them that change is needed within the organization. Referring this to SEV case, SEV is aware that its organization is lacking the assets such as irregular flow of electricity and water, lack of skilled employees who are well versed on the green activities, as well as lacking in maintenance capability where their architectures are easily worn out by natural processes due ti its geographical factors (e.g. Brunei’s humid climate and saline wind from the salt-water river beside SEV). It is also stated that SEV feels threatened by the neighbouring ecotourism businesses which may offer better services. 2.1.1 BruTEL's 'Going Paperless' Initiative 2) Creating the guiding coalition – this is a process where the organization finds the most influential person or people (within or outside the organization) to create the most effective way to telling people on the importance of the change. In this case, it is stated that SEV is trying to get connected with green building schemes to help them prolonging its architectures and facilities’ endurance. It is also understood that SEV founder intends to urge the local community especially the nearby community to support SEV as it is mentioned that the nearby community are against SEV change initiative to run the ecotourism business. 3) Developing a vision and strategy – this phase involves the creation of the change vision in order to provide the organization members the clear idea or the rationale of the planned change. The development of strategies also takes place in this phase. Referring to the case, SEV founder explained that she intends to make its business more attractive (in architecture and services wise) in order to attract more customers, to be competitive with the other neighbouring competitors, as well as to change the community’s mind sets on the ecotourism. 4) Communicating the change – this is where the organization members gather as many people in the organization to ensure all organization members are aware of the new change and clear of its vision. As for this stage onwards, it is not stated in the case of whether SEV has conducted the strategies based on these stages of Kotter’s eight step model. Thus, the writer assumes that SEV has not gone to this stage yet. Hence, the writer will discuss on this stages relating to SEV case study in the Alternative Solution section of this report. 5) Empowering the broad-based action – this phase omits as much barriers as possible e.g. hierarchical barriers, in order to promote freedom in communication and it enables the members of the organization to do their task or jobs well. 6) Generating short-term wins – this process involves the structuring of the short term steps towards the long term goals. It is to create the sense of winning the goals often in which this action will promote the organization members to be motivated and can perform better. 7) Consolidating gains and producing more change – this phase involves in the constant effort put after success in order to produce more successful changes. This process is mainly to achieve the organization’s long term vision. 7) Consolidating gains and producing more change – this phase involves in the constant effort put after success in order to produce more successful changes. This process is mainly to achieve the organization’s long term vision. 8) Anchoring new approaches in the culture – this final stage of the planned change model consists of the conveyed connections between the new planned changes. 8) Anchoring new approaches in the culture – this final stage of the planned change model consists of the conveyed connections between the new planned changes. 2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project 2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project As for the SCOT case, it is assumed that SCOT uses the Positive model because it is stated in the case study that SCOT is focusing on its success activities to develop to a much better outcome. Positive model “focuses on positive dynamics in organizations that give rise to extraordinary outcomes” (Cummings & Worley, 2009). It means that the organization focuses on its good side as a driver to develop to a better outcome. Positive model has five stages: 1) Initiate the inquiry, 2) Inquire into Best Practice, 3) Discover the themes, 4) Envision a preferred future, and 5) Design and deliver ways ti create the future. 1) Initiate the Inquiry – this is a process where the organization members identifies the main organization issue. This is when SCOT identifies that they are facing financial issue where they highly reliant to the government and private organization’s funding and donations for most of its conducted projects. 2) Inquire into Best Practices – in this stage, communicated information amongst the organization members are gathered; it involves the topic on what is their best attributes. It is understood that SCOT has communicated to its staffs and volunteers that their Green Xchange project is the most successful project and that has the most potential to overcome its financial issue. 3) Discover the Themes – this is when the organization members shares ideas or innovation of change and create the theme of change. In this phase, SCOT founder itself belief that their Green Xchange project has benefitted them financially since it is fund-driven. This project at the same time enables them to achieve its mission on the green initiatives (raising green awareness and reducing poverty). financially since it is fund-driven. This project at the same time enables them to achieve its mission on the green initiatives (raising green awareness and reducing poverty). 4) Envision a Preferred Future – after all information are gathered, the members identifies which theme is the most mutually agreed. The members also envision its change initiative’s vision; it involves on the long term goal of the intended change. At this phase, SCOT has visualized the possible goals of its Green Xchange project where they strongly belief that this project would give them an internal financial source. 5) Design and Deliver Ways to Create the Future – this is the final stage of the Positive model. 2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project It involves the set of activities that are to be conducted to form and implement on the change strategies. As stated in the case study, SCOT has successfully innovate changes to achieve an even better outcome. They are also able to generate its own finance that is sufficient enough to be allocated to their next projects. Additionally, this project has also successfully promoted the public to the green awareness which is aligned with SCOT’s mission. This indicates that SCOT has fully utilized this change model. 2.2 Alternative Solutions In this section, the writer will suggest some solutions in order to overcome the issues faced in each case. 2.2.1 BruTEL's 'Going Paperless' Initiative 2.2.1 BruTEL's 'Going Paperless' Initiative  It is recommended that BruTel communicate to the elderly on the actual benefit of the ‘go paperless’ change. This is to ensure that the elder customers are aware of the green initiative – not only to the organization itself, but to the world as a whole.  BruTel should not completely implement the change of going paperless as according to Kotter & Schlesinger (2008), some people are limited to change that caused them to resist the change. To overcome this issue, BruTel may provide employees to guide them on how to use the technology. However, this strategy may not be as effective thus, BruTel could hire a specific employee to be assigned as the guider or helper whenever these non-techno-savvy people struggles on the new change. 2.2.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District  Since it is assumed that the SEV has not fully executed its change strategies, it is recommended for the SEV to continue its strategies from developing its change vision and strategies to communicating the change. It is crucial to communicate to the whole organization of the intended change. This is to aid and guide all members of the organization to conduct and execute its respective jobs. Additionally, this step is crucial in order to avoid any uncertainty experiences of the organization members. When they are uncertain, the change strategy would have not successfully executed.  Next is empowering the broad-based action; this is where the organization members of SEV, especially the manager, should avoid any barriers. The barriers include, allowing rumours, strict hierarchical structures during meeting and etc. This hinders the freedom of its member to express their feedback on the intended change which may affect the effectiveness of change strategies.  SEV also have to create appreciation more often for its employees. This is to create motivation to the employees. Thus, the employees could be motivated to exert its expertise in their jobs (e.g. survival skills for the jungle camping programme).  As for the seventh stage of Kotter’s eight step model, SEV should make continuous efforts in its strategies as SEV is an ecotourism business which most of its income are of the interaction with human. SEV should keep track of its customers’ feedback on the business as a whole. SEV should also pay attention to the nearby community where one of the SEV’s activity is jungle camping. 2.2.1 BruTEL's 'Going Paperless' Initiative This activity could or may interrupt the nearby community’s routines which eventually cause them to oppose to the establishment of SEV.  Last but not least, SEV should portray the necessity and the benefit of the implemented change. This is to help the process of adaptation and the acceptation of the new change from the respective affected parties to the SEV. 2.2.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project 2.2.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project  It is recommended that SCOT would have at least, another fund-generated project in order to generate its own self-financed green projects. This is to avoid limited options for its financial resources. Seeing the possibilities, if the Green Xchange project is no longer plausible to the public, then SCOT would have again dependent of other parties’ funds and donations.  SCOT should also communicate to the public in order to have an optimal effect on raising its green awareness. This action will eventually help the public to engage or participate to SCOT’s green activities and thus, enabling them to accomploish their missions (i.e. raising green awareness, promoting green behaviour and reducing poverty by teaching the underprivileged people to do entrepreneurship though managing waste materials). 3.0 Recommendation Through the analyses and the proposed solutions, it is concluded that communication is the best befitting strategy to be implemented in all three cases as this the most basic type of organizational development and change strategy. According to Kotter and Schleisinger (2008), communication is the best strategy to use when dealing with changes, especially when there is resistance to change. Communication provides people to give and take the rationale of changes. It also aids the organization member or any parties to have the clear view of what and why there is the needs to be change. Elving (2005) also mentioned in his study that communication promtes the sense of community where organization members can work in harmony. This will eventually enhances the execution of its organizational change strategies. While there may be several other solutions to overcome organizational change implementation, the writer believes that communication is the most common and the best solution to these three given cases. The 8-Step Process for Leading Change | Dr. John Kotter. Kotter. Retrieved 5 May 2020, from https://www.kotterinc.com/8-steps-process-for-leading-change/. References Cummings, T. G., Worley, C. G. (2009): Organization Development & Change (9th ed.). Cengage Learning. Elving, W. (2005). The role of communication in organisational change. Corporate Communications: An International Journal. 10. 129-138. 10.1108/13563280510596943. Kotter, J., & Schlesinger, L. (2008). Choosing Strategies for Change. Harvard Business Review. Retrieved 4 May 2020, from https://hbr.org/2008/07/choosing-strategies-for- change. The 8-Step Process for Leading Change | Dr. John Kotter. Kotter. Retrieved 5 May 2020, from https://www.kotterinc.com/8-steps-process-for-leading-change/.
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PUBLIC COMMUNICATION ON SOCIAL MEDIA: CONNECTING GOVERNMENTS AND CITIZENS ONLINE
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PUBLIC COMMUNICATION ON SOCIAL MEDIA: CONNECTING GOVERNMENTS AND CITIZENS ONLINE Author: Priscila Minussii PUBLIC COMMUNICATION ON SOCIAL MEDIA: CONNECTING GOVERNMENTS AND CITIZENS ONLINE Author: Priscila Minussii PUBLIC COMMUNICATION ON SOCIAL MEDIA: CONNECTING GOVERNMENTS AND CITIZENS ONLINE Author: Priscila Minussii Author: Priscila Minussii Social media use is growing worldwide. Although research shows that they are used primarily for leisure, it has been increasingly used for political and public communication. On the one hand, many experts have been condemning social media for undermining democracy and enhancing political polarisation. On the other hand, these networks potentially bring positive outcomes, such as greater transparency from public institutions and direct communication between governments and citizens. However, the institutions have a long way to go in tailoring their communications efforts to convey public information while appealing to the public successfully — and ultimately strengthening people's trust and interest in politics. If entertainment seems to be the most popular content on social media, how could public agencies compete for people's attention online? How could public communication be adapted to social media? This public service must not simply replicate the content designed for traditional broadcast media. Instead, it should balance different needs: the audience's demands and the states' commitment to informing citizens while maximizing each platform's particular demographics, features and algorithms. BACKGROUND authors found that 45% of all posts regarded symbolic and presentational exchanges, which m that the Facebook posts had messages containing a favourable presentation of the departm political positioning, symbolic acts and/or marketing. Further, they found that 17.5% of the p FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power of the Internet and how it could hinder citizen participation and engagement. Among the several criticisms, it’s argued that the same political elite tendencies of the pre-Internet age continue evolving in the new media age; digital communication is used predominantly as “a new technical system for distributing old-fashioned mass communications content” according to Meyer and Hinchman [6]. In the end of the day, the public only gets further from the political centre [7], and the expansion of new media remains far from achieving its potential for effectively engaging citizens. Regardless of such criticisms, governments worldwide are increasingly investing in and professionalizing their social media communication strategies. The reasons behind this approach include: a) reaching out to citizens and stakeholders; b) sharing information across and within political agencies and governments; c) disseminating information about the governmental operation; d) promoting citizen participation and government transparency; among others [8][9][10][11]. Despite its flaws, the potential for connecting people and governments should not be underestimated: on a global scale, people are spending more time on social media than ever, increasing the likelihood of coming across government’s news and statements on social media first and, then, on other media, such as TV or radio. BACKGROUND Surveys show citizens’ growing scepticism towards politics: their concerns range from dissatisfaction with the democracies they live in [1] to utter distrust in political institutions [2]. Nevertheless, their expectations towards democratic institutions and demands for accountability and transparency only increase [3]. "Transparency is now proposed as the solution to one of the most intangible problems of democratic governance: citizens' increasing mistrust of government", argues Grimmelikhuijsen and Porumbescu [4]. 1 rnment", ucer and ation are f Manuel In parallel, social media flourishes, and virtually anyone can simultaneously be a producer and consumer of information or become an influencer [5]. Thus, public and political communication are transformed: what was once limited to mass communication becomes, in the words of Manuel Castells, "mass self-communication" [5] — fertile soil for misinformation. 1 interagency.institute N.1 APR 2022 ISSN 2789-8040 interagency institute This paradigm shift inevitably le Internet and how it could hin criticisms, it’s argued that the evolving in the new media age; system for distributing old-fash Hinchman [6]. In the end of the the expansion of new media re citizens. Regardless of such criticisms professionalizing their social me include: a) reaching out to citiz political agencies and governm operation; d) promoting citize [8][9][10][11]. Despite its flaws, the potent underestimated: on a global sc increasing the likelihood of comi and, then, on other media, such a Studies show that effective publi and political activism; and that affects the latter's perception of Case studies on government soci the E.U. found that these netw gestures and information provis Self-presentation is connected to be used to counteract a governm Marketing is used in communic separate it from the informativ promote a municipality or vacc resorted to present government or condolences due to an eve governmental activities and fact teach citizens about an importan DePaula, Dincelli and Harrison which they used to analyze Am authors found that 45% of all po that the Facebook posts had me political positioning, symbolic ac FINDINGS interagency.institute N 1 AP This paradigm shift inevitably led to discussions Internet and how it could hinder citizen part criticisms, it’s argued that the same political el evolving in the new media age; digital communic system for distributing old-fashioned mass com Hinchman [6]. In the end of the day, the public o the expansion of new media remains far from citizens. BACKGROUND Despite its flaws, the potential for connecting people and governments should not be underestimated: on a global scale, people are spending more time on social media than ever, increasing the likelihood of coming across government’s news and statements on social media first and, then, on other media, such as TV or radio. Studies show that effective public communication in social media can increase citizen engagement and political activism; and that online interaction between political representatives and citizens affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S. and the E.U. found that these networks are used mostly for self-presentation, marketing, symbolic gestures and information provision purposes [8][9][10][11], which are described below. Self-presentation is connected to impression management and public relations efforts, which could FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power of th Internet and how it could hinder citizen participation and engagement. Among the severa criticisms, it’s argued that the same political elite tendencies of the pre-Internet age continu evolving in the new media age; digital communication is used predominantly as “a new technica system for distributing old-fashioned mass communications content” according to Meyer an Hinchman [6]. In the end of the day, the public only gets further from the political centre [7], an the expansion of new media remains far from achieving its potential for effectively engagin citizens. Regardless of such criticisms, governments worldwide are increasingly investing in an professionalizing their social media communication strategies. The reasons behind this approac include: a) reaching out to citizens and stakeholders; b) sharing information across and withi political agencies and governments; c) disseminating information about the governmenta operation; d) promoting citizen participation and government transparency; among other [8][9][10][11]. Despite its flaws, the potential for connecting people and governments should not b underestimated: on a global scale, people are spending more time on social media than eve increasing the likelihood of coming across government’s news and statements on social media firs and, then, on other media, such as TV or radio. FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power o Internet and how it could hinder citizen participation and engagement. BACKGROUND Among the sev criticisms, it’s argued that the same political elite tendencies of the pre-Internet age cont evolving in the new media age; digital communication is used predominantly as “a new tech system for distributing old-fashioned mass communications content” according to Meyer Hinchman [6]. In the end of the day, the public only gets further from the political centre [7], the expansion of new media remains far from achieving its potential for effectively enga citizens. Regardless of such criticisms, governments worldwide are increasingly investing in professionalizing their social media communication strategies. The reasons behind this appr include: a) reaching out to citizens and stakeholders; b) sharing information across and w political agencies and governments; c) disseminating information about the governme operation; d) promoting citizen participation and government transparency; among ot [8][9][10][11]. Despite its flaws, the potential for connecting people and governments should not underestimated: on a global scale, people are spending more time on social media than e increasing the likelihood of coming across government’s news and statements on social media and, then, on other media, such as TV or radio. Studies show that effective public communication in social media can increase citizen engagem and political activism; and that online interaction between political representatives and citi affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S the E.U. found that these networks are used mostly for self-presentation, marketing, symb gestures and information provision purposes [8][9][10][11], which are described below. Self-presentation is connected to impression management and public relations efforts, which c be used to counteract a government's low popularity or push citizens' interest in public institut Marketing is used in communications in many ways and for many purposes — it's often har separate it from the informative content. In plain words, marketing is useful, for instanc promote a municipality or vaccination campaign. Symbolic presentation is emblematic, usu resorted to present governments taking a stance on a political issue or expressing congratulat or condolences due to an event. Information provision regards the traditional reportin governmental activities and factual information and educational campaigns, which are intende teach citizens about an important issue or simply to make public service announcements [12]. DePaula, Dincelli and Harrison created a typology of government social media communica which they used to analyze American local government departments' Facebook pages [12]. BACKGROUND Regardless of such criticisms, governments w professionalizing their social media communicat include: a) reaching out to citizens and stakeho political agencies and governments; c) dissem operation; d) promoting citizen participation [8][9][10][11]. Despite its flaws, the potential for connecti underestimated: on a global scale, people are s increasing the likelihood of coming across govern and, then, on other media, such as TV or radio. Studies show that effective public communication and political activism; and that online interactio affects the latter's perception of government tran Case studies on government social media commun the E.U. found that these networks are used m gestures and information provision purposes [8][ Self-presentation is connected to impression man be used to counteract a government's low popular Marketing is used in communications in many w separate it from the informative content. In pl promote a municipality or vaccination campaig resorted to present governments taking a stance or condolences due to an event. Information governmental activities and factual information a teach citizens about an important issue or simply DePaula, Dincelli and Harrison created a typolo which they used to analyze American local gove authors found that 45% of all posts regarded sym that the Facebook posts had messages containin political positioning, symbolic acts and/or marke FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power of the Internet and how it could hinder citizen participation and engagement. Among the several criticisms, it’s argued that the same political elite tendencies of the pre-Internet age continue evolving in the new media age; digital communication is used predominantly as “a new technical system for distributing old-fashioned mass communications content” according to Meyer and Hinchman [6]. In the end of the day, the public only gets further from the political centre [7], and the expansion of new media remains far from achieving its potential for effectively engaging citizens. Regardless of such criticisms, governments worldwide are increasingly investing in and professionalizing their social media communication strategies. The reasons behind this approach include: a) reaching out to citizens and stakeholders; b) sharing information across and within political agencies and governments; c) disseminating information about the governmental operation; d) promoting citizen participation and government transparency; among others [8][9][10][11]. FINDINGS Ultimate collaboration with citizens, promote intera Public institutions still have a long way to While these interactive channels have the underused, serving as a replica of the mess channels — that is, one-way communicatio Changing the public communication para Although using social media isn't exclusive reason behind most governments' decisio the core of transparency efforts, they need all and, most importantly, effectively reach Effective online communication is not onl that interests them but also following an certain topics and formats to the detrimen the case mentioned above studies must b and political communication on social me online platforms, types of content or medi Although social media's features, populari useful for public agencies and government CONCLUSIONS RECOMMENDATIONS regard employ Minuss nation of post operat to the C Sobaci local p i f regarded fav employees w Minussi used national gove of posts conc operations an to the Covid- regarded favour employees were Mi i d h regarded favourable presentation alone, in which achievements of the organization or one of its employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Facebook posts published by the national governments of Portugal, Spain and Denmark [10][11]. It was found that the great majority of posts concerning information provision, that is, messages about public service announcements, operations and events that these governments joined or organized, and policies - especially related o the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, participatory and citizen-oriented local public services in Turkey. The analysis revealed that Turkish mayors use it mostly to share information, send personal messages and share their location and activities. Promoting transparent, participatory and citizen-oriented public service wasn't the goal. Indeed, self- promotion and political marketing were the main purposes for using Twitter. Looking at all research findings described above, it was fairly usual to find social media posts that overlap two or more purposes. Ultimately, the research findings agree that posts that foster collaboration with citizens, promote interactivity, or ask for citizens' input are rare. CONCLUSIONS Public institutions still have a long way to maximize their communication efforts on social media. While these interactive channels have the potential to bridge politics and citizens, they have been underused, serving as a replica of the messages and strategy designed for the old media, broadcast channels — that is, one-way communication of news, marketing and symbolic acts. FINDINGS Changing the public communication paradigm presents both new challenges and opportunities. Although using social media isn't exclusively for promoting public transparency, this is the alleged reason behind most governments' decision to join these networks. If institutions aim at making it the core of transparency efforts, they need to start by ensuring their communications are open to all and, most importantly, effectively reach citizens. Effective online communication is not only a matter of providing social media users with content that interests them but also following and adapting to each platform’s algorithms, which boosts certain topics and formats to the detriment of others. On the whole, findings and conclusions from the case mentioned above studies must be taken with a grain of salt: not only research on public and political communication on social media is still at an early stage, but the popularity of certain online platforms, types of content or media formats are debatable and constantly changing. 3 could be In short, FINDINGS Public institutions still have a long way to maximize their communication efforts on socia While these interactive channels have the potential to bridge politics and citizens, they ha underused, serving as a replica of the messages and strategy designed for the old media, br channels — that is, one-way communication of news, marketing and symbolic acts. Changing the public communication paradigm presents both new challenges and oppor Although using social media isn't exclusively for promoting public transparency, this is the reason behind most governments' decision to join these networks. If institutions aim at m the core of transparency efforts, they need to start by ensuring their communications are all and, most importantly, effectively reach citizens. Effective online communication is not only a matter of providing social media users with that interests them but also following and adapting to each platform’s algorithms, which certain topics and formats to the detriment of others. On the whole, findings and conclusio the case mentioned above studies must be taken with a grain of salt: not only research o and political communication on social media is still at an early stage, but the popularity of online platforms types of content or media formats are debatable and constantly changing CONCLUSIONS regarded favourable presentation alone, in which achievements of the employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Faceboo national governments of Portugal, Spain and Denmark [10][11]. It was fo of posts concerning information provision, that is, messages about pub operations and events that these governments joined or organized, and to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, particip local public services in Turkey. The analysis revealed that Turkish ma information, send personal messages and share their location a transparent, participatory and citizen-oriented public service wasn promotion and political marketing were the main purposes for using Tw Looking at all research findings described above, it was fairly usual to f overlap two or more purposes. FINDINGS Further, they found that 17.5% of the posts 2 N.1 APR 2022 ISSN 2789-8040 regarded favourable presentation alone, in which achievements of the organization or one of its employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Facebook posts published by the national governments of Portugal, Spain and Denmark [10][11]. It was found that the great majority of posts concerning information provision, that is, messages about public service announcements, operations and events that these governments joined or organized, and policies - especially related to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, participatory and citizen-oriented local public services in Turkey. The analysis revealed that Turkish mayors use it mostly to share information, send personal messages and share their location and activities. Promoting transparent, participatory and citizen-oriented public service wasn't the goal. Indeed, self- promotion and political marketing were the main purposes for using Twitter. Looking at all research findings described above, it was fairly usual to find social media posts that overlap two or more purposes. Ultimately, the research findings agree that posts that foster collaboration with citizens, promote interactivity, or ask for citizens' input are rare. regarded favourable presentation alone, in which achievements of the organization or on employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Facebook posts published national governments of Portugal, Spain and Denmark [10][11]. It was found that the great m of posts concerning information provision, that is, messages about public service announc operations and events that these governments joined or organized, and policies - especially to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, participatory and citizen-o local public services in Turkey. The analysis revealed that Turkish mayors use it mostly t information, send personal messages and share their location and activities. Pro transparent, participatory and citizen-oriented public service wasn't the goal. Indee promotion and political marketing were the main purposes for using Twitter. Looking at all research findings described above, it was fairly usual to find social media po overlap two or more purposes. Ultimately, the research findings agree that posts tha collaboration with citizens, promote interactivity, or ask for citizens' input are rare. FINDINGS Studies show that effective public communication in social media can increase citizen engagement and political activism; and that online interaction between political representatives and citizens affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S. and the E U found that these networks are used mostly for self presentation marketing symbolic Studies show that effective public communication in social media can increase citizen engagement and political activism; and that online interaction between political representatives and citizens affects the latter's perception of government transparency, efficiency, and corruption [13]. and political activism; and that online interaction between political representatives and citizens affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S. and the E.U. found that these networks are used mostly for self-presentation, marketing, symbolic gestures and information provision purposes [8][9][10][11], which are described below. Case studies on government social media communication at local and national levels in the U.S. and the E.U. found that these networks are used mostly for self-presentation, marketing, symbolic gestures and information provision purposes [8][9][10][11], which are described below. Self-presentation is connected to impression management and public relations efforts, which could be used to counteract a government's low popularity or push citizens' interest in public institutions. Marketing is used in communications in many ways and for many purposes — it's often hard to separate it from the informative content. In plain words, marketing is useful, for instance, to promote a municipality or vaccination campaign. Symbolic presentation is emblematic, usually resorted to present governments taking a stance on a political issue or expressing congratulations or condolences due to an event. Information provision regards the traditional reporting of governmental activities and factual information and educational campaigns, which are intended to teach citizens about an important issue or simply to make public service announcements [12]. 2 DePaula, Dincelli and Harrison created a typology of government social media communication, which they used to analyze American local government departments' Facebook pages [12]. The authors found that 45% of all posts regarded symbolic and presentational exchanges, which means that the Facebook posts had messages containing a favourable presentation of the departments, political positioning, symbolic acts and/or marketing. FINDINGS Ultimately, the research findings ag collaboration with citizens, promote interactivity, or ask for citizens' inp Public institutions still have a long way to maximize their communicat While these interactive channels have the potential to bridge politics a underused, serving as a replica of the messages and strategy designed fo channels — that is, one-way communication of news, marketing and sym Changing the public communication paradigm presents both new cha Although using social media isn't exclusively for promoting public trans reason behind most governments' decision to join these networks. If in the core of transparency efforts, they need to start by ensuring their co all and, most importantly, effectively reach citizens. Effective online communication is not only a matter of providing socia that interests them but also following and adapting to each platform’s certain topics and formats to the detriment of others. On the whole, find the case mentioned above studies must be taken with a grain of salt: n and political communication on social media is still at an early stage, bu online platforms, types of content or media formats are debatable and c Although social media's features, popularity and algorithms aren't defin CONCLUSIONS RECOMMENDATIONS regarded favourable presentation alone, i employees were the main content of the p Minussi used the typology mentioned ea national governments of Portugal, Spain an of posts concerning information provision operations and events that these governm to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's im local public services in Turkey. The analy information, send personal messages transparent, participatory and citizen-o promotion and political marketing were th Looking at all research findings described overlap two or more purposes. RECOMMENDATIONS Public institutions from local to national levels sh strategies that would boost their reach and governments could work on promoting citizen p projects and meetings with political represen governments plan on using social media prima more effective if the posted contents concern th see as impactful on their lives" [14]. Since local and regional governments seem institutions could focus on wider campaigns sh lives. Otherwise, citizens might continue to feel have a unique opportunity to benefit from s respective national identities and, consequently, Governmental agencies need to use social med ongoing communication that creates awareness encouraging the population to get vaccinated a with bureaucratic-tone news or strong self-prom algorithms nor interest people. In the end, accountable and informing: it's also about st representatives. [1] Alliance of Democracies; Rasmussen Global; Index 2018. [2] Ferrín-Pereira, M. (2012). What is Democr Evaluations of Democratic Systems in Contem University Institute, Florence, Italy). [3] Coicaud, J. M. (2019). The Paradoxical Pe Question of its Future. Global Policy, 10(1), 110– [4] Grimmelikhuijsen, S., & Porumbescu, G. ( REFERENCES Every country and region has its more popular platforms (Facebook, Instagram, TikTok, Twitter, etc.), and each platform requires different communication strategies, supports certain media formats (photos, videos, text, etc.) and has different user demographics. Thus, publishing content tailored for each network is quintessential. Public institutions from local to national levels should collaborate and liaise digital communications strategies that would boost their reach and efficacy online. For instance, local and regional governments could work on promoting citizen participation, such as volunteering for community projects and meetings with political representatives. Bonsón and Ratkai argue that “if local governments plan on using social media primarily for citizen engagement, their efforts might be more effective if the posted contents concern the citizens' topics of interest — that is, topics they see as impactful on their lives" [14]. p [ ] Since local and regional governments seem more approachable to citizens, national-level institutions could focus on wider campaigns showing their impact on the population's everyday lives. Otherwise, citizens might continue to feel detached from representative institutions. States have a unique opportunity to benefit from social media communication to strengthen their respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and ongoing communication that creates awareness and advocates for the public good. RECOMMENDATIONS For instance, encouraging the population to get vaccinated and vote. Uncoordinated efforts to inform citizens with bureaucratic-tone news or strong self-promotional messages won't cut through the networks' algorithms nor interest people. In the end, public communication is not only about being accountable and informing: it's also about strengthening citizens' trust in politics and their representatives. Since local and regional governments seem more approachable to citizens, national-level institutions could focus on wider campaigns showing their impact on the population's everyday lives. Otherwise, citizens might continue to feel detached from representative institutions. States have a unique opportunity to benefit from social media communication to strengthen their respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and ongoing communication that creates awareness and advocates for the public good. For instance, encouraging the population to get vaccinated and vote Uncoordinated efforts to inform citizens Since local and regional governments seem more approachable to citizens, national-level institutions could focus on wider campaigns showing their impact on the population's everyday lives. Otherwise, citizens might continue to feel detached from representative institutions. States have a unique opportunity to benefit from social media communication to strengthen their respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and ongoing communication that creates awareness and advocates for the public good. For instance, encouraging the population to get vaccinated and vote. Uncoordinated efforts to inform citizens with bureaucratic-tone news or strong self-promotional messages won't cut through the networks' algorithms nor interest people. In the end, public communication is not only about being accountable and informing: it's also about strengthening citizens' trust in politics and their representatives. REFERENCES RECOMMENDATIONS Although social media's features, popularity and algorithms aren't definitive, the findings could be useful for public agencies and governments reshape and improve online communications. In short, 3 interagency.institute N.1 APR 2022 ISSN 2789-8040 online communications can't be limited to providing old-fashioned mass communications news and information. Every country and region has its more popular platforms (Facebook, Instagram, TikTok, Twitter, etc.), and each platform requires different communication strategies, supports certain media formats (photos, videos, text, etc.) and has different user demographics. Thus, publishing content tailored for each network is quintessential. Public institutions from local to national levels should collaborate and liaise digital communications strategies that would boost their reach and efficacy online. For instance, local and regional governments could work on promoting citizen participation, such as volunteering for community projects and meetings with political representatives. Bonsón and Ratkai argue that “if local governments plan on using social media primarily for citizen engagement, their efforts might be more effective if the posted contents concern the citizens' topics of interest — that is, topics they see as impactful on their lives" [14]. Since local and regional governments seem more approachable to citizens, national-level institutions could focus on wider campaigns showing their impact on the population's everyday lives. Otherwise, citizens might continue to feel detached from representative institutions. States have a unique opportunity to benefit from social media communication to strengthen their respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and ongoing communication that creates awareness and advocates for the public good. For instance, encouraging the population to get vaccinated and vote. Uncoordinated efforts to inform citizens with bureaucratic-tone news or strong self-promotional messages won't cut through the networks' algorithms nor interest people. In the end, public communication is not only about being accountable and informing: it's also about strengthening citizens' trust in politics and their representatives. REFERENCES online communications can't be limited to provid information. Every country and region has its more popular etc.), and each platform requires different co formats (photos, videos, text, etc.) and has diffe tailored for each network is quintessential. REFERENCES [1] Alliance of Democracies; Rasmussen Global; & Dalia Research. (2018). Democracy Perception Index 2018. [1] Alliance of Democracies; Rasmussen Global; & Dalia Research. (2018). Democracy Perception Index 2018. [2] Ferrín-Pereira, M. (2012). What is Democracy to Citizens? Understanding Perceptions and Evaluations of Democratic Systems in Contemporary Europe (Doctoral dissertation, European University Institute, Florence, Italy). [2] Ferrín-Pereira, M. (2012). What is Democracy to Citizens? Understanding Perceptions and Evaluations of Democratic Systems in Contemporary Europe (Doctoral dissertation, European University Institute, Florence, Italy). 4 and the Trust in w, 73(4), y. [3] Coicaud, J. M. (2019). The Paradoxical Perception of Contemporary Democracy, and the Question of its Future. Global Policy, 10(1), 110–121. y ( ) [4] Grimmelikhuijsen, S., & Porumbescu, G. (2013). The Effect of Transparency on Trust in Government: A Cross-National Comparative Experiment. Public Administration Review, 73(4), 575–586. [5] Castells, M. (2007). Communication, Power and Counter-Power in the Network Society. International Journal of Communication, 1, 238–266 [5] Castells, M. (2007). Communication, Power and Counter-Power in the Network Society. International Journal of Communication, 1, 238–266 interagency.institute interagency.institute N.1 APR 2022 ISSN 2789-8040 N.1 APR 2022 ISSN 2789-8040 [6] Meyer, T., & Hinchman, L. (2002). Media Democracy: How the Media Colonize Politics. Malden: Blackwell. [7] Davis, A. (2010). Political Communication and Social Theory. London: Routledge. [7] Davis, A. (2010). Political Communication and Social Theory. London: Routledge. [8] Sobaci, M. Z., & Karkin, N. (2013). The Use of Twitter by Mayors in Turkey: Tweets for Better Public Services? Government Information Quarterly, 30(4), 417–425. [ ] , ( ) y g [8] Sobaci, M. Z., & Karkin, N. (2013). The Use of Twitter by Mayors in Turkey: Tweets for Better Public Services? Government Information Quarterly, 30(4), 417–425. [9] Bellström, P., Magnusson, M., Pettersson, J. S., & Thorén, C. (2016). Facebook Usage in a Local Government: A Content Analysis of Page Owner Posts and User Posts. Transforming Government, 10(4), 548–567. ( ) [10] Minussi, P. (2020). Political Communication on Facebook: Comparing the Regeringen and La Moncloa Pages (Master dissertation, University of Porto, Porto, Portugal). [11] Minussi, P. (2021). Political Communication on Facebook: Comparing the República Portuguesa and LaMoncloa Pages. Journal of Education, Innovation, and Communication, 3(2), 39– 51. [12] DePaula, N., Dincelli, E., & Harrison, T. (2018). Toward a Typology of Government Social Media Communication: Democratic Goals, Symbolic Acts and Self-Presentation. Government Information Quarterly, 35(1), 98–108. [13] Valle-Cruz, D., Sandoval-Almazan, R., & Gil-Garcia, R. (2016). i Master in Political Communication from the University of Porto. E-mail: priscilaminussi@gmail.com REFERENCES Citizens' perceptions of the impact of information technology use on transparency, efficiency and corruption in local governments. Information Polity, 21(3), 321–334. Retrieved from y ( ) https://content.iospress.com/articles/information-polity/ip393 https://content.iospress.com/articles/information-polity/ip393 [14] Bonsón, E., Royo, S., & Ratkai, M. (2014). Citizens' Engagement on Local Governments' Facebook Sites. An Empirical Analysis: The Impact of Different Media and Content Types in Western https://content.iospress.com/articles/information polity/ip393 [14] Bonsón, E., Royo, S., & Ratkai, M. (2014). Citizens' Engagement on Local Governments' Facebook Sites. An Empirical Analysis: The Impact of Different Media and Content Types in Western Europe. Government Information Quarterly 32(1), 52–62. 5 interagency.institute interagency.institute N.1 APR 2022 ISSN 2789-8040
https://openalex.org/W2565762286
https://www.frontiersin.org/articles/10.3389/fpsyg.2016.02032/pdf?isPublishedV2=False
English
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Commentary: From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition
Frontiers in psychology
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cc-by
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GENERAL COMMENTARY published: 04 January 2017 doi: 10.3389/fpsyg.2016.02032 Peter Kramer* and Paola Bressan Keywords: sense of number, sense of magnitude, numerosity estimation, occupancy, statistical learning A commentary on From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition by Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). Behav. Brain. Sci. [Epub ahead of print]. doi: 10.1017/S0140525X16000960 From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition by Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). Behav. Brain. Sci. [Epub ahead of print]. doi: 10.1017/S0140525X16000960 Unlike abstract ones in mathematics, concrete sets of elements in the real world have continuous physical properties, such as overall area and density. The dominant view has it that humans can estimate the discrete numerosities of such sets independently of the co-varying continuous magnitudes; i.e., that humans have a “sense of number.” It has indeed been claimed that various animals, ranging from monkeys to tiny fish, have this sense too. A recent paper by Leibovich et al. (2016) questions all of this (see also Morgan et al., 2014; Gebuis et al., 2016) and argues convincingly that numerosity estimation is not independent from continuous magnitudes but relies on them; that we have not a “sense of number” but a “sense of magnitude.” Edited by: Bert Reynvoet, KU Leuven, Belgium Reviewed by: Marcus Lindskog, Uppsala University, Sweden *Correspondence: Peter Kramer peter.kramer@unipd.it Edited by: Bert Reynvoet, KU Leuven, Belgium Reviewed by: Marcus Lindskog, Uppsala University, Sweden *Correspondence: Peter Kramer peter.kramer@unipd.it Edited by: Bert Reynvoet, KU Leuven, Belgium Reviewed by: Marcus Lindskog, Uppsala University, Sweden Yet the authors fail to cite a classic article that made the very same argument 25 years ago, and—unlike Leibovich et al.—supported it with a quantitative model (Allik and Tuulmets, 1991). Although neither density, nor overall area, nor any other single continuous magnitude can provide reliable information about numerosity, Leibovich et al. imply that all of them together can; they suggest that “statistical learning” will take care of extracting this information and turn numerosity estimates out of it. How statistical learning achieves this feat and whether the resulting numerosity estimates will fit observed ones remains, unfortunately, unclear. Allik and Tuulmets’s alternative “occupancy” model has its limits (e.g., Kramer et al., 2011; Bertamini et al., 2016) but it is specific, it is quantitative, and it predicts observed numerosity estimation surprisingly well with just a single free parameter. *Correspondence: Peter Kramer peter.kramer@unipd.it Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology To understand the occupancy model, consider a set of identical dots and imagine that each of them is covered with a larger disk, as in Figure 1. The model posits that the total area occupied by the disks (occupancy) will be linearly related to the estimated numerosity of the dots. As long as dot densities remain relatively low (Durgin, 1995), the model accounts for nearly 90% of the variance in human data (Allik and Tuulmets, 1991). The closer together the dots are and thus the more the disks overlap, the smaller occupancy is and thus the smaller the estimated numerosity of the dots is predicted to be—as indeed observed (e.g., DeWind et al., 2015). Notably, because large numerosities tend to be dense, the occupancy model predicts they will tend to be underestimated. This prediction has been corroborated repeatedly too (e.g., Izard and Dehaene, 2008). How Leibovich et al.’s statistical learning could make the same prediction is hard to tell. Received: 31 October 2016 Accepted: 14 December 2016 Published: 04 January 2017 FIGURE 1 | Effect of item spacing on occupancy. The “occupancy” of a dot may be represented as a larger, concentric virtual disk that covers it, shown here in gray. (The radius of the disk is a free parameter that, during model fitting, Allik and Tuulmets estimated to be 0.33◦of visual angle.) The three black dots in the bottom row are closer together than those in the top row. As a result, the virtual disks in the bottom row overlap, making for a smaller occupancy (total gray area) and therefore a smaller predicted numerosity estimate. For illustration purposes only a few dots are shown; note, however, that numerosity estimation concerns numerosities outside the “subitizing” range of one to four items. The occupancy model—even in Durgin’s (1995) variant—is too simple to work under all conditions (Kramer et al., 2011). Still, if the degree of overlap of the virtual disks covering the dots is interpreted as an inverse measure of the mutual discriminability of the dots, the model makes an intuitive point. The point is that the less discriminable from one another items are, the lower their estimated numerosity ought to be. If so, the occupancy model could be expanded and improved (Kramer et al., 2011) by taking into account anything that affects the items’ (1) discriminability (their distance from one another and from fixation, for example), (2) retention across saccades (if presentation times allow eye movements), and (3) retention in memory (if the stimulus is presented or inspected sequentially or compared to another one). The occupancy model deserves at the very least discussion— if not revival and development. It has the potential to challenge not only “sense of number” models but also Leibovich et al.’s statistical-learning alternative to them. Unlike the latter, the occupancy model is a “sense of magnitude” model that does not require learning, is deterministic, quantitative, perception- rather than cognition-based, and not inconsistent with the idea that numerosity estimation might be innate and so simple that even fish could do it. AUTHOR CONTRIBUTIONS Although people are under the impression they are blessed with a high-resolution image of the world around them, careful experimentation has revealed that this is largely an illusion (e.g., Durgin, 1995). In numerosity-estimation experiments the PK wrote the first draft of the manuscript; both authors discussed it, critically revised it, and agreed on the final version. dispersion. Atten. Percept. Psychophys. 78, 1460–1471. doi: 10.3758/s13414-01 6-1100-0 DeWind, N. K., Adams, G. K., Platt, M. L., and Brannon, E. M. (2015). Modeling the approximate number system to quantify the contribution of visual stimulus features. Cognition 142, 247–265. doi: 10.1016/j.cognition.2015.05.016 Durgin, F. H. (1995). Texture density adaptation and the perceived numerosity and distribution of texture. J. Exp. Psychol. Hum. Percept. Perform. 21, 149–169. Gebuis, T., Cohen Kadosh, R., and Gevers, W. (2016). Sensory-integration system rather than approximate number system underlies numerosity processing: Citation: Kramer P and Bressan P (2017) Commentary: From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition. Front. Psychol. 7:2032. doi: 10.3389/fpsyg.2016.02032 January 2017 | Volume 7 | Article 2032 Frontiers in Psychology | www.frontiersin.org Item Discriminability and Numerosity Estimation Kramer and Bressan stimuli tend to be randomly placed items, usually dots, and some of them end up clumped together. These items may be impossible to discriminate from one another, even when they can all be perceptually detected. This crowding effect (Whitney and Levi, 2011) is a serious concern, because the opportunities for crowding necessarily grow with numerosity and have been shown to affect its estimation (Valsecchi et al., 2013; Anobile et al., 2015). So far, studies that claim that numerosity estimation does not fully rely on continuous magnitudes, or that it is more cognitive than perceptual in nature, have not been very specific about how the perceptual system extracts numerosity from the stimulus (see also Morgan et al., 2014). These studies have considered a wide range of perceptual factors such as density, overall area, and item size. None of these factors, however, has ever been claimed to be crucial in numerosity estimation. The one factor that has for as long as 25 years (i.e., occupancy) is instead consistently ignored, including now by Leibovich et al. (2016). Izard and Dehaene (2008), for example, do cite Allik and Tuulmets (1991) and control for various factors including “occupied area,” but they use the term to mean overall area (which nobody considers critical to numerosity estimation) and not occupancy (which Allik and Tuulmets themselves consider critical). Izard and Dehaene dismiss occupancy as “a complex combination” of two parameters (density and occupied area). Occupancy does not strike us as complex, but even if it were, the fact that it reflects well-established perceptual discriminability limitations that have been demonstrated to affect numerosity estimation—rather than hypothetical cognitive ones that continue to be debated—should give the occupancy model an edge over any “sense of number” model. In defense of their own “sense of magnitude” model, which Leibovich et al. fail to cite too, Morgan et al. (2014) make a similar point. dispersion. Atten. Percept. Psychophys. 78, 1460–1471. doi: 10.3758/s13414-01 6-1100-0 DeWind, N. K., Adams, G. K., Platt, M. L., and Brannon, E. M. (2015). Modeling the approximate number system to quantify the contribution of visual stimulus features. Cognition 142, 247–265. doi: 10.1016/j.cognition.2015.05.016 Frontiers in Psychology | www.frontiersin.org January 2017 | Volume 7 | Article 2032 REFERENCES Allik, J., and Tuulmets, T. (1991). Occupancy model of perceived numerosity. Percept. Psychophys. 49, 303–314. doi: 10.3758/BF03205986 Anobile, G., Turi, M., Cicchini, G. M., and Burr, D. C. (2015). Mechanisms for perception of numerosity or texture-density are governed by crowding-like effects. J. Vis. 15, 1–12. doi: 10.1167/15.5.4 Bertamini, M., Zito, M., Scott-Samuel, N. E., and Hulleman, J. (2016). Spatial clustering and its effect on perceived clustering, numerosity, and January 2017 | Volume 7 | Article 2032 Frontiers in Psychology | www.frontiersin.org 2 Item Discriminability and Numerosity Estimation Kramer and Bressan Whitney, D., and Levi, D. M. (2011). Visual crowding: a fundamental limit on conscious perception and object recognition. Trends Cogn. Sci. 15, 160–168. doi: 10.1016/j.tics.2011. 02.005 a critical review. Acta Psychol. 171, 17–35. doi: 10.1016/j.actpsy.2016. 09.003 Izard, V., and Dehaene, S. (2008). Calibrating the mental number line. Cognition 106, 1221–1247. doi: 10.1016/j.cognition.2007.06.004 Kramer, P., Di Bono, M. G., and Zorzi, M. (2011). Numerosity estimation in visual stimuli in the absence of luminance-based cues. PLoS ONE 6:e17378. doi: 10.1371/journal.pone.0017378 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). From ‘sense of number’ to ‘sense of magnitude’ - the role of continuous magnitudes in numerical cognition. Behav. Brain. Sci. doi: 10.1017/S0140525X16000960. [Epub ahead of print]. Copyright © 2017 Kramer and Bressan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Morgan, M. J., Raphael, S., Tibber, M. S., and Dakin, S. C. (2014). A texture- processing model of the ‘visual sense of number’. Proc. Biol. Sci. 281:20141137. doi: 10.1098/rspb.2014.1137 Valsecchi, M., Toscani, M., and Gegenfurtner, K. R. (2013). Perceived numerosity is reduced in peripheral vision. J. Vis. 13, 1–16. doi: 10.1167/13.13.7 January 2017 | Volume 7 | Article 2032 Frontiers in Psychology | www.frontiersin.org 3
https://openalex.org/W2050186163
https://researchonline.lshtm.ac.uk/id/eprint/2130250/1/pone.0119271.pdf
English
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Pertussis Post-Exposure Prophylaxis among Household Contacts: A Cost-Utility Analysis
PloS one
2,015
cc-by
9,212
RESEARCH ARTICLE Nisha Thampi1,2*, Ipek Gurol-Urganci3, Natasha S. Crowcroft4,5, Beate Sander5,6,7,8 1 Division of Infectious Diseases, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada, 2 University of Ottawa, Ottawa, ON, Canada, 3 Department of Health Services Research and Policy, London School of Hygiene and Tropical Medicine, London, United Kingdom, 4 Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, 5 Public Health Ontario, Toronto, ON, Canada, 6 Institute of Health Policy, Management & Evaluation, University of Toronto, Toronto, ON, Canada, 7 Institute for Clinical Evaluative Sciences, Toronto, ON, Canada, 8 Toronto Health Economics and Technology Assessment Collaborative, Toronto, ON, Canada * nthampi@cheo.on.ca OPEN ACCESS OPEN ACCESS Citation: Thampi N, Gurol-Urganci I, Crowcroft NS, Sander B (2015) Pertussis Post-Exposure Prophylaxis among Household Contacts: A Cost- Utility Analysis. PLoS ONE 10(3): e0119271. doi:10.1371/journal.pone.0119271 OPEN ACCESS Citation: Thampi N, Gurol-Urganci I, Crowcroft NS, Sander B (2015) Pertussis Post-Exposure Prophylaxis among Household Contacts: A Cost- Utility Analysis. PLoS ONE 10(3): e0119271. doi:10.1371/journal.pone.0119271 Methods A Markov model was constructed to examine 4 mutually exclusive strategies: erythromycin, azithromycin, clarithromycin, or no intervention, stratified by age group of contacts (“infant”, “child”, and “adult”). Transition probabilities, costs and quality-adjusted life years (QALYs) were derived from the literature. Chronic neurologic sequelae were modeled over a lifetime, with costs and QALYs discounted at 5%. Associated health outcomes and costs were com- pared, and incremental cost-effectiveness ratios (ICER) were calculated in 2012 Canadian dollars. Deterministic and probabilistic sensitivity analyses were performed to evaluate the degree of uncertainty in the results. Academic Editor: T. Mark Doherty, Glaxo Smith Kline, DENMARK Copyright: © 2015 Thampi 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. * nthampi@cheo.on.ca Background Recent pertussis outbreaks have prompted re-examination of post-exposure prophylaxis (PEP) strategies, when immunization is not immediately protective. Chemoprophylaxis is recommended to household contacts; however there are concerns of clinical failure and significant adverse events, especially with erythromycin among infants who have the high- est disease burden. Newer macrolides offer fewer side effects at higher drug costs. We sought to determine the cost-effectiveness of PEP strategies from the health care payer perspective. Pertussis Post-Exposure Prophylaxis among Household Contacts: A Cost-Utility Analysis Nisha Thampi1,2*, Ipek Gurol-Urganci3, Natasha S. Crowcroft4,5, Beate Sander5,6,7,8 Findings Azithromycin offered the highest QALYs in all scenarios. While this was the dominant strate- gy among infants, it produced an ICER of $16,963 per QALY among children and $2,415 per QALY among adults. Total QALYs with azithromycin were 19.7 for a 5-kg infant, 19.4 for a 10-year-old child, and 18.8 for a 30-year-old adult. The costs of azithromycin PEP among infants, children and adults were $1,976, $132 and $90, respectively. While results were sensitive to changes in PEP effectiveness (11% to 87%), disease transmission (variable Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. 1 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model among age groups) and hospitalization costs ($379 to $59,644), the choice of strategy remained unchanged. among age groups) and hospitalization costs ($379 to $59,644), the choice of strategy remained unchanged. Interpretation Pertussis PEP is a cost-effective strategy compared with no intervention and plays an im- portant role in contact management, potentially in outbreak situations. From a healthcare payer perspective, azithromycin is the optimal strategy among all contact groups. Introduction In the pre-vaccine era, pertussis was a major childhood illness affecting children primarily under 10 years of age, and a major cause of death among infants under 1 year [1]. Caused by Bordetella pertussis, the disease was pandemic throughout the 20th century, with cyclical epi- demic peaks every 2 to 5 years [1]. Widespread immunization of children has not lengthened the epidemic cycle as much as expected [2], suggesting that adults serve as a reservoir for dis- ease in young children [3]; pertussis is common and endemic among this group [4,5]. Prolonged pertussis outbreaks have recently been reported across North America [6–9], prompting re-examination of control and prevention strategies [10]. In 2010, 101 cases of pertus- sis were reported in Ontario (0.8 cases per 100,000 population) [6]. Following a 2011–2012 out- break, 792 cases were reported across both years, or 5.9 per 100,000 population. Immunization coverage in Ontario among 7- and 17-year olds in 2012 was 76% and 68%, respectively [6]. The resurgence of pertussis has been attributed to waning immunity in older children and poor vaccine efficacy against other Bordetella species associated with pertussis-like illnesses [7,11,12]. A randomized, controlled trial (RCT) of post-exposure prophylaxis (PEP) with erythromycin compared with placebo found PEP to be effective in preventing secondary cases in 67.5% of adult and pediatric household contacts [13]. Practice guidelines in Canada, the United States and the United Kingdom recommend chemoprophylaxis with a macrolide for household and close contacts of pertussis cases of all ages, irrespective of immunization status [14–16]. While studies have examined erythromycin as a treatment and PEP agent, the newer macrolides, azithromycin and clarithromycin, have also been shown to be active against B. per- tussis in vitro [17], and have been recommended as better-tolerated, equally effective [18,19] but more costly treatment options [15,16]. Economic evaluations can provide “value-for-money” information to support health care decision-making [20]. A cost-utility analysis was performed to compare the additional cost per QALY gained across the 4 strategies of erythromycin, azithromycin, clarithromycin and no prophylaxis, with an aim to estimate the effects, costs and cost-effectiveness of alternative strat- egies for the prevention of pertussis among household contacts. This analysis is especially time- ly in informing clinical practice and health care resource allocation given the recent outbreaks among under-immunized populations in countries that have well-established immunization programs. Cost-utility analysis In accordance with established guidelines for economic evaluations [20,21], a cost-utility analy- sis was performed to determine whether PEP with a macrolide was cost-effective compared to 2 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model no PEP (i.e. treatment of cases as they arise) in household contacts exposed to pertussis. Specif- ic PEP regimens considered were erythromycin and azithromycin in all age groups (infants, children and adults) and clarithromycin in children and adults only. The primary outcome was the incremental cost-effectiveness ratio (ICER), derived from estimated quality-adjusted life years (QALYs) and direct healthcare cost over a lifetime time horizon. The analysis was con- ducted from the healthcare payer perspective (i.e. Ontario’s Ministry of Health and Long-Term Care), with a commonly-used cost-effectiveness threshold of $50,000 per QALY gained [22]. Healthcare costs for PEP and the treatment of cases are included. All costs were expressed in 2012 Canadian dollars (1 US dollar was 0.99958 Canadian dollars) [23–26]. Costs and health outcomes were discounted at 5% per year as recommended for health economic evaluations in Canada [20]. Data Probabilities of disease-specific outcomes and medication-related gastrointestinal effects were drawn from RCTs and observational cohort studies. All data are shown in Table 1. Secondary attack rates of pertussis are highest among young infants at 65% [28], with a 10-fold decrease among children and adults [13]. Similarly, more than two-thirds of infants with pertussis are managed in hospital [29], compared to only 7% of children [30] and 3.5% of adult contacts [29]. Encephalitis is an infrequent but serious complication of pertussis, affecting 0.5% infants [31], 0.08% children [30] and 0.05% adults who are hospitalized [30]. Early economic evalua- tions and population studies reported residual effects in one-third of survivors of pertussis en- cephalitis [1,32,33]. Death is least likely to occur among adult patients with pertussis, at 0.01%, followed by children at 0.06%. The probability of death is highest among infants, at 0.6% [29]. Model and assumptions A Markov model was constructed to evaluate the lifetime health outcomes and costs of devel- oping pertussis among household contacts following expectant management or PEP. Most events occurred in the first year. The Markov model had a cycle length of 1 year and incorpo- rated 2 health states: survival and death. The duration of acute events in the first year was taken into account. Household contacts were stratified into 3 age groups: less than 1 year of age (“infant”), 1 to 12 years old (“child”) and greater than 12 years old (“adult”). The model structure was the same for all contacts (Fig. 1A and B). Infant contacts had a higher frequency of hospitalization for severe disease, including significant respiratory or neu- rologic complications. Children and adult contacts with respiratory symptoms were managed in the outpatient setting depending on severity, although all patients with neurologic presenta- tions required hospitalization. Household contacts that developed mild symptoms went to their general practitioner (GP) rather than an emergency department. Mild-to-moderate cough resulted in an additional GP visit and antibiotics, and those with severe cough or pneu- monia had 2 additional GP visits, antibiotics and a chest x-ray. Survivors of respiratory disease requiring hospitalization did not experience long-term sequelae. In all groups, pertussis-related deaths were assumed to occur during hospitalization. Age-specific mortality was obtained from Statistics Canada [27]. Given that the total duration of illness is 6–8 weeks [1], all costs at- tributable to pertussis were assumed to have been incurred in the first year unless patients ex- perienced neurological sequelae, which were assumed to require lifetime healthcare services. Intervention An RCT of erythromycin prophylaxis against pertussis demonstrated 67.5% effectiveness (95% CI 7.6% to 88.7%) in preventing culture-positive pertussis among household contacts [13]. 3 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Fig 1. Markov model for post-exposure prophylaxis strategy. The square represents a decision node, and circle represents a chance node. The triangle represents the final outcome for that event pathway. Consequences associated with the chance node are mutually exclusive. PEP: post-exposure prophylaxis; GI: gastrointestinal. Fig 1. Markov model for post-exposure prophylaxis strategy. The square represents a decision node, and circle represents a chance node. The triangle represents the final outcome for that event pathway. Consequences associated with the chance node are mutually exclusive. PEP: post-exposure prophylaxis; GI: gastrointestinal. Fig 1. Markov model for post-exposure prophylaxis strategy. The square represents a decision node, and circle represents a chance node. The triangle represents the final outcome for that event pathway. Consequences associated with the chance node are mutually exclusive. PEP: post-exposure prophylaxis; GI: gastrointestinal. doi:10.1371/journal.pone.0119271.g001 doi:10.1371/journal.pone.0119271.g001 With no subsequent studies re-examining this intervention, this point estimate of effectiveness was extrapolated to azithromycin and clarithromycin based on their comparatively similar effi- cacy with erythromycin in treatment studies [18,19] which is in keeping with current pertussis PEP guidelines [15,16]. PEP was assumed to have been given within 21 days of onset of cough in the index case and before a secondary case had occurred [28]. Clarithromycin has not been studied for those under 1 month of age and so was not included as an option for the infant group [19]. g p All contacts who receive an antibiotic have a likelihood of developing a gastrointestinal ad- verse event. These probabilities were derived from case series and clinical trials involving eryth- romycin, clarithromycin and/or azithromycin. Neonates may be more than 10 times at risk for infantile hypertrophic pyloric stenosis (IHPS) in the month following orally administered erythromycin, compared to unexposed neonates, with a peak incidence of 1.28% in under 3 month olds [34,35]. As the risk of IHPS with erythromycin has had an impact on recommen- dations for infant PEP, this was explicitly examined in the model. There were 2 case reports of azithromycin-associated IHPS [36]. PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Intervention With over 4,000 cases noted over a 10-year period in the US [29] and azithromycin having been adopted as the preferred agent for young infants [16], a PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 4 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Table 2. Utility values and duration of relevant health states. Health State Utility value for base case (SD) Days in first year Reference (utility; duration) INFANT Mild illness, outpatient 0.67 (0.30) 76 [40,41] Hospitalization 0.58 (0.37) 8 [38,39] Respiratory complications 0.58 (0.37) 8 [38] Encephalitis without sequelae 0.51 (0.38) 14 [38] Chronic neurologic sequelae1 0.77 (0.25)2 365 [38] IHPS 0.51 (0.38)3 6 Assumption; [34,43] CHILD Mild cough, outpatient 0.85 (0.26)4 76 [38,41] Moderate-severe cough, outpatient 0.81 (0.30) 76 [38,41] Hospitalization with recovery 0.67 (0.33) 3 [38,42] Encephalitis without sequelae 0.51 (0.38) 14 [38]; Assumption Chronic neurologic sequelae 0.77 (0.25) 365 [38]; Assumption GI adverse event 0.70 (0.15)5 7 [84]; Assumption ADULT Mild cough 0.85 (0.26) 87 [38,40] Moderate cough 0.81 (0.30) 87 [38,40] Hospitalization for respiratory complications 0.62 (0.40) 3 [38,42] Encephalitis without sequelae 0.51 (0.38) 14 [38]; Assumption Chronic neurologic sequelae 0.77 (0.25) 365 [38]; Assumption GI adverse event 0.70 (0.15)5 7 [84]; Assumption 1 Utility value and duration in health state assumed to be the same for all age groups 2 Interpreted as willing to give up 84 days of life to prevent 1 year of neurologic sequelae 3 Assumed to be same as for encephalitis 4 Interpreted as willing to give up 8 days of life to prevent 8 weeks of mild cough that does not require hospitalization 5 Standard error Table 2. Utility values and duration of relevant health states. Utility value for base case (SD) Days in first year Reference (utility; duration) Utility value for base case (SD) Days in first year 1 Utility value and duration in health state assumed to be the same for all age groups 1 Utility value and duration in health state assumed to be the same for all age groups 2 Interpreted as willing to give up 84 days of life to prevent 1 year of neurologic sequelae 3 Assumed to be same as for encephalitis 4 Interpreted as willing to give up 8 days of life to prevent 8 weeks of mild cough that does not require hospitalization 5 Standard error GI: gastrointestinal IHPS: infantile hypertrophic pyloric stenosis PEP: post-exposure prophylaxis doi:10.1371/journal.pone.0119271.t002 IHPS: infantile hypertrophic pyloric stenosis Health outcomes. QALYs combine quality of life and duration of life, or life years, into a single measure. Health states were valued with preferences (Table 2) drawn from individuals with pertussis [38]. Pertussis Post-Exposure Prophylaxis: A Decision Model Table 1. Event probabilities for decision model. Contact group Parameter Base case value Range examined References ALL Prophylaxis effectiveness 0.675 0.076–0.887 [13] INFANT Intervention IHPS following erythromycin 0.0128 0.0026–0.0275 [35] IHPS following azithromycin 0.0005 0.0001–0.0010 Assumption1 Pertussis Develops pertussis post-exposure 0.65 0.25–0.81 [28,70] Hospitalization for severe disease 0.69 0.59–0.82 [29,30] Develops complication in hospital 0.14 0.10–0.19 [42,66] Death 0.006 0.001–0.009 [29,66,82] Develops encephalitis 0.005 0.002–0.009 [29,31,42] Develops chronic neurologic sequelae 0.33 0.25–0.50 [32,33] CHILD Intervention GI adverse event with erythromycin 0.34 0.27–0.44 [13,19,28] GI adverse event with azithromycin 0.19 0.12–0.20 [18] GI adverse event with clarithromycin 0.32 0.20–0.402 [19] Pertussis Acquires pertussis post-exposure 0.061 0.048–0.440 [13,28] Develops moderate to severe respiratory pertussis 0.25 0.10–0.32 [42,83] Hospitalization for severe disease 0.07 0.04–0.08 [29,30,58] Death 0.0006 0.0004–0.0010 [29,30,70] Develops encephalitis/ encephalopathy 0.0008 0.0005–0.0040 [29,30,32] Develops chronic neurologic sequelae 0.33 0.25–0.50 [1,32,33] ADULT Intervention GI adverse event with erythromycin 0.34 0.27–0.44 [13,19,28] GI adverse event with azithromycin 0.19 0.12–0.20 [18] GI adverse event with clarithromycin 0.32 0.2–0.402 [19] Pertussis Acquires pertussis post-exposure 0.061 0.048–0.200 [13,68] Develops moderate to severe respiratory pertussis 0.035 0.021–0.040 [29,30] Hospitalization for severe disease 0.027 0.008–0.060 [28,30,73] Death 0.0001 0–0.0010 [29,30,75] Develops encephalitis/ encephalopathy 0.0005 0.0002–0.0040 [29,30,32] Develops chronic neurologic sequelae 0.33 0.25–0.50 [32,33] 1 A ti b d 2 t f ith i i t d IHPS [36] Table 1. Event probabilities for decision model. 1 Assumption based on 2 case reports of azithromycin-associated IHPS [36] 2 Assumption of range GI: gastrointestinal IHPS: infantile hypertrophic pyloric stenosis PEP: post-exposure prophylaxis doi:10.1371/journal.pone.0119271.t001 point estimate of 0.05%, or 2 cases among 4,000 infants treated with azithromycin, was assumed. If no PEP was received, or no gastrointestinal event occurred on chemoprophylaxis, then erythromycin was used for treatment of pertussis as the least expensive option [37]. If IHPS oc- curred during erythromycin PEP, infants who subsequently developed pertussis received azi- thromycin for treatment [16]. However, if IHPS followed azithromycin PEP, then infants with subsequent pertussis were treated with erythromycin. As azithromycin and clarithromycin have fewer side effects than erythromycin, child and adult contacts that developed pertussis after experiencing PEP-related side effects were treated with either of these agents. PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 5 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 doi:10.1371/journal.pone.0119271.t002 In this study, time trade-off and contingent valuation methods were used to determine utilities of short-term health states among adult patients and parents of adoles- cent patients diagnosed with pertussis [38]. Infant utilities were derived from the adult respon- dents, who were presented with the scenario of a 1-month old who developed pertussis, and asked to value the prevention of short-term health states, that is respiratory or neurologic com- plications lasting 8 weeks’ duration, as compared to long-term health states, which reflect neu- rologic sequelae [38]. The time spent in each health state was derived from studies examining the natural course of pertussis in under-immunized populations [39–43], and was multiplied by the correspond- ing preference value to calculate the QALYs. The literature search did not identify a study re- porting a preference value for IHPS, and so it was assumed to be the same as for encephalitis without sequelae, given the need for hospitalization and short-term disability. 6 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Costs. The direct medical costs were expressed in 2012 Canadian dollars, and were associated with exposure to and treatment of pertussis. These costs include contact tracing by a public health nurse to review the need for PEP within households, medications, GP visits, diag- nostic testing, hospitalization and long-term medical costs associated with neurologic sequelae. While there is no universal drug coverage in Ontario, medication costs were included in this analysis as Canadian public health policy makers have recommended that prophylaxis, where appropriate, should be supplied by public health [15]. Cost estimates are summarized in Table 3. Costs of health care visits and procedures were obtained from the Ontario Health In- surance Plan Schedule of Benefits [44], and prices for generic formulations from the Ontario Drug Benefit Formulary [37,45]. The doses and dispensing costs for each macrolide are outlined in Table 4. Given the infant contact is at highest risk for complications with pertussis in the first 4 months of life [9,46], the model assumed a 6-week old infant whose weight was at the 50th percentile-for-age, or 5 kg, as per the CDC Growth Charts for the United States [47]. Thus, the cost of erythromycin was $2 and azithromycin $8.90. Similarly, a child was assumed to have completed the primary im- munization series and prefer taking liquid preparations of antibiotics, so that a 10-year old child whose weight was at the 50th percentile-for-age at 34 kg had erythromycin at a cost of $13.58, azithromycin at $42.87 and clarithromycin at $8.16. An adult was assumed to be 30 years old and weigh 70 kg, so that erythromycin cost $10.24, azithromycin $7.84 and clari- thromycin $11.54, respectively. The duration of prophylaxis and treatment of pertussis was as per Health Canada guidelines [15]. Medications, GP visits and diagnostic testing are set by the Ministry for all practitioners and pharmacies. Older children and adults with PEP-related gastrointestinal symptoms were as- sumed to visit their GP to discuss discontinuation of therapy. Acquiring the disease incurred further costs. Hospitalization costs for pertussis and encephalitis were drawn from the Ontario Case Cost- ing Initiative (OCCI), which collects case costing data for acute inpatient, day surgery and am- bulatory care cases, complex continuing care and community care cases [39]. The mean cost per hospitalized case includes costs associated with ward and intensive care, and diagnostic and therapeutic resources. During the fiscal year 2010–2011, there were 10 pertussis-related hospitalizations and fewer than 5 associated encephalitis cases. Fifty-one cases of encephalitis associated with other infectious etiologies were reported to the OCCI and those costs were re- viewed. Physician costs are not part of the OCCI, but an estimate of 5% of hospitalization costs was used based on previous studies of influenza analyzing health administrative data (expert opinion, based on [48,49]). There are no data on long-term outcomes of patients with pertussis encephalitis. Direct medical costs associated with the long-term neurologic sequelae of pertussis encephalitis were extrapolated from a review of direct medical costs for children with complex medical condi- tions in Ontario. Excluding initial hospitalization costs, the average costs of care for a child with neurological impairment and technology assistance (including cerebrospinal fluid ventric- ular shunt, gastrostomy, or tracheostomy) was $34,574 (interquartile range: $10,178-$97,063) over a 2-year period [50]. This population was comparable with respect to burden of disease and resource utilization patterns. Pertussis Post-Exposure Prophylaxis: A Decision Model Table 3. Estimated direct medical costs per contact. Costs in Canadian dollars, 2012 valuation. Parameter Base case value Standard Error Reference GENERAL Contact tracing (public health) 37.74 - Assumption1 Visit to GP 33.70 - [44] Chest X-ray (professional and technical cost) 32.65 - [44] No pertussis 0 - Assumption MEDICATIONS Erythromycin2 Infant 2.00 - [45] Child 13.58 - [37] Adult 10.24 - [37] Azithromycin3 Infant 8.90 - [37] Child 42.87 - [37] Adult 7.84 - [37] Clarithromycin4 Child 8.16 - [37] Adult 11.54 - [37] PERTUSSIS GI symptoms in child or adult contact 33.705 [44] Outpatient with mild illness GP + treatment Assumption Outpatient with moderate-severe illness 2 GP visits + 1 chest x-ray + treatment Assumption Hospitalization6 12,160 5,689 [39] IHPS 10,340 550 [43] COMPLICATIONS Encephalitis 27,643 8370 [39] Chronic neurologic sequelae7 103,652 148,867 [50] Table 3. Estimated direct medical costs per contact. Costs in Canadian dollars, 2012 valuation. 1 Public health nurse with 4 years of seniority paid an hourly rate of CAD 37.74 (personal communication, Public Health Ontario, July 2012) 2 4 clarithromycin at 15 mg/kg/day divided twice daily, maximum 1000g/day, dispensed for a child at $0.5712 per 250mg/5mL; and an adult at $0.4122 per 250mg tablet p p 6 Hospitalization includes the infant with an uncomplicated admission; infant with respiratory complications; child and adult hospitalizations; death in all groups 6 Hospitalization includes the infant with an uncomplicated admission; infant with respiratory complications; child a all groups 7 Mean cost and standard deviation over 2-year period; includes initial hospitalization GI: gastrointestinal GP: general practitioner IHPS: infantile hypertrophic pyloric stenosis PEP: post-exposure prophylaxis effectiveness. The ranges examined in the deterministic sensitivity analysis were derived either from the confidence intervals of the point estimate used for the base case; or, if not available, minimum and maximum values in the model were derived from point estimates in other ob- servational studies (Table 1). Probabilistic sensitivity analysis was performed to address uncer- tainty involving parameters with an underlying probability distribution, varying all selected effectiveness. The ranges examined in the deterministic sensitivity analysis were derived either from the confidence intervals of the point estimate used for the base case; or, if not available, minimum and maximum values in the model were derived from point estimates in other ob- servational studies (Table 1). Analysis Base case analysis was conducted for a previously healthy 6-week old infant at 5 kg; 10-year old child at 34 kg; and 30-year old adult [51,52]. Deterministic sensitivity analysis was performed to explore uncertainty in the point estimates, and a threshold analysis was performed for PEP 7 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 doi:10.1371/journal.pone.0119271.t003 Pertussis Post-Exposure Prophylaxis: A Decision Model Table 4. Macrolide strategies for pertussis PEP among household contacts. Strategy Dose (oral) Cost Duration (days) Erythromycin 40 mg/kg/day divided three times daily (maximum 2,000 mg/day) $0.0713 per 50 mg/mL and $0.1828 per 250 mg tablet 7 Azithromycin 10 mg/kg (maximum 500 mg) on day 1 followed by 5 mg/kg (maximum 250 mg) once daily $5.9347 per 100mg/5mL and $1.3070 per 250mg tablet 5 Clarithromycin1 15 mg/kg/day divided twice daily (maximum 1,000 mg/day) $0.5712 per 250mg/5mL and $0.4122 per 250mg tablet 7 1 Not included as strategy among infants Table 4. Macrolide strategies for pertussis PEP among household contacts. variables simultaneously [53]. In this assessment, beta distributions for probabilities and utili- ties and gamma distributions for costs were derived, based on mean and standard error [54]. The model was run 10,000 times so that each event of interest may occur at least once in the simulation. Results were summarized as cost-effectiveness acceptability curves. Probabilistic sensitivity analysis was performed to address uncer- tainty involving parameters with an underlying probability distribution, varying all selected PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 8 / 17 Results From the model, PEP with azithromycin prevents 439 secondary cases per 1,000 infant con- tacts, and 41 secondary cases per 1,000 child and adult contacts. The results for the base case analysis are shown in Table 5. A previously-healthy 5-kg infant contact who did not receive PEP experienced 19.22 QALYs, whereas a macrolide resulted in at least 0.45 more expected QALYs, or 165 quality-adjusted life days. Between the macrolides, azithromycin dominated erythromycin with lower expected costs ($1,976 versus $2,096) and outcomes (19.6661 versus 19.6660 QALYs). These findings were sensitive to costs of hospitalization, costs of encephalitis and probability of hospitalization, death, PEP effectiveness and acquiring disease following ex- posure, and not sensitive to the probability of complications, death, annual costs of neurologic sequelae, probability of an IHPS event with either macrolide, and changes in utility on PEP or with any pertussis health state. However, in all scenarios in univariate and probabilistic sensi- tivity analyses, azithromycin dominated erythromycin and no PEP. Table 5. Costs, effects and cost-effectiveness of prophylaxis with erythromycin, azithromycin or clarithromycin compared with no intervention for household contacts of cases of pertussis, stratified by age group, discounted at 5%. Age group Strategy Average expected cost ($) Average expected QALY Difference in cost ($) Difference in QALY ICER ($ per QALY) Infant azithromycin 1,975.87 19.66612 - - erythromycin 2,095.70 19.66602 - - dominated none 5,815.10 19.21593 - - dominated Child none 35.13 19.37751 - - - clarithromycin 101.98 19.40780 66.86 0.0303 2,207 erythromycin 107.85 19.40753 - - dominated azithromycin 132.04 19.40957 30.06 0.0018 16,963 Adult none 14.22 18.72856 - - - azithromycin 90.25 18.76004 76.02 0.0315 2,415 erythromycin 97.74 18.75800 - - dominated clarithromycin 98.37 18.75827 - - dominated doi:10 1371/journal pone 0119271 t005 Table 5. Costs, effects and cost-effectiveness of prophylaxis with erythromycin, azithromycin or clarithromycin compared with no intervention for household contacts of cases of pertussis, stratified by age group, discounted at 5%. 5. Costs, effects and cost-effectiveness of prophylaxis with erythromycin, azithromycin or clarithromycin c usehold contacts of cases of pertussis, stratified by age group, discounted at 5%. ess of prophylaxis with erythromycin, azithromycin or clarithromycin compared with no intervention sis, stratified by age group, discounted at 5%. Results PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 9 / 17 Pertussis Post-Exposure Prophylaxis: A Decision Model The lifetime expected health outcomes for a previously healthy 10-year-old contact were 19.38 QALYs without intervention and increased by 12 days (0.032 QALYs) with azithromycin PEP, with a $97 cost increase for an ICER of $16,963 per QALY when compared with clarithro- mycin. While clarithromycin was less expensive than azithromycin, there were fewer QALYs gained, thus making it the non-preferred option in the base case scenario and sensitivity analy- ses. In the probabilistic sensitivity analysis, the median ICER of azithromycin compared to clarithromycin was $16,709 per QALY (mean $16,966 per QALY, 95% CI: $16,900-$16,972 per QALY), and was sensitive to the probability of a GI adverse event with clarithromycin and azi- thromycin, the probability of acquiring disease, and PEP effectiveness, but not sensitive to the complications and costs of pertussis, including probability and cost of hospitalization, the probability and costs of encephalitis and its sequelae, probability of death, and changes in utili- ty on PEP or with any pertussis health state (Fig. 2A). No PEP had a 95% probability of being cost-effective when the willingness-to-pay threshold was below $2,000 per QALY, whereas azi- thromycin had a 95% probability of being cost-effective at the threshold of $50,000 per QALY (Fig. 3A). Among adult contacts, the quality-adjusted life expectancy was 18.73 years without PEP. Among adult contacts, the quality-adjusted life expectancy was 18.73 years without PEP. Azithromycin was the dominant strategy compared to the other macrolides, with more QALYs and lower costs. Compared to no intervention, which was the least costly option, PEP with azi- thromycin resulted in an additional 0.031 QALYs, or 11 quality-adjusted life days, and $76 in incremental costs, for an ICER of $2,415 per QALY. The median ICER was $2,574 per QALY (mean $2,426 per QALY, 95% CI $2,425-$2,426 per QALY) and was sensitive to PEP effective- ness, probability of acquiring disease and probability of encephalitis, and not sensitive to the probability of an adverse GI event with azithromycin, sequelae and death, costs of hospitaliza- tion and encephalitis, and changes in utility on PEP or with any pertussis health state; however, azithromycin remained the preferred strategy (Fig. 2B). Among adult contacts, azithromycin was the preferred strategy, with a 95% probability of being cost-effective at $4,000 per QALY (Fig. 3B). Results A threshold analysis for the effectiveness of PEP in preventing a secondary case of pertussis is shown in Table 6. Among infants, when the PEP effect was 0.2% or greater, azithromycin of- fered the highest net benefit of all strategies. Among children and adults, this threshold proba- bility was 10% and 9%, respectively. Below these values, none of the macrolides offered a higher net benefit than no post-exposure prophylaxis. PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Fig 2. Tornado diagram of the univariate sensitivity analysis for the azithromycin PEP strategy for child (A) and adult (B) contacts. Azithromycin remained the dominant strategy among infants. The bars represent the variation in cost-effectiveness ratios from the base case scenario in response to sequential changes in model parameters, with the vertical axis reflecting the base case ICER. The maximal and minimal values were tested according to ranges outlined in Table 1. (A) Axis at $16,963 per QALY. (C) Axis at $2,415 per QALY. Fig 2. Tornado diagram of the univariate sensitivity analysis for the azithromycin PEP strategy for child (A) and adult (B) contacts. Azithromycin remained the dominant strategy among infants. The bars represent the variation in cost-effectiveness ratios from the base case scenario in response to sequential changes in model parameters, with the vertical axis reflecting the base case ICER. The maximal and minimal values were tested according to ranges outlined in Table 1. (A) Axis at $16,963 per QALY. (C) Axis at $2,415 per QALY. Fig 2. Tornado diagram of the univariate sensitivity analysis for the azithromycin PEP strategy for child (A) and adult (B) contacts. Azithromycin remained the dominant strategy among infants. The bars represent the variation in cost-effectiveness ratios from the base case scenario in response to sequential changes in model parameters, with the vertical axis reflecting the base case ICER. The maximal and minimal values were tested according to ranges outlined in Table 1. (A) Axis at $16,963 per QALY. (C) Axis at $2,415 per QALY. doi:10.1371/journal.pone.0119271.g002 doi:10.1371/journal.pone.0119271.g002 A recent systematic review found only 2 RCTs related to pertussis PEP [13,61], and insuffi- cient evidence to support erythromycin for all household contacts [62]; although, they may not have been sufficiently powered to detect a significant difference. An argument against PEP is that widespread immunization has been associated with a milder clinical course among sec- ondary cases [41,63–65]. Most chemoprophylaxis studies occurred in the context of an under- immunized adult population [3,5,66,67] and large cohort studies have demonstrated some ben- efit in offering PEP to household contacts [27,28,68,69]. However, a recent study among a highly-immunized population of health care professionals found no difference in rates of symptomatic pertussis between those who received PEP compared to daily symptom monitor- ing [64]. Discussion A cost-utility analysis was undertaken to examine 4 strategies against pertussis transmission to household contacts. The decision model synthesized evidence from a broad range of published literature and used QALYs as a preference-based outcome measure. Azithromycin offered the most QALYs in all age groups. Among infants, PEP with azithromycin had the lowest expected cost and most QALYs. This strategy was cost-effective among children and adult contacts at $16,963 and $2,415 per QALY gained, respectively, compared to no PEP, which was well under the cost-effectiveness threshold of $50,000 per QALY gained. The findings were robust in sen- sitivity analyses. In particular, when the probability of disease acquisition and disease severity were minimized (reflecting an immune population), disease-related costs were still significant, especially among infants, and azithromycin remained the preferred strategy. Pertussis transmission has been mitigated historically through immunization and chemo- prophylaxis. While numerous studies have analyzed the impact of varying immunization strat- egies [32,38,40,55–60], this is the first study to comparatively evaluate the benefits, risks and costs associated with antibiotic PEP options among household contacts. PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 10 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Pertussis Post-Exposure Prophylaxis: A Decision Model doi:10.1371/journal.pone.0119271.g002 Pertussis Post-Exposure Prophylaxis: A Decision Model Fig 3. Cost-effectiveness acceptability curves for child (A) and adult (B) contacts. These curves reflect the proportion of times each intervention is likely to be cost-effective for a given cost-effectiveness threshold, up to $50,000 per additional QALY. Clarithromycin post-exposure prophylaxis was never a preferred strategy for adult contacts, and so does not feature for clarity. Fig 3. Cost-effectiveness acceptability curves for child (A) and adult (B) contacts. These curves reflect the proportion of times each intervention is likely to be cost-effective for a given cost-effectiveness threshold, up to $50,000 per additional QALY. Clarithromycin post-exposure prophylaxis was never a preferred strategy for adult contacts, and so does not feature for clarity. doi:10.1371/journal.pone.0119271.g003 associated with contact tracing, evaluation and counseling for PEP. However, all participants had been immunized against pertussis within the previous 2 years, suggesting higher rates of immunity than what may be found in the general community [67]. Thus, the marginal benefits of PEP in this study may have been smaller compared to what may be found in the general population, particularly among infants and young children who are at highest risk for severe disease [29,42,66]. The analysis has several limitations. The model does not account for changes in disease inci- dence as it is not an infectious disease transmission model; however the probability of acquir- ing pertussis and the impact of prophylaxis were varied in sensitivity analyses. The healthcare payer perspective includes only direct medical costs related to health care resource utilization, such as medication, laboratory, procedures and personnel, hospitalizations and physician visits [55]. Numerous studies have highlighted the significant indirect medical and non-medical costs of pertussis [32,40,59,60,70–75]. Given that the antibiotic strategies had similar effective- ness, the differences in non-medical costs between each regimen may be minimal. However, the indirect economic benefit of PEP in avoiding pertussis and its complications may have been underestimated, as the model did not account for QALY loss of caregivers looking after contacts with pertussis or with neurologic sequelae following pertussis encephalitis. Table 6. Threshold analysis of post-exposure prophylaxis effectiveness. PEP effect Contact Group 0 0.001 0.002 0.005 0.01 0.08 0.09 0.1 0.9 1 Infant no no A A A A A A A A Child no no no no no no no A A A Adult no no no no no no A A A A “no” indicates no PEP as the preferred option. The authors questioned whether antibiotic PEP could be eliminated in such popula- tions, given the potential for reduced prescriptions, rates of adverse events, and labor costs PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 11 / 17 “A” indicates azithromycin. When PEP has zero effect, the net benefit of no prophylaxis exceeds that of azithromycin and other macrolides. At or above 10% effectiveness, PEP with azithromycin offers a higher net benefit than no intervention. Table 6. Threshold analysis of post-exposure prophylaxis effectiveness. “no” indicates no PEP as the preferred option. p p “A” indicates azithromycin. When PEP has zero effect, the net benefit of no prophylaxis exceeds that of azithromycin and other macrolides. At or above 10% effectiveness, PEP with azithromycin offers a higher net benefit than no intervention. PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 12 / 17 Pertussis Post-Exposure Prophylaxis: A Decision Model The costs incurred during lifeyears gained from the intervention were not included in this model, in keeping with Canadian guidelines on economic evaluations [20]. Whether to include future unrelated healthcare costs, that is costs solely due to changes in survival, has been con- troversial. Theoretical arguments for [76] and against inclusion [77] of unrelated costs have been advanced and debated [78].To date no consensus has been reached, and to the best of our knowledge, no current guideline on economic evaluation calls for inclusion of unrelated cost as a requirement. The Canadian guideline recommends including unrelated costs only in sensitiv- ity analysis, if at all [20]. While substantial survival benefits may have a major impact on spending, in the case of pertussis, where the highest risk of mortality is among infants and at 0.6%, offering chemoprophylaxis arguably does not have substantial effects on survival. Since these costs were excluded, the cost-effectiveness of the intervention was likely overestimated. Existing clinical and cost data were applied to each age group used in this model, and so nu- merous assumptions were made regarding the calculation and application of costs and proba- bilities where published data were not available. It is arguable that the populations from which secondary attack rates are estimated may differ from the best available data, which is from Que- bec during the 1990s. Despite subsequent changes in the immunization schedules, including a booster dose during adolescence and among adults in contact with children, and in vaccine product, from an adsorbed whole cell vaccine to acellular vaccine, there is no prospective sur- veillance on immunization rates or household secondary attack rates. PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 References 1. Cherry JD. The epidemiology of pertussis and pertussis immunization in the United Kingdom and the United States: a comparative study. Curr Probl Pediatr. 1984; 14: 1–78. PMID: 6098424 1. Cherry JD. The epidemiology of pertussis and pertussis immunization in the United Kingdom and the United States: a comparative study. Curr Probl Pediatr. 1984; 14: 1–78. PMID: 6098424 2. Fine PE, Clarkson JA. The recurrence of whooping cough: possible implications for assessment of vac- cine efficacy. Lancet. 1982; 1: 666–669. PMID: 6121976 3. Wendelboe AM, Njamkepo E, Bourillon A, Floret DD, Gaudelus J, Gerber M, et al. Transmission of Bor- detella pertussis to young infants. Pediatr Infect Dis J. 2007; 26: 293–299. PMID: 17414390 4. Cherry JD. Pertussis in adults. Ann Intern Med. 1998; 128: 64–66. PMID: 9424983 5. Mink CM, Cherry JD, Christenson P, Lewis K, Pineda E, Shlian D, et al. A search for Bordetella pertus- sis infection in university students. Clin Infect Dis. 1992; 14: 464–471. PMID: 1554832 6. Public Health Ontario. Monthly Infectious Diseases Surveillance Report—June. 2012. Available: http:// www.publichealthontario.ca/en/DataAndAnalytics/Documents/2012_June_PHO_Monthly_Report.pdf. 7. Rodgers L, Martin SW, Cohn A, Budd J, Marcon M, Terranella A, et al. Epidemiologic and Laboratory Features of a Large Outbreak of Pertussis-Like Illnesses Associated With Cocirculating Bordetella hol- mesii and Bordetella pertussis—Ohio, 2010–2011. Clin Infect Dis. 2013; 56: 322–331. doi: 10.1093/cid/ cis888 PMID: 23087388 8. Roehr B. Whooping cough outbreak hits several US states. BMJ. 2010; 341: c4627. doi: 10.1136/bmj. c4627 PMID: 20736256 9. Winter K, Harriman K, Zipprich J, Schechter R, Talarico J, Watt J, et al. California pertussis epidemic, 2010. J Pediatr. 2012; 161: 1091–1096. doi: 10.1016/j.jpeds.2012.05.041 PMID: 22819634 10. Clark TA. Responding to pertussis. J Pediatr. 2012; 161: 980–982. doi: 10.1016/j.jpeds.2012.07.014 PMID: 22906510 11. Klein NP, Bartlett J, Rowhani-Rahbar A, Fireman B, Baxter R. Waning protection after fifth dose of acel- lular pertussis vaccine in children. N Engl J Med. 2012; 367: 1012–1019. doi: 10.1056/ NEJMoa1200850 PMID: 22970945 12. Cherry JD. Why do pertussis vaccines fail? Pediatrics. 2012; 129: 968–970. doi: 10.1542/peds.2011- 2594 PMID: 22529282 13. Halperin SA, Bortolussi R, Langley JM, Eastwood BJ, De Serres G. A randomized, placebo-controlled trial of erythromycin estolate chemoprophylaxis for household contacts of children with culture-positive bordetella pertussis infection. Pediatrics. 1999; 104: e42. PMID: 10506267 14. Dodhia H, Crowcroft NS, Bramley JC, Miller E. UK guidelines for use of erythromycin chemoprophylaxis in persons exposed to pertussis. J Public Health Med. 2002; 24: 200–206. Moreover, more than half of pertussis cases continue to occur among children and adults reported as being fully im- munized for their age [7,9,28,79], and an increasing lack of protection has been found among children within 5 years of completing the childhood immunization series against pertussis [11]. In this economic analysis, to explore the uncertainty in secondary attack rates, the proba- bility of acquiring disease was varied broadly, and did not result in a change in the preferred strategy for contacts in any of the age groups. The assumption that respiratory and neurologic diseases are mutually exclusive does not necessarily reflect clinical experience; however the associated costs and QALYs of both compli- cations are likely to overlap, and thus be potentially overestimated in this analysis. The utility scores for infants were derived from adolescent and adult respondents, which may have im- pacted the validity of the QALYs measured. Direct elicitation of preferences can be especially challenging in children due to evolving developmental maturity and their cognitive abilities at various ages to value health; parents may also not reliably report on subjective outcomes of clinical conditions, all of which potentially impact the validity and generalizability of the QALYs measured.[80,81] Furthermore, only respiratory and neurologic complications were in- cluded, while other serious health states in the disease spectrum were not assessed, including apneic spells with spontaneous and complete recovery, generalized illness with outpatient care, and long-term neurologic sequelae following encephalitis. All cases reported to the OCCI were less than 18 years of age, and the costs of encephalitis and long-term neurologic sequelae have been extrapolated from other disease processes. The cost of death was the same as for hospitalization in this model; however end-of-life care is asso- ciated with higher costs [46,49]. Uncertainty in the parameter estimates was tested through univariate and probabilistic sensitivity analyses, and did not shift the preferred strategy from the base case scenario. Alongside policy recommendations for immunization of infants, children, adolescents and adults, there remains an important role for chemoprophylaxis in the prevention of pertussis, especially in outbreaks, when immunization is not immediately protective, and among infants, who are at highest risk for complicated disease. Our analysis suggests azithromycin PEP to be cost-effective when compared with other macrolides and no intervention, protecting house- hold contacts from acquiring infection at acceptable costs. 13 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Disclaimer The opinions, results and conclusions reported in this paper are those of the authors. No en- dorsement by Public Health Ontario is intended or should be inferred. Acknowledgments The authors wish to thank Dr. Chaim Bell for his critical review of the manuscript Author Contributions Conceived and designed the experiments: NT IGU NC BS. Performed the experiments: NT BS. Analyzed the data: NT BS. Wrote the paper: NT IGU NC BS. PLOS ONE | DOI:10.1371/journal.pone.0119271 March 6, 2015 References PMID: 12831090 15. Health Canada. National Consensus Conference on Pertussis. Canada Communicable Disease Re- port. 2003; 2953: 1–28. 16. Tiwari T, Murphy TV, Moran J. Recommended antimicrobial agents for the treatment and postexposure prophylaxis of pertussis: 2005 CDC Guidelines. MMWR Recomm Rep. 2005; 54: 1–16. PMID: 16382216 17. Hoppe JE, Eichhorn A. Activity of new macrolides against Bordetella pertussis and Bordetella paraper- tussis. Eur J Clin Microbiol Infect Dis. 1989; 8: 653–654. PMID: 2550234 18. Langley JM, Halperin SA, Boucher FD, Smith B. 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Comprehensive analysis of the ionospheric response to the largest geomagnetic storms from solar cycle 24 over Europe
Frontiers in astronomy and space sciences
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TYPE Original Research PUBLISHED 24 April 2023 DOI 10.3389/fspas.2023.1092850 TYPE Original Research PUBLISHED 24 April 2023 DOI 10.3389/fspas.2023.1092850 TYPE Original Research PUBLISHED 24 April 2023 DOI 10.3389/fspas.2023.1092850 geomagnetic storms, space weather, ionosphere, ionospheric storm, midlatitude ionospheric trough, GNSS TEC, ionosphere-plasmasphere coupling, Swarm observations geomagnetic storms, space weather, ionosphere, ionospheric storm, midlatitude ionospheric trough, GNSS TEC, ionosphere-plasmasphere coupling, Swarm observations Comprehensive analysis of the ionospheric response to the largest geomagnetic storms from solar cycle 24 over Europe OPEN ACCESS EDITED BY Joseph Huba, Syntek Technologies, United States REVIEWED BY Shun-Rong Zhang, Haystack Observatory, United States Bapan Paul, Vivekananda B.Ed and D.El.Ed College, India *CORRESPONDENCE K. A. Berényi, berenyi.kitti@epss.hu RECEIVED 08 November 2022 ACCEPTED 11 April 2023 PUBLISHED 24 April 2023 CITATION Berényi KA, Heilig B, Urbář J, Kouba D, Kis Á and Barta V (2023), Comprehensive analysis of the ionospheric response to the largest geomagnetic storms from solar cycle 24 over Europe. Front. Astron. Space Sci. 10:1092850. doi: 10.3389/fspas.2023.1092850 OPEN ACCESS doi: 10.3389/fspas.2023.1092850 COPYRIGHT © 2023 Berényi, Heilig, Urbář, Kouba, Kis and Barta. 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. Frontiers in Astronomy and Space Sciences OPEN ACCESS EDITED BY Joseph Huba, Syntek Technologies, United States REVIEWED BY Shun-Rong Zhang, Haystack Observatory, United States Bapan Paul, Vivekananda B.Ed and D.El.Ed College, India *CORRESPONDENCE K. A. Berényi, berenyi.kitti@epss.hu RECEIVED 08 November 2022 ACCEPTED 11 April 2023 PUBLISHED 24 April 2023 REVIEWED BY Shun-Rong Zhang, Haystack Observatory, United States Bapan Paul, Vivekananda B.Ed and D.El.Ed College, India K. A. Berényi1,2,3*, B. Heilig3,4, J. Urbář5, D. Kouba5, Á. Kis3 and V. Barta3 1ELKH-ELTE Space Research Group, Budapest, Hungary, 2Doctoral School of Environmental Sciences, ELTE Eötvös Loránd University, Budapest, Hungary, 3Institute of Earth Physics and Space Science, Sopron, Hungary, 4Space Research Group, Eötvös Loránd University, Budapest, Hungary, 5Institute of Atmospheric Physics CAS, Prague, Czechia CITATION Berényi KA, Heilig B, Urbář J, Kouba D, Kis Á and Barta V (2023), Comprehensive analysis of the ionospheric response to the largest geomagnetic storms from solar cycle 24 over Europe. Front. Astron. Space Sci. 10:1092850. doi: 10.3389/fspas.2023.1092850 A multi-instrumental analysis of the meridional ionospheric response is presented over Europe during the two largest ICME-driven geomagnetic storms of solar cycle #24 maximum. Data from 5 European digisonde stations, ground-based Global Navigation Satellite System, Total Electron Content (GNSS TEC), the ratio of the TEC difference (rTEC), as well as Swarm and Thermosphere, Ionosphere, Mesosphere, Energetics and Dynamics (TIMED) satellite observations have been used for the investigation of selected intervals (11–17 November, 2012, and 16–25 March, 2015). The storm evolution is monitored by digisonde foF2 critical frequency (related to the maximum electron density of F2-layer) and GNSS TEC data. Moreover, Global Ultraviolet Imager (GUVI) measurements from the TIMED satellite are used to investigate the changes in the thermospheric O/N2 ratio. Our main focus was on the main phase of the geomagnetic storms, when during the nighttime hours extremely depleted plasma was detected. The extreme depletion is observed in foF2, TEC and rTEC, which is found to be directly connected to the equatorward motion of the midlatitude ionospheric trough (MIT) on the nightside. We demonstrate a method (beside the existing ones) which allows the monitoring of the storm-time evolution of the disturbances (e.g., MIT, SAPS, SED) in the thermosphere-ionosphere-plasmasphere system by the combined analysis of the worldwide digisonde system data (with the drift measurements and the ionospheric layer parameters with 5–15 min cadence), with rTEC and GNSS TEC data, and with the satellite data like Swarm, TIMED/GUVI. Front. Astron. Space Sci. 10:1092850. doi: 10.3389/fspas.2023.1092850 Front. Astron. Space Sci. 10:1092850. Frontiers in Astronomy and Space Sciences frontiersin.org 1 Introduction In addition, several other influencing factors must be taken into consideration, such as: geomagnetic storm size, local time (LT) of the sudden storm commencement (SSC), time of the day, season, geomagnetic latitude and longitude (Immel and Mannucci, 2013; Mendillo and Narvaez, 2010; Mendillo and Narvaez, 2009). Within the Earth’s plasma environment (ionosphere, plasmasphere, outer magnetosphere), all regions are closely related to each other. In the presence of an external forcing (e.g., ICME or CIR/HSSWS), perturbations can be observed in each plasma layer. The F-layer of the ionosphere has the highest electron density, so electron density changes are the most pronounced there. The electric fields generated during geomagnetically disturbed periods (e.g., prompt penetration of magnetospheric convection electric field-PPEF and disturbance dynamo electric field-DDEF) map along geomagnetic field lines through all these regions and can interact with all of them (see e.g., Nava et al., 2016). The Earth’s plasma environment is a very complex, tightly coupled system, and the effects cannot be studied and explained in their entirety if we do not consider them as part of the system. Geomagnetic storms generate so-called ionospheric storms, which have similar evolution and phases to those of geomagnetic storms, but with a faster procession. Ionospheric storms have already been studied using several types of observations of the F2-layer: ionosonde data of the maximum electron density (NmF2); measurements of the total electron content; incoherent scatter radar measurements of electron and ion densities, temperatures and plasma dynamics; satellite measurements of ionospheric/thermospheric parameters along their orbits (see e.g., Kane and Makarevich, 2010). Comprehensive reviews of storm effects in NmF2 and TEC have been given by Prölss (1995) and Mendillo (2006), for incoherent scatter radar results by Buonsanto (1999), and for in-situ satellite data by Prölss and Zahn (1974). Long-duration positive ionospheric storm phase can be generated by the enhanced storm-induced equatorward meridional winds because they cause the downwelling of the neutral atomic oxygen and the uplifting of the F-layer along the magnetic field lines due to wind induced vertical ExB drift (Prölss, 1995; Danilov, 2013). A similar drift can also be caused by an increase in E-fields of other origin (e.g., from the magnetosphere) (Danilov, 2013). As the loss-rate decreases with altitude, the density increases. 1 Introduction Previous studies concluded that at high and middle latitudes this negative phase type occurs more often, and has much more dangerous effect on HF propagation (Danilov, 2013). the geomagnetic activity. For intense storms, it could be found below L = 2.5 (Mendillo and Narvaez, 2009). There are two major consequences of high-latitude heating: composition changes (specifically, decrease in the O/N2 ratio) and the intensification of equatorward winds that can carry the composition change toward lower latitudes (Buonsanto, 1999). Both processes contribute to the formation of long- duration negative storm phases. The electron density near the F2-layer maximum is, approximately, directly proportional to the O/N2 ratio (Rishbeth and Barron, 1960; Pirog, 2006). This means, if other conditions are unchanged (constant pressure), we should detect depletion in electron density (negative phase) in all regions where O/N2 ratio has been decreased at F2-layer heights (Danilov, 2013). Under geomagnetic storm conditions this so-called compositional disturbance zone at F2-layer heights reaches lower latitudes because of the intensified heating induced equatorward meridional winds. Negative phase is most common in the summer hemisphere (both day and night) due to the seasonal variation of the background thermospheric wind circulation, but during intense geomagnetic storms it can be detected even in winter months. It favors the postmidnight and morning sectors, because then the background and the storm- induced circulation coincide and add up and therefore can reach much lower latitudes (Prölss, 1995; Buonsanto, 1999; Danilov, 2013). This equatorward expansion of the negative phase has about 50–300 m/s velocity (Danilov and Belik, 1991). Besides the O/N2 ratio depletion, the increased temperature of the heated thermospheric gas itself is an important factor in forming a negative storm phase in the ionosphere (Mikhailov and Foster, 1997). The increase in temperature leads to an increase in the recombination coefficient causing a further decrease in electron density (see Mikhailov et al., 1995). Previous studies concluded that at high and middle latitudes this negative phase type occurs more often, and has much more dangerous effect on HF propagation (Danilov, 2013). perturbations. Besides there are several processes that have to be taken into consideration during the examination of the mid- and low latitude ionosphere, as follows: photo-production, chemical loss, and transport by thermal expansion, neutral winds, waves, tides and electric fields of internal and external origin (Mendillo and Narvaez, 2009). Frontiers in Astronomy and Space Sciences frontiersin.org 1 Introduction The physical processes of the Earth’s plasma environment responsible for the perturbations of the system and the exact mechanisms of action of solar events affecting the near-Earth space have been of interest to researchers for decades. The most remarkable disturbing solar activity events are interplanetary coronal mass ejections (ICMEs) and high- speed solar wind streams (HSSWS)/corotating interaction regions (CIRs). When they collide with the Earth’s magnetic field, they trigger geomagnetic storms. The two types of geomagnetic storms have different time courses and result in different magnitudes of Frontiers in Astronomy and Space Sciences 01 frontiersin.org frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. the geomagnetic activity. For intense storms, it could be found below L = 2.5 (Mendillo and Narvaez, 2009). There are two major consequences of high-latitude heating: composition changes (specifically, decrease in the O/N2 ratio) and the intensification of equatorward winds that can carry the composition change toward lower latitudes (Buonsanto, 1999). Both processes contribute to the formation of long- duration negative storm phases. The electron density near the F2-layer maximum is, approximately, directly proportional to the O/N2 ratio (Rishbeth and Barron, 1960; Pirog, 2006). This means, if other conditions are unchanged (constant pressure), we should detect depletion in electron density (negative phase) in all regions where O/N2 ratio has been decreased at F2-layer heights (Danilov, 2013). Under geomagnetic storm conditions this so-called compositional disturbance zone at F2-layer heights reaches lower latitudes because of the intensified heating induced equatorward meridional winds. Negative phase is most common in the summer hemisphere (both day and night) due to the seasonal variation of the background thermospheric wind circulation, but during intense geomagnetic storms it can be detected even in winter months. It favors the postmidnight and morning sectors, because then the background and the storm- induced circulation coincide and add up and therefore can reach much lower latitudes (Prölss, 1995; Buonsanto, 1999; Danilov, 2013). This equatorward expansion of the negative phase has about 50–300 m/s velocity (Danilov and Belik, 1991). Besides the O/N2 ratio depletion, the increased temperature of the heated thermospheric gas itself is an important factor in forming a negative storm phase in the ionosphere (Mikhailov and Foster, 1997). The increase in temperature leads to an increase in the recombination coefficient causing a further decrease in electron density (see Mikhailov et al., 1995). 1 Introduction In early studies, two different phases of ionospheric storms were distinguished: positive ionospheric storm phase, when the electron density is increased, and negative ionospheric storm phase, when the electron density is decreased with respect to its expected value. Earlier studies during the past few decades have already found clear and unambiguous patterns in the ionosphere during geomagnetic storms, see for example, the great reviews of Sato (1957), Matsushita (1959), Prölss (1995), Danilov (2013) (for ionosonde data) and Mendillo (2006) (for TEC data). (2) The ionospheric F- region plasma is the base of the plasmasphere (Mendillo et al., 1974), i.e., the plasmasphere is filled from the dayside ionosphere and empties into the nightside F-region through diffusion along the geomagnetic field lines. Under quiet conditions the midlatitude region maps into the plasmasphere corotating with the Earth. During storms the footprint of the plasmapause (PP) often moves to midlatitudes, especially after sunset (Mendillo and Narvaez, 2009) as the plasmapause moves inward due to the increased geomagnetic activity, while the plasmasphere can still remain conjugated with the midlatitude ionosphere during The various perturbations in the midlatitude ionosphere during geomagnetic storms can be linked to various processes detailed below. For case studies of ionospheric storm effects, it is necessary to determine the key drivers of the actual events. (1) During geomagnetic storms, the Joule dissipation of currents and the absorption of precipitating particles in the auroral region of the lower thermosphere (100–140 km) are the cause of the so called auroral heating of the thermosphere (Prölss, 1995; Danilov, 2013). The location of both the precipitation boundary and the auroral currents depends on 02 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 10.3389/fspas.2023.1092850 daytime hours. Thus, from local noon to dusk the dynamical plasmaspheric processes may contribute to daytime positive- phase ionospheric storms (Lanzerotti et al., 1975), which is followed after sunset by a sharp transition to a depleted state beyond the footprint of the plasmapause (Mendillo et al., 1974). After sunset, both the above discussed dynamics and chemistry cause the onset of a negative phase. Furthermore, multi-instrumental comprehensive analysis of the thermosphere-ionosphere-plasmasphere coupling processes during individual geomagnetic storms are rare in the literature. Most studies are based on one or two types of observational data, however the effects cannot be studied separately since the Earth’s plasma environment is a very complex, multiplied coupled system. 1 Introduction In this study our aim is to determine the exact connections between the thermosphere-ionosphere-plasmasphere system during two intense ICME-caused geomagnetic storm events. We analyzed the effects through a meridional chain of digisondes (foF2, h’F2 and drift data) across Europe (Northern Hemisphere) complemented by GNSS TEC, Swarm satellite observations of the topside electron density, electron temperature and PP location data, as well as O/N2 ratio observations from TIMED satellite. Another aim is to identify the processes responsible for the extreme decrease during the night in the main phase of the geomagnetic storm. The rest of the paper is organized as follows. First, the utilized data and the applied methods are introduced. In Section 3 the observations are presented, followed by the discussion in Section 4. Finally, main conclusions are summarized in Section 5. (3) During intense geomagnetic storm events, mainly in the main phase, at night, a drastic “peeling off” of the magnetospheric plasma can be seen, and the ionospheric projection of the PP can penetrate into the midlatitude region also on the dayside. In such a case, an all-day negative phase storm is expected. Based on earlier studies like Heilig et al. (2022), it is stated that the ionospheric footprint of the PP is tightly coupled to the minimum of the midlatitude (main) ionospheric trough (MIT). The poleward edge of the MIT in the evening sector has been observed to coincide with the equatorward boundary of the soft electron precipitation, and the precipitation has therefore been proposed as a major source of electrons building up the poleward wall of the MIT in the evening sector (Turunen and Liszka, 1972; Rodger et al., 1986; Voiculescu et al., 2010). During the night the ionospheric F-layer on the equatorward side of the plasmapause is maintained by the plasmasphere, but not on the global convection dominated poleward side (Schunk and Banks, 1975). Consequently, an MIT minimum could be bounded by a plasmasphere-maintained and the precipitation- maintained ionosphere. The minimum could be sharpened by storm-time developed subauroral polarization streams (SAPS) due to SAPS E-field, and by plasma heating (frictional heating by the drifting plasma) and its consequences (Rodger et al., 1992; Horváth and Lovell, 2016), and by enhancing recombination due to molecular composition changes by neutral winds or diffusion from the aurora oval = composition disturbance zone (1) (Schunk and Banks, 1975). 2 Data and method Its typical latitudinal extent (width) is 5°–10° (Voiculescu et al., 2010), 10°–15° (Liu and Xiong, 2020), but it can be as wide as 20°. MIT is typically found somewhere between 55° and 75° geographic latitude depending on the geomagnetic activity and MLT (e.g., Whalen, 1989; Werner and Prölss, 1997; Voiculescu et al., 2006; Deminov and Shubin, 2018; Karpachev et al., 2019; Aa et al., 2020; Liu and Xiong, 2020). However, as we will show, in severely disturbed cases it can move even further equatorward. MIT typically occurs in the dark hemisphere, thus, it is most regularly observed during winter months and equinoxes, while in summer, it is mainly restricted to the midnight sector (Rodger et al., 1992; Voiculescu et al., 2006). We analyzed individual events to see in detail the processes and to determine the key drivers of the geomagnetic storm generated ionospheric perturbations. Using the meridional station chain, the latitudinal evolution of the effects can be followed. With the digisonde drift measurements the directions of the associated plasma drifts can be determined. Using also satellite data, the thermospheric (with TIMED, GUVI measurements) and plasmaspheric (with Swarm, Langmuir probe measurements) processes can be linked to the perturbations observed in the ionosphere. The measurements and data utilized are listed below: Over the years, the general behavior of the ionosphere during storms has become well studied. However, each space weather event and the involved processes are unique, and in many cases the magnitude of the effects cannot yet be predicted. Consequently, case studies of extraordinary/high magnitude events are still important as they can deepen/refine our understanding of the extent of change that can be expected in the individual layers of the Earth’s plasma in response to a geomagnetic storm. Frontiers in Astronomy and Space Sciences 2 Data and method In the present study our main focus is on the analysis of the effects in the ionospheric F2-layer during two intense (Dstmin > −100 nT) ICME related geomagnetic storms. The storms considered are from the winter/equinox of 2012 and 2015 (the maximum of #24 solar cycle), respectively, with the following characteristics: 11–17/11/2012 (Dstmin = −108 nT, Kpmax = 6.33) and 16–25/03/2015 (Dstmin = −223 nT, Kpmax = 7.67). For the investigation, intervals covering all the pre-storm (24 h before the SSC), initial, main and recovery phase were selected. We used the geomagnetic Dst-index to identify the storm phases. A sudden sharp increase in the data is observed when an SSC occurs, this is followed by a major decrease in the data (main phase), and then a slower recovery lasting for several days. The magnitude of the storm itself is given by the minimum value of Dst. The AE-index (auroral electrojet index - mainly characterizing the polar region) increases as the substorm activity intensifies. MIT is a longitudinally elongated and latitudinally restricted region of depleted plasma in the ionosphere (He et al., 2011). Its typical latitudinal extent (width) is 5°–10° (Voiculescu et al., 2010), 10°–15° (Liu and Xiong, 2020), but it can be as wide as 20°. MIT is typically found somewhere between 55° and 75° geographic latitude depending on the geomagnetic activity and MLT (e.g., Whalen, 1989; Werner and Prölss, 1997; Voiculescu et al., 2006; Deminov and Shubin, 2018; Karpachev et al., 2019; Aa et al., 2020; Liu and Xiong, 2020). However, as we will show, in severely disturbed cases it can move even further equatorward. MIT typically occurs in the dark hemisphere, thus, it is most regularly observed during winter months and equinoxes, while in summer, it is mainly restricted to the midnight sector (Rodger et al., 1992; Voiculescu et al., 2006). Over the years, the general behavior of the ionosphere during storms has become well studied. However, each space weather event and the involved processes are unique, and in many cases the magnitude of the effects cannot yet be predicted. Consequently, case studies of extraordinary/high magnitude events are still important as they can deepen/refine our understanding of the extent of change that can be expected in the individual layers of the Earth’s plasma in response to a geomagnetic storm. MIT is a longitudinally elongated and latitudinally restricted region of depleted plasma in the ionosphere (He et al., 2011). 2.2 Ionosonde data For comparison, the National Institute of Information and Communications Technology (NICT) Ratio of the TEC difference (rTEC) global maps constructed using the RINEX files obtained from thousands of GNSS receivers all over the world were applied for the European region with a grid of 0.5 × 0.5° and smoothed with a 5 × 5 boxcar. The rTEC value was defined as the difference between the observed TEC and the monthly average quiet TEC value (mean of 10 geomagnetically quietest days), normalized by the average TEC. A meridional ionosonde station chain across Europe was selected to represent the latitudinal changes. The following 5 stations were chosen for the analysis (from North to South): Juliusruh (JR), Pruhonice (PQ), Sopron (SO), Rome (RO), and Athens (AT). In Table 1, information on the ionosonde stations is given. LT = UT +1. Most of the stations are operating with a DPS-4D type of ionosonde, a digisonde. Only SO is an exception where a VISRC-2 type ionosonde operated until 2018, when the same DPS-4D type digisonde was installed also here (Bór et al., 2020). Old ionosonde/digisonde data has typically 30/15 min resolution, respectively, however, in some cases (like at Athens station) data are available at 5-min cadence. Frontiers in Astronomy and Space Sciences 2.3 TEC data The Dst index shows the intensity of the ring current and as we mentioned above are generally used to separate the geomagnetic storm’s phases case to case. The AE index describe the evolution of the auroral electrojets, so it inform us about the processes at high latitudes. GNSS TEC IONEX (IONosphere Map EXchange) maps data (http://ftp.aiub.unibe.ch/ionex/draft/ionex11.pdf) International GPS Service for Geodynamics (IGSG) with 2-hourly cadence were used to determine vertical TEC (vTEC) development in areas surrounding the respective digisondes, each map area of size 2.5 (latitude) x 5 (longitude) degrees. 2.4 TIMED satellite, global ultraviolet imager (GUVI) O/N2 measurements During this investigation, two ionospheric parameters of the F2- layer were examined, namely, foF2, h’F2. The foF2 parameter represent the F2 layer critical frequency associated with the maximum plasma (electron) density of the F2 layer, while the h’F2 parameter reflects the changes in the virtual height of the layer. All digisonde data were manually checked and corrected with SAO Explorer. Besides, modern digital ionosondes provide also routine ionospheric drift measurements in addition to classical vertical ionospheric sounding (for more see the Supplementary Data section and Kouba et al., 2008; Kouba and Koucka Knizova, 2012; Kouba and Koucka Knizova, 2016). The ionospheric drift data for the selected storms of this study were manually processed for the PQ station. Nowadays, tens of digisondes worldwide measure ionospheric drifts routinely and store their data in Global Ionosphere Radio Observatory (GIRO). The TIMED satellite has been operating at 625 km altitude since December of 2001 and focuses its measurements to the appr. 60–180 km altitude range (neutral thermosphere and ionosphere) below the satellite (Christensen et al., 2003). TIMED is orbiting on a circular polar orbit with an inclination of 74.1° corresponding to 97.8 min period, which means that a global map of the measurements can be derived from 14.9 daily orbits. GUVI measurements provide dayside O/N2 composition (note that this is a column integrated value for an altitude range) and temperature profile of the Mesosphere and Lower Thermosphere/Ionosphere (MLTI) region, as well as the auroral energy inputs (Christensen et al., 2003; Crowley et al., 2006; http://guvitimed.jhuapl.edu/home_background). Reference values were needed to determine the magnitude of the storm-time deviations from the nominal state. For this, we chose the 3 closest geomagnetically quiet days (QDs) preceding the storms investigated (8, 9, 10 November 2012 and 10, 13, 14 March 2015, respecti721vely) based on the International Q-days (QD) list, and averaged them. These reference values appear as green lines in Figure 3; Figure 4; Figure 5; Figure 6. 2.1 Solar and geomagnetic indices During case studies we typically identify and characterize the storm events considering the magnitude of geomagnetic indices such as Kp, Dst and AE. The Kp index represents the geomagnetic activity of the midlatitudal regions, and it has logarithmic scale. 03 frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. TABLE 1 In this table, we can see information about the used 5 digisonde stations. For the geomagnetic coordinates, International Geomagnetic Reference Field (IGRF-13)-model (for year 2015) based calculator was used. Name of the station Station ID Geomagnetic latitude (deg) Geomagnetic longitude (deg) Geographic latitude (deg) Geographic longitude (deg) High mid- latitude Juliusruh JR055 53.95° N 99.48°E 54.6° N 13.4° E Middle latitude Pruhonice PQ052 49.32° N 98.61° E 50° N 14.6° E Sopron SO148 46.67° N 99.75° E 47.63° N 16.72° E Low mid- latitude Rome RO041 41.7° N 93.76° E 41.8° N 12.5° E Athens AT138 36.17° N 103.33° E 38° N 23.5° E 2.5 Swarm satellite measurements The three Swarm satellites were launched into a polar low- Earth orbit (LEO) in November 2013. The altitude of Swarm A Frontiers in Astronomy and Space Sciences 04 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 C (flying side-by-side with ca 1 5° longitudinal difference) 3 Observations GURE 1 he geomagnetic Dst, Kp and AE indices are plotted for the 2012 November storm (A), and for the 2015 March storm (B). In the upper diagrams the aily variation of the Dst index is presented, in the middle panel the Kp index, while in the bottom the daily variation of the AE index is shown. The UT of the C was at 23:12 for the 2012 storm marked with a red dotted line. In the 2015 storm, the first storm commenced at UT 04:45, while the second SSC was 20:54 (red dotted lines). FIGURE 1 The geomagnetic Dst, Kp and AE indices are plotted for the 2012 November storm (A), and for the 2015 March storm (B). In the upper diagrams the daily variation of the Dst index is presented, in the middle panel the Kp index, while in the bottom the daily variation of the AE index is shown. The UT of the SSC was at 23:12 for the 2012 storm marked with a red dotted line. In the 2015 storm, the first storm commenced at UT 04:45, while the second SSC was at 20:54 (red dotted lines). Frontiers in Astronomy and Space Sciences 3 Observations and C (flying side-by-side with ca. 1.5° longitudinal difference) was around 460 km in March 2015, while Swarm B was operating at around 520 km altitude, with a 91 min orbiting period. Swarm satellites provide in-situ measurements of the electron density and temperature observed by Langmuir probes, as well as the location of the MIT from which the footprint of the nightside plasmapause can be derived. and C (flying side-by-side with ca. 1.5° longitudinal difference) was around 460 km in March 2015, while Swarm B was operating at around 520 km altitude, with a 91 min orbiting period. Swarm satellites provide in-situ measurements of the electron density and temperature observed by Langmuir probes, as well as the location of the MIT from which the footprint of the nightside plasmapause can be derived. In our previous article (Berényi et al., 2018), the characteristics of the 2012 November and 2015 March storm were described in detail, the course of the storms was investigated in several aspects using data from the Sopron ionosonde station. In this article, we discuss the meridional evolution and characteristics of the two Frontiers in Astronomy and Space Sciences 05 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 2 The interplanetary magnetic field (IMF) Bz component and the solar wind speed data can be seen for the two investigated storm intervals. (A) Is for the 2012 November, (B) is for the 2015 March storm. The SSC times are with red dotted lines (Supplementary Figure S1 is the same, except that it is with 1- min resolution). FIGURE 2 FIGURE 2 The interplanetary magnetic field (IMF) Bz component and the solar wind speed data can be seen for the two investigated storm intervals. (A) Is for the 2012 November, (B) is for the 2015 March storm. The SSC times are with red dotted lines (Supplementary Figure S1 is the same, except that it is with 1- min resolution). investigated storms using several types of measurements, detailed in Section 2. Right after that a negative polarity coronal hole associated high speed solar wind stream (HSSWS) arrived. The main phase of the geomagnetic storm started on 14 November (Dstmin = −108 nT, Kpmax = 6.33), this can be distinctly seen in Figure 1A in the evolution of the geomagnetic Dst, Kp and AE indices. Frontiers in Astronomy and Space Sciences 3 Observations According to the ACE satellite data, in the case of storm from 2012 November, the geomagnetic disturbance started with two ICMEs shock arrivals at 22:16 UT on 12 November (source: SWPC PRF 1942, 19 November, 2012; https://izw1.caltech.edu/ ACE/ASC/DATA/level3/icmetable2.htm). The storm started with a Sudden Storm Commencement (SSC) at 23:12 UT (00:12 LT) on 12 November On Figure 2A the evolution of the interplanetary magnetic field (IMF) Bz component and the solar wind (SW) speed data are plotted (1-min resolution is on Supplementary Figure S1A). During the SSC time, short southward turning of the Bz appeared which 1 h later turned northward and lasted until ca. 13 h. In the meanwhile the SW speed from 300 km/s after the SSC went up to 420 km/s and it oscillated around this value through the whole storm interval. Early on 14 November the storm reached its maximum magnitude (major storm), which was caused by a prolonged period of negative Bz (Figure 2A) from 8 h to 18 h, attributed to a combination of lingering ICME effects and a solar sector boundary crossing that occurred at approximately 02:45 UT. The 2015 March geomagnetic storm started with an SSC at 04:45 UT (05:45 LT) on 17 March, around equinox. The magnitude of this storm was (on day 17 March): Dstmin = −234 nT, Kpmax = 7.67 (see also Figure 1B). More information about this ICME generated geomagnetic storm can be found in the article of Wu et al. (2016). On Figure 2B the IMF Bz and SW speed values were displayed (1-min resolution is on Supplementary Figure S1B). The most significant episodes: right after the SSC the northward Bz turned southward around 7 UT, this followed by a northward turning around 10, then from noon until midnight a prolonged southward Bz can be observed. The SW speed values increased from an initial 400 km/s to 600 km/s around noon on17 March, and peaked at 700 km/s on the night of 18 March. On the days after the ICME the Earth was inside the flow of HSSWS (Nava et al., 2016). A subsequent geomagnetic 06 frontiersin.org Berényi et al. 3 Observations 10.3389/fspas.2023.1092850 FIGURE 3 On the upper diagrams the Dst-index values and on the lower diagrams the storm-time foF2 (with red line, A)) and h’F2 (with light blue line, B)) with the reference foF2, h’F2 values of selected 3 quiet days [with dotted green (A) and pink line (B)] are portrayed for storm 2012 over the meridional station chain. FIGURE 3 On the upper diagrams the Dst-index values and on the lower diagrams the storm-time foF2 (with red line, A)) and h’F2 (with light blue line, B)) with the reference foF2, h’F2 values of selected 3 quiet days [with dotted green (A) and pink line (B)] are portrayed for storm 2012 over the meridional station chain. frontiersin.org Frontiers in Astronomy and Space Sciences 3.1.2 Geomagnetic storm 16-25/03/2015 The evolution of the 2015 March geomagnetic storm started on day 17th. The local time of the SSC was at 05:45 on 17 March (these are marked with a red dotted line in Figure 4). After the SSC, during the main phase of the ionospheric and geomagnetic storm, a positive phase of the ionospheric storm started at all stations. These kinds of storms are called Regular Positive Phase (RPP) storms because in the main phase of the storm, the electron density (foF2) increases, with no delay (Mendillo and Narvaez, 2010). At the same time, there is also a significant increase in height of F2-layer (h’F2) during daylight hours at all stations, but much less pronounced at RO and AT (see the right panel of Figure 4). During the daytime hours in the main phase of the geomagnetic storm (14 November) a negative ionospheric storm phase was observed at all stations. In the meantime, the F2 layer was extremely uplifted, up to 680 km at stations SO, PQ and JR (Figure 3, right panel). In this case, we observe a negative ionospheric storm phase during a relatively weak (i.e., closer to moderate) geomagnetic storm. This event can be considered as an atypical storm, since the main phase of the geomagnetic storm is delayed by 1 day. In a regular storm, the main phase typically starts with a significant positive or negative ionospheric storm phase within a few hours after the SSC. The most significant increase in the virtual height of the F2-layer occurred from around 20:00 UT (21 LT) on 17 March. The highest peak of the h’F2 parameter was observed at Pruhonice at 587.6 km at 22:45 UT. This main phase pattern in h’F2 parameter lasts until 21 March, and appears significant at SO, PQ and JR stations (see Figure 4). Note. during this storm the ionosonde at Sopron provided observations only up to 8 MHz, but the negative ionospheric response during the main phase of the geomagnetic storm clearly shows up. Besides, during the evening/night on 17/18 March, a sharp decrease in the electron density was observed, the start of the negative phase began at noon at JR and it occurred later and later with decreasing latitude. The depletion was the most pronounced at PQ, SO and RO (see Figure 4 and Supplementary Table S2). 3.1.1 Geomagnetic storm 11-17/11/2012 The first phenomenon known from previous studies, which can appear in foF2 even 24 h before the SSC during the daytime hours, is called the pre-storm enhancement (Kane, 2005; Burešová and Laštovička, 2007; Danilov, 2013). This can be also observed in all the 5 digisonde stations, though it is not very well pronounced (see Figure 3). On the contrary, during the night on 11/12 November all station data show negative phase, except for AT where no change can be detected. From 15 November, during the early recovery phase, a so-called early recovery phase enhancement effect was detected in foF2 at stations AT, RO, SO and PQ (see Figure 3). This effect with increased electron density around noon lasts 3-day long, most significantly at Rome and Athens station, but observable at the other stations, too (see Figure 3). On the other hand, during the nighttime hours of 15 November a negative phase appears at all stations, while on 16 November only JR and PQ are negative, SO does not show any clear trend, and RO and AT are positive. On the right panel of Figure 3, the virtual height of the F2-layer (h’F2) is presented, not any significant daytime change at any station can be seen on 12 November, however a quite significant increase can be observed during the night starting at 21:00 UT, just before the SSC. During the recovery phase, no deviation from the reference quiet time h’F2 was seen during daytime, however there was a quite significant increase during the night, first of all at the PQ station, but less pronounced also at SO and JR. Right after the SSC at 23:12 UT, an increase in electron density (foF2 parameter) can be seen during that night and then during the day on 13 November at all stations. This is the main phase of the ionospheric storm, but the geomagnetic storm main phase starts only later on the following day. At the beginning of the following night, still positive phase at all stations can be seen, but around midnight it turns negative at JR, PQ and SO. Besides, a significant increase in h’F2 appears already around 20:00 UT which lasts even throughout the next day (14 November). 3.1 Digisonde data over Europe In Supplementary Table S1 the exact time and duration of the fade-outs, the start time of the decrease/increase and the minimum/maximum values are tabulated (the foF2 value in MHz). ionospheric storm phase can be observed, while over lower latitudes (closer to the equator) this turns into a positive phase (the exact latitude of the phase reversal depends e.g., on the season, LT, the strength of the geomagnetic storm, meridional winds, etc.). First, we present ionosonde observations for the extended 2012 and 2015 storm periods. ionograms) at these stations. Along with this electron density drop, the virtual height of the F2-layer increased up to 400 km (from the 250–280 km QD value) before the observed fade-out (see Figures 1, 3, as well as the Supplementary Table S1). On the contrary, at RO and AT stations, a significant increase of the foF2 parameter (electron density) with respect to the reference days could be seen during the night. Furthermore, the h’F2 was not as increased at RO and AT during this period as at the other stations. In Supplementary Table S1 the exact time and duration of the fade-outs, the start time of the decrease/increase and the minimum/maximum values are tabulated (the foF2 value in MHz). Frontiers in Astronomy and Space Sciences 3.1 Digisonde data over Europe disturbance which is connected to a HSSWS (see Figure 1B) started at 20:54 UT on 21 March, which does not appear in the value of the Dst index, but is clearly visible in the Kp and AE index (see Figure 1B) (Nava et al., 2016). Several articles have been published in recent years about this storm, also known as St. Patrick’s Day storm, which is the largest storm of the solar cycle 24 (e.g., Astafyeva et al., 2015; Cherniak and Zakharenkova, 2015; Zhang J.J. et al., 2015; Zhang S.R. et al., 2015; Li et al., 2016; Wu et al., 2016; Nayak et al., 2016; Nava et al., 2016; Kalita et al., 2016; Tulasi et al., 2016; Hairston et al., 2016; Jin et al., 2017; Polekh et al., 2017; Zhang S.R. et al., 2017a; Zhang S.R. et al., 2017b and references therein; Berényi et al., 2018; Huang et al., 2018; Habarulema et al., 2018; Kumar and Kumar, 2019; Ratovsky et al., 2019; Lu et al., 2020). The main results presented in the following were derived from ionosonde and digisonde measurements across a meridional station chain introduced in Section 2. In this section, the evolution of the ionospheric foF2, h’F2 is shown. Using data of European ionosonde stations for comparison, our primary goal is to check whether the ionosonde recorded effects at Sopron station (Berényi et al., 2018) were local or regional/global. Furthermore, we would like to illustrate how an ionospheric storm develops from North to South, along a geomagnetic meridian. Through this demonstration, the turning point of the ionospheric phase development can be determined (see Buonsanto, 1999; Kane, 2005). In a general case, over the auroral region a negative Frontiers in Astronomy and Space Sciences 07 frontiersin.org frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 10.3389/fspas.2023.1092850 10.3389/fspas.2023.1092850 ionograms) at these stations. Along with this electron density drop, the virtual height of the F2-layer increased up to 400 km (from the 250–280 km QD value) before the observed fade-out (see Figures 1, 3, as well as the Supplementary Table S1). On the contrary, at RO and AT stations, a significant increase of the foF2 parameter (electron density) with respect to the reference days could be seen during the night. Furthermore, the h’F2 was not as increased at RO and AT during this period as at the other stations. frontiersin.org 3.1.2 Geomagnetic storm 16-25/03/2015 The electron density decreased below the detectability level (disappeared from the ionograms) for a short period at RO and SO station. In Supplementary Table S2 the exact time and duration of the fade-outs, the start time of the negative phase and the time of the observed minimum value are tabulated. Along with this electron density drop, the virtual height of the F2-layer increased up to 483 km (from the 250–280 km QD value) before the fade-out at Sopron (see Figure 4). This storm can be identified as a Regular Positive Phase (RPP) storm type following the nomenclature introduced by Mendillo and Narvaez (2010) according to the meridional evolution of foF2. Their classification is based on the local time of the SSC, which was in this case around midnight (00:12 LT) on 13 November The significant increase of foF2 (electron density) lasted from around 10–11:00 to 20:00 LT at all stations, and this positive phase was followed by a negative phase on 14 November. In Figure 3 during the main phase of the geomagnetic storm at night hours (18:00–02:30 UT), a negative ionospheric storm phase can be observed at the SO, PQ and JR stations. The electron density decreased below the detectability level (disappeared from the Depletions in the foF2 parameter value (negative phase) compared to the main phase value can be observed on the next 08 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 4 In the upper diagrams the Dst index values and in the other diagrams (A) the storm-time foF2 (red line) and (B) the storm-time h’F2 (light blue line) and the corresponding reference foF2 and h’F2 values (green (A) and pink (B), respectively) are portrayed for the 2015 storm along the meridional chain. FIGURE 4 In the upper diagrams the Dst index values and in the other diagrams (A) the storm-time foF2 (red line) and (B) the storm-time h’F2 (light blue line) and the corresponding reference foF2 and h’F2 values (green (A) and pink (B), respectively) are portrayed for the 2015 storm along the meridional chain. days (during the early recovery phase) at SO, PQ and JR stations (middle - and high midlatitude), during both days and nights. Even at AT and RO, for a short period positive phase occurred around 9 UT, then it is turned into a significant negative phase around 11 UT. 3.2 GNSS TEC data To provide a wider context for the ionosonde observations, changes in the total electron content (TEC) data was compared with the foF2 values measured by the ionosondes. TEC data are presented similarly to Figures 3, 4 for a better comparison. The TEC represents the total electron content as an integrated value between the receiver on the ground and the GNSS satellite (MEO orbit, at around 20,000 km altitude), therefore it contains not just the whole ionosphere, but also most of the plasmasphere (Hofmann- Wellenhof and Lichtenegger, 2001). g g y A subsequent geomagnetic disturbance, associated with a HSSWS (see Figures 1, 4) started at 20:54 UT on 21 March, occurred during the recovery phase of the first storm and generated a positive ionospheric storm. Decreased electron density during the daytime and nighttime of 21th can be observed at all stations, but most significantly at RO. In the h’F2 parameter just a slight increase can be seen right after the SSC during the night, mostly at AT and RO stations. Meanwhile on 22 March no change was observed at JR, and only a slight increase in foF2 at PQ, SO, RO, AT. During the night the storm turns negative at all stations, except for RO and AT, where no change was observed. During the remaining days in the recovery phase: on 23 March no changes at JR and PQ were detected, but a slight increase in foF2 at SO, RO, AT can be seen around noon. During the night just JR showed no changes, the others were positive. On 24 March no changes were observed in foF2 at all stations during the day, but at night just JR did not show changes, the other stations had positive A subsequent geomagnetic disturbance, associated with a HSSWS (see Figures 1, 4) started at 20:54 UT on 21 March, occurred during the recovery phase of the first storm and generated a positive ionospheric storm. Decreased electron density during the daytime and nighttime of 21th can be observed at all stations, but most significantly at RO. In the h’F2 parameter just a slight increase can be seen right after the SSC during the night, mostly at AT and RO stations. Meanwhile on 22 March no change was observed at JR, and only a slight increase in foF2 at PQ, SO, RO, AT. 3.1.2 Geomagnetic storm 16-25/03/2015 At RO and AT stations (low mid-latitude) the depletion became really significant (see Figure 4) only on 20 March. It can be stated that in the early recovery phase the low mid-latitude and the high mid-latitude regions behave differently. phase. On 25 March the situation was the same as on day 23. The h’F2 parameter did not show any deviation from the QD curve during these days. Frontiers in Astronomy and Space Sciences frontiersin.org 3.2.1 rTEC maps of the storms storm (14 November) all stations turned negative except for RO, where there until 12 UT the TEC variation followed the mean variation. Later a slight decrease started and lasted until 18 UT. Also AT behaved differently, because here a TEC enhancement took place during the whole day. In the recovery phase (15–17 November) during the day positive phase was detected. On the contrary, during the nighttime hours on 15 November negative phase, and on 16 November no change was observed at all stations. In Figure 7 the Ratio of the TEC difference (rTEC) maps are shown for (geomagnetic) main phase of the two storms during the nighttime hours (18–04:00 UT). These maps show the relative deviation of TEC from its QD value (10 quietest days of the month). These rTEC maps clearly present the evolution of the nighttime negative phase along the examined stations in Europe. In the case of the 2012 storm as shown in Figure 7A, the reduced electron density region moves from higher to lower latitudes, reaching its minimum latitude around midnight near the Athens- Rome line. Meanwhile, it is also nicely seen that the low-midlatitude Athens-Rome region remains in a positive phase throughout the night. In the 00:00 and 02:00 UT maps the depleted electron density zone is nicely drawn out in blue, and it can be seen that JR, PQ, SO stations are all located within it. In Figure 6 the 2015 March storm can be seen in the TEC data. On the pre-storm day and night (16 March) no deviation from the reference values can be seen at any stations. In the main phase of the storm (17 March) significant positive phase is detected during the day at all stations, but during the night the storm phase is turned negative at all stations, except at JR where first a positive phase and then after midnight no deviation can be seen. During the early recovery phase between 18 and 20 March, a negative effect in TEC is detected during both day and night at all stations. Interestingly at RO and AT on 18 March between around 06–12 UT the TEC is increased with respect to the QD value, then rapidly decreases and stays decreased during the following 3 days. Frontiers in Astronomy and Space Sciences 3.2 GNSS TEC data During the night the storm turns negative at all stations, except for RO and AT, where no change was observed. In Figure 5 the TEC values (red) along with their corresponding references (green) for the 2012 November storm are plotted. Before the SSC time, on 11 and 12 November a small positive deviation can be seen during the day at all stations, while during the night on 11 November no significant deviation was observed. On the following night (12 November) a slight negative phase appeared at RO, SO and PQ. On 13 November, the day after the SSC, very impressive positive ionospheric storm phase developed at all stations during both day and night. In the main phase of the geomagnetic During the remaining days in the recovery phase: on 23 March no changes at JR and PQ were detected, but a slight increase in foF2 at SO, RO, AT can be seen around noon. During the night just JR showed no changes, the others were positive. On 24 March no changes were observed in foF2 at all stations during the day, but at night just JR did not show changes, the other stations had positive 09 frontiersin.org frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 5 The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC derived from selected 3 quiet days (with green line) are portrayed for storm 2012. SSC time is marked with a red dashed line. FIGURE 5 FIGURE 5 The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC derived from selected 3 quiet days (with green line) are portrayed for storm 2012. SSC time is marked with a red dashed line. Frontiers in Astronomy and Space Sciences 3.2.1 rTEC maps of the storms On 21 March the same effect was repeated during the day, but at night JR station showed no deviation, while all the other station data was negative. On 22 March (the day after the 2nd SSC) a positive ionospheric storm phase appeared in TEC during the day at all stations, but during the nighttime hours there was no effect. Between 23 and 25 March a slight positive phase can be seen during the day and night at all stations, except at JR where there is no effect during the night of 23 March. For the 2015 storm, a very strong positive phase (red color in Figure 7B) is visible at 18 UT on 17 March spreading in a NW-SE direction, its boundary shows up in yellow along the line connecting the Black See and North-France (see Figure 7B). SO, PQ to the North is green, indicating that there is no deviation from the expected value, while JR is in negative phase. Interestingly, there is a narrow but strong positive-phase region at high latitudes (over Scandinavia). As we move into the night, at 22:00 UT the negative phase moves towards the equator and the positive phase region also decreases in strength in the Athens-Rome line. At 00:00, the depleted plasma has gone even lower, and this is when both Rome and Athens go into negative phase at night, so the low mid- latitude region is also under the influence of this negative storm effect, which lasts until 04:00 UT. The more specific connections are explained in the Discussion Section 4 below. 10 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 6 The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC of selected 3 quiet days (with green line) are portrayed for storm 2015. SSC time is marked with red dashed line. FIGURE 6 FIGURE 6 The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC of selected 3 quiet days (with green line) are portrayed for storm 2015. SSC time is marked with red dashed line. 3.3 GUVI measurements most significantly over the JR station. This effect also happens on the two following days just the same way, with one difference. On 20 March a slight depletion (light blue) showed up also at RO and AT stations (see Supplementary Figure S3). On 21 March, when the second SSC happened, another slight decrease follows in the GUVI data over JR, PQ, SO. During the next day, on 22 March, right over the JR station a slight decrease can be observed. On 23–25 March SO, PQ and JR stations are in the slightly depleted O/N2 region (light blue) during the day. The TIMED satellite GUVI measurements were used to examine the variation of the daytime thermospheric O/N2 composition during the evolution of the considered ionospheric storms. In Figures 8, 9 the three QDs as a reference are shown (Figure 8A; Figure 9A) and also 3 days from the storm (Figure 8B; Figure 9B) to present the main features. The whole storm intervals are plotted in Supplementary Figure S2 and Supplementary Figure S3 (note that time runs from right to left). For the 2012 November storm the GUVI data presented in Figure 8 do not show any deviation in the O/N2 ratio from the QD level over the examined stations during the daytime hours in the pre- storm phase (12 November). On 13 November (the day after the SSC) around noon (in LT) a slight increase in the O/N2 (yellow) can be seen over Europe. In the main phase (when the Dst minimum value occurred), on 14 November around 12 LT, a significant decrease (blue) in the GUVI measured O/N2 ratio can be observed all over Europe. On 15 November there is no deviation (green), but on 16 and 17 November a slight increase appears in GUVI data marked with yellow (see Supplementary Figure S2). 3.4 Swarm satellite measurements While ground ionosondes are good at monitoring the temporal variation of ionospheric parameters at fixed locations, LEO observations can provide pole-to-pole latitudinal profiles of the considered parameters recorded during around 45 min. The location of the plasmapause (PP), more precisely the location of ionospheric phenomena conjugated with PP, such as the MIT can also be monitored by LEO satellites (e.g., Heilig and Lühr, 2013; 2018; Heilig et al., 2022). Swarm observations are only available for the 2015 storm event. Figure 10 shows the change in the topside electron density (top panel) for three orbits of Swarm B, each separated by 9.5 h (in UT). All measurements were taken at around 21 h MLT but along different meridians. Thus this plot represents primarily the temporal evolution of the topside electron density at 21 h MLT, but the profiles are also influenced by longitudinal effects. St. Patrick’s Day storm 2015 (Figure 9): during the pre-storm phase was on 16 March, the O/N2 ratio shows a decrease (blue) in the subauroral region, e.g., over JR station. After the SSC, during the main phase of the geomagnetic and ionospheric storm, on 17 March in the daytime GUVI data do not show any significant departure from the reference values. On the contrary, during the following day, on 18 March, a quite deep depletion was observed in the O/N2 ratio at JR, PQ and SO, but 11 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 7 The rTEC values are plotted for the nighttime hours (18:00–04:00 UT) on 14 November, 2012 (A), and on 17 March, 2015 (B). FIGURE 7 The rTEC values are plotted for the nighttime hours (18:00–04:00 UT) on 14 November, 2012 (A), and on 17 March, 2015 (B). A deepening density minima (marked by vertical dashed lines on the Northern Hemisphere) can be observed in both hemispheres, moving progressively equatorward as the geomagnetic storm main phase develops. These are the minima of the MIT that is typically well observed in the nighttime ionosphere during equinoxes. Poleward of MIT, the erosion by the increased ExB drift continued along the whole day, while at lower latitudes at the altitude of Swarm B (505–525 km) a general increase was observed. The presented profiles were all taken following the SSC (04:45 UT). The latest profile corresponds to the time of the maximum of the plasmasphere erosion (most equatorward MIT). Frontiers in Astronomy and Space Sciences 3.4 Swarm satellite measurements In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of 3 selected days from the storm interval are presented, namely, the day of SSC (12 November), the main phase of the ionospheric storm (13 November), and the main phase of the geomagnetic storm (14 November) (Note: a plot of GUVI data for the whole storm can be found in Supplementary Figure S1). FIGURE 9 The GUVI measured O/N2 ratio for the 2015 March storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of 3 selected days from the storm interval are presented, namely, the day of pre-storm phase (16 March), the main phase of the ionospheric and geomagnetic storm (17 March), and 1 day from the recovery phase (18 March) (Note: a plot of GUVI data for the whole storm can be found in Supplementary Figure S2). The GUVI measured O/N2 ratio for the 2015 March storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of 3 selected days from the storm interval are presented, namely, the day of pre-storm phase (16 March), the main phase of the ionospheric and geomagnetic storm (17 March), and 1 day from the recovery phase (18 March) (Note: a plot of GUVI data for the whole storm can be found in Supplementary Figure S2). As shown in the lower panel, the MLT difference between Swarm B (red) and C (blue) observations was fairly stable, around 1.5 h on the average. The MIT position is well-known to have a strong MLT dependence (e.g., Heilig et al., 2022). The MIT latitude decreases during afternoon and also during nighttime until 4–6 h MLT. Here this dependence is reflected in the latitude difference between Swarm B and C observations, Swarm B observations are consequently equatorward of Swarm C observations by 2.3° on the average. The time evolution of the MIT minimum position based on Swarm in-situ observations made on the Northern Hemisphere is presented in Figure 11. The two colors depict observations of Swarm B and C satellites. Right after the SSC, MIT started to shift equatorward. This decrease lasted the whole day, and by the end of the day MIT approached 45° magnetic latitude. Frontiers in Astronomy and Space Sciences 3.4 Swarm satellite measurements Sharp MIT minima are accompanied by an enhanced electron temperature (bottom panel). This is a well-known feature of sub- auroral dynamics and appears mainly as a result of frictional heating by the intense westward drift that in turn are driven by an intense substorm (precipitation) associated poleward electric field (e.g., Heilig et al., 2022). MIT minimum in this MLT sector is tightly coupled to the footprint of the plasmapause (Heilig et al., 2022). Thus the equatorward movement of MIT reflects the shrinking of the plasmasphere due to the increased magnetospheric convection and intensified substorm-related sub-auroral electric field (see also in Supplementary Figure S4). 12 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 The time evolution of the MIT minimum position based on As shown in the lower panel, the MLT difference between FIGURE 8 The GUVI measured O/N2 ratio for the 2012 November storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of 3 selected days from the storm interval are presented, namely, the day of SSC (12 November), the main phase of the ionospheric storm (13 November), and the main phase of the geomagnetic storm (14 November) (Note: a plot of GUVI data for the whole storm can be found in Supplementary Figure S1). FIGURE 9 The GUVI measured O/N2 ratio for the 2015 March storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of 3 selected days from the storm interval are presented, namely, the day of pre-storm phase (16 March), the main phase of the ionospheric and geomagnetic storm (17 March), and 1 day from the recovery phase (18 March) (Note: a plot of GUVI data for the whole storm can be found in Supplementary Figure S2). Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 8 The GUVI measured O/N2 ratio for the 2012 November storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of 3 selected days from the storm interval are presented, namely, the day of SSC (12 November), the main phase of the ionospheric storm (13 November), and the main phase of the geomagnetic storm (14 November) (Note: a plot of GUVI data for the whole storm can be found in Supplementary Figure S1). FIGURE 8 The GUVI measured O/N2 ratio for the 2012 November storm. 3.4 Swarm satellite measurements Using these adjustments the MIT at Sopron could have moved down as low as to magnetic latitude 44.6°, maybe even below 42°. The width of the most equatorward MIT was 3.6° as observed by Swarm B. This width is shared between the equatorward wall (1.9°) and the poleward wall (1.7°). This means that on 17 March during the night MIT (but at least its equatorward wall) should have reached the Sopron station. Thus, the Sopron ionosonde observed the depleted plasma associated with the MIT structure. It is also clear that the MIT could not approach Athens, located at 36.1° magnetic latitude. The MIT position is also known to depend on the geographic longitude (e.g., Heilig et al., 2022). In the American sector MIT appears a few degrees higher than in the European or Asian sector. The Equation 5 of Deminov and Shubin (2018) estimates this difference to 0.98°, while according to the statistical results of Karpachev et al. (2019), this difference could easily be 2°–3°. Using these adjustments the MIT at Sopron could have moved down as low as to magnetic latitude 44.6°, maybe even below 42°. The quality of the skymap is very good and corresponds to the assumption of one drift velocity vector in the area above the station. Estimated vectors are determined reliably. The width of the most equatorward MIT was 3.6° as observed by Swarm B. This width is shared between the equatorward wall (1.9°) and the poleward wall (1.7°). This means that on 17 March during the night MIT (but at least its equatorward wall) should have reached the Sopron station. Thus, the Sopron ionosonde observed the depleted plasma associated with the MIT structure. It is also clear that the MIT could not approach Athens, located at 36.1° magnetic latitude. On 14 November around 00:00 UT, a significantly increased value of the vertical drift velocity component (to the tens of m/s) and its very rapid changes is added to the above-described effect. The drift measurement cadence was 15 min. The dramatic changes are visible in every consequent measurement, it is obvious that the period of the vertical drift velocity variation is significantly smaller than 15 min and cannot be studied (described) in more detail due to the limitations of the drift measurements. During the day-time on 14 November both described manifestations practically disappeared. 3.4 Swarm satellite measurements The value of the vertical velocity component no longer fluctuates quickly, and the value of the horizontal (westward) component drops to small values. Here it is necessary to note that the quality of measured skymaps during the Frontiers in Astronomy and Space Sciences frontiersin.org 3.4 Swarm satellite measurements The lowest latitude of the MIT position actually observed (by Swarm B) was 46.3°. This observation took place on 18 March at 00:15 UT (21.9 h MLT) at −43.2° longitude. 13 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 10 Meridional profiles of the topside electron density (top panel) and electron temperature (bottom panel) observed by Swarm B on 17 and 18 March, 2015. Vertical dashed lines indicate the location of MIT on the Northern hemisphere. FIGURE 10 Meridional profiles of the topside electron density (top panel) and electron temperature (bottom panel) observed by Swarm B on 17 and 18 March, 2015. Vertical dashed lines indicate the location of MIT on the Northern hemisphere. At the time of the minimum observation, MLT at Sopron was 0.72 h. Estimating from the MLT difference between Sopron and Swarm B (1.1 h) and the rate of decrease (2.3°/1.5 h = 1.53°/h), the MIT minimum at 0.72 h MLT was located around 45.6°. The formula (their Equation 4) of Deminov and Shubin (2018) for the MLT dependence yields a somewhat higher MIT latitude (45.9°). However, these values are already lower than the latitude of Pruhonice and very close to that of Sopron. vertical motion of the F-layer during the two storm intervals. For the detailed analysis method of the manual correction see the Supplementary section. Now we are going to detail the phenomena found in the manual drift data for each storm separately. The first significant manifestations of the 2012 November storm event can be seen on the drifts in the night from 13 to 14 November (Figure 12A). On 13 November around 20:30 UT a pronounced episode of westward drift begins. Roughly until 14 November 10: 00 UT we observe dominantly horizontal westward drifts. The speed of the observed drifts is significantly higher than the horizontal speeds observed under quiet conditions (<100 m/s, according to Kouba and Koucka Knizova, 2012). The detected maximum speeds exceed even 350 m/s. The MIT position is also known to depend on the geographic longitude (e.g., Heilig et al., 2022). In the American sector MIT appears a few degrees higher than in the European or Asian sector. The Equation 5 of Deminov and Shubin (2018) estimates this difference to 0.98°, while according to the statistical results of Karpachev et al. (2019), this difference could easily be 2°–3°. 3.5 Digisonde drift measurements Digisonde drift measurements from Pruhonice station were analyzed manually in order to determine the true horizontal and 14 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 11 Evolution of the MIT minimum position (quasi dipole magnetic latitude) on the course of the 2015 event (A); magnetic longitude (B) and MLT (C) of the in-situ Swarm observations. Red and blue colors depict Swarm B and C observations, respectively. FIGURE 11 Evolution of the MIT minimum position (quasi dipole magnetic latitude) on the course of the 2015 event (A); magnetic longitude (B) and MLT (C) of the in-situ Swarm observations. Red and blue colors depict Swarm B and C observations, respectively. During day-time 18 March no significant manifestations of the storm are observed in the drift measurements. During day-time 18 March no significant manifestations of the storm are observed in the drift measurements. day-time is significantly lower than during the night-time. Therefore, information about the value of westward drift should be taken with a large margin. In most cases, the daytime skymap quality is not sufficient to determine the horizontal drift components with good reliability. The next episode is coming at night 18/19 March. Rapid changes occur in the vertical component between 23:00 UT and 06:00 UT, and the amplitude of the vertical drift is significantly greater than values under normal conditions. In the horizontal component, we observe a pronounced westward peak between about 18:00 UT and 06:00 UT. The maximum detected values significantly exceed 200 m/s. The second significant episode is observed during the night of 14/15 November This episode has similar characteristics, again between approx. 21:00 UT and 03:00 UT there is increased activity of the vertical component – larger values and rapid variations. In the horizontal components, a significant increase in the westward component can be seen again in the time interval between 23:00 – 08:00 UT. In this episode, the maximal measured values of the westward component exceed 250 m/s. It can be seen that the observed manifestations in the case of the second episode are smaller than in the case of the first. The last significant episode is observed the following night. On 20 March at around 00:00 – 04:00 UT the features described for the previous episode are repeated. It is interesting that this episode is significantly shorter than the previous one (roughly half) but the manifestations are more pronounced (on both the westward and the vertical components). Frontiers in Astronomy and Space Sciences 3.5 Digisonde drift measurements Drift activity affected by the storm is apparently observable also the following night (21 March, approx. 00:00 – 05:00 UT) on the westward component. The maximum detected velocity value is close to 200 m/s. In this case, however, the insufficient number of high-quality measurements does not allow proving a clear connection to the storm. In the drift measurements of 2015 March storm (Figure 12B), the first significant manifestations of the storm are observed on 17 March around 20:00 UT. The measurements show very quick variations of all observed parameters. The variations are significantly faster than the cadence of measurements and therefore the fluctuation period cannot be resolved. The value of the vertical drift velocity component exceeds the extreme values of +/- 50 m/s. frontiersin.org 4.1 Pre-storm phase In the pre-storm phase of the 2012 storm (11–12 November - Dstmin = 0 nT; Kpmax = 1,33; AEmax = 134 nT) foF2 and TEC show a slight positive deviation at all stations, while by the 2015 storm (16 March - Dstmin = 0 nT; Kpmax = 3,77; AEmax = 300 nT) no deviation from the reference value is detected. However, the O/N2 data for this storm shows a decrease in the subauroral region already in this phase (16 March). Earlier studies (e.g., Kane, 1973a; Araujo-Pradere and Fuller-Rowell, 2002; Kane, 2005; Burešová and Laštovička, 2007) found a pre-storm feature (positive effect) in foF2 24 h before the SSC, however other authors doubt its presence (Mikhailov and Perrone, 4 Discussion In this section, we attempt to define the cause-effect relationships in order to gain a more detailed understanding of the response of the terrestrial ionosphere-plasmasphere system to geomagnetic storms. Our main focus was on the main phase of the geomagnetic storm, when during the night extremely depleted Unfortunately, in this phase of the storm, the actual extreme ionospheric situation (the values of foF2 and h’F2) does not allow successful realization of drift measurement in most cases. Between 22:00 UT and 01:00 UT successful measurements are rare. 15 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 12 The manually corrected digisonde drift measurement of F-region for the 2012 November (A) and 2015 March (B) storm at Pruhonice station. FIGURE 12 The manually corrected digisonde drift measurement of F-region for the 2012 November (A) and 2015 March (B) storm at Pruhonice station. FIGURE 12 The manually corrected digisonde drift measurement of F-region for the 2012 November (A) and 2015 March (B) storm at Pruhonice station. 2009). Burešová and Laštovička (2007) examined 65 strong geomagnetic storms from the period of 1995–2005 and found that about 20%–25% of the storms had a significantly strong pre-storm effect. Similarly to the finding of Liu et al. (2008), we found a slight pre- storm enhancement in both TEC and foF2 in the 2012 storm case. Other authors suggested that this effect could be related to some different channel of penetration of the energy from the interplanetary environment and magnetosphere to the ionosphere (Danilov and Belik, 1991; 1992; Blagoveshchensky and Kalishin, 2009), but this needs verification. plasma was detected. Case studies are still important to shed light on deeper relationships that cannot be easily extracted from multi- event-based statistical investigations. Frontiers in Astronomy and Space Sciences 4.2 The main phase of ionospheric and geomagnetic storm The upward ion drift could also contribute to the daytime positive phase (Zhang S.R. et al., 2017a). The diurnal evolution of the positive phase was different, dual peak can be seen at all stations. The first peak was at noon with maximum half hour delay, the second was around 18 h (UT). The second peak was observed (station-hour): JR-17 h, PQ and SO-17:30, RO and AT-18 h (UT). This could connected to the propagation of storm-induced electric field from north to south (e.g., PPEFs). After Kumar and Kumar (2022), eastward/westward PPEFs are associated with the southward/ northward IMF Bz. The southward turnings of the IMF Bz component (induce eastward PPEF) can cause electron density increase during the daytime hours (Kumar and Kumar, 2022). There were two significant southward Bz episodes: around 8 and 14 (UT) during this time, which could support the above detailed assumption, but this needs verification. g g p y y y In the main phase of the geomagnetic storm in 2012 (14 November) during the daytime extremely depleted electron density was observed in foF2 at all stations. A similar decrease was observed also in TEC at all stations but with lower magnitude, except for AT and RO where it turned into negative only after 12 UT (see Figures 3, 5). Kane (2005) also found a deep decrease in electron density over the surrounding stations at midlatitude during the main phase of a similar storm (Dstmin = −589 nT). He also found that the magnitude of the Dst is not proportional to the magnitude of the negative effect in the ionosphere at middle and high-latitude. Pirog et al. (2006) strengthened it, where the analyzed geographic latitude range was similar to our case. The decrease of foF2 during the main phase of a geomagnetic storm is a known response of the ionosphere in winter, but usually during intense storms (Dstmin<= −100 nT) can reach lower latitudes (Danilov, 2013). According to the GUVI measurements around 12 LT a significant decrease of O/N2 ratio was observed over Europe by GUVI (marked by blue in Figure 8). 4.2 The main phase of ionospheric and geomagnetic storm Generally, the main cause of a negative ionospheric storm is related to the depletion in the O/N2 ratio, which is generated by the storm- time Joule-heating of the auroral thermospheric neutral gas leading to the formation of composition disturbance zone (with decreased O/N2 rate) this zone is transported by the enhanced thermospheric meridional winds (TADs) equatorward (Seaton, 1956; Prölss and von Zahn, 1974; Mikhailov et al., 1989; Mikhailov et al., 1995; Prölss et al., 1995; Bunonsanto, 1999; Astafyeva et al., 2015). The third peak in AE-index with 1,000 nT happened at ~01:00 UT. This energy input could generate also Joule-heating of the thermosphere and this strengthen the possibility of our hypothesis. This is strengthening our earlier assumption that the main reason for the negative phase is due to the decreased O/N2 ratio transported to lower latitudes (up to Sopron station) by meridional neutral winds (Berényi et al., 2018). 4.2 The main phase of ionospheric and geomagnetic storm On the day right after the SSC (on 13 November 2012 - Dstmin = −29nT; Kpmax = 4,77; AEmax = 674 nT and on 16 frontiersin.org frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. 10.3389/fspas.2023.1092850 17 March, 2015 - Dstmin = −234nT; Kpmax = 7,7; AEmax = 1570 nT) we observed a positive ionospheric storm phase at all station in both foF2 and TEC in both storms. This behavior is consistent with the average winter ionospheric response (in foF2 and TEC) to geomagnetic storms observed in previous studies (e.g., Matsuhita, 1959; Prölss, 1995; Buonsanto, 1999; Mendillo, 2006; Danilov, 2013). Astafyeva et al. (2015) examined the St. Patrick’s Day storm’s main phase (17–18 March) globally with multi-instrument measurements (e.g., Swarm VTEC, GUVI, GNSS TEC), and they found that the daytime positive phase during the day of 17 March at lower latitudes was due to increased O/N2 ratio. This finding was also investigated and supported by Nayak et al. (2016); Kalita et al. (2016). The AE- index mostly exceeded 500 nT and increased often up to 1,500 nT during the day, which indicates the possibility of intense Joule heating of the thermosphere, which can cause positive storm phase by two ways: 1) the uplift of the layer through enhanced meridional winds, and then lift the ionization to greater heights (Prölss and Zahn, 1974). 2) the downwelling of atomic [O] causing the increase of O/N2 ratio (see also in Introduction). Nava et al. (2016); Polekh et al. (2017) found also that the positive ionospheric effects at middle and low latitudes are related to different inputs of energy (as we see in AE-index; Figure 1B), and as they described the high latitude heating of the thermosphere travelled toward the equator with the velocity of 460 m/s during 17 March (along 120°E, see Polekh et al., 2017). Besides, Zhang S.R. et al. (2017a) found PPEF signatures on dayside over American sector, appearing as poleward/upward ion drift, and the observations showed meridional wind equatorward surges during also daytime hours. The upward ion drift could also contribute to the daytime positive phase (Zhang S.R. et al., 2017a). The diurnal evolution of the positive phase was different, dual peak can be seen at all stations. The first peak was at noon with maximum half hour delay, the second was around 18 h (UT). The second peak was observed (station-hour): JR-17 h, PQ and SO-17:30, RO and AT-18 h (UT). Frontiers in Astronomy and Space Sciences frontiersin.org 4.2 The main phase of ionospheric and geomagnetic storm This could connected to the propagation of storm-induced electric field from north to south (e.g., PPEFs). After Kumar and Kumar (2022), eastward/westward PPEFs are associated with the southward/ northward IMF Bz. The southward turnings of the IMF Bz component (induce eastward PPEF) can cause electron density increase during the daytime hours (Kumar and Kumar, 2022). There were two significant southward Bz episodes: around 8 and 14 (UT) during this time, which could support the above detailed assumption, but this needs verification. main phase of the geomagnetic storm, but by the 2012 storm, the geomagnetic storm’s main phase was delayed by 1 day. 17 March, 2015 - Dstmin = −234nT; Kpmax = 7,7; AEmax = 1570 nT) we observed a positive ionospheric storm phase at all station in both foF2 and TEC in both storms. This behavior is consistent with the average winter ionospheric response (in foF2 and TEC) to geomagnetic storms observed in previous studies (e.g., Matsuhita, 1959; Prölss, 1995; Buonsanto, 1999; Mendillo, 2006; Danilov, 2013). Astafyeva et al. (2015) examined the St. Patrick’s Day storm’s main phase (17–18 March) globally with multi-instrument measurements (e.g., Swarm VTEC, GUVI, GNSS TEC), and they found that the daytime positive phase during the day of 17 March at lower latitudes was due to increased O/N2 ratio. This finding was also investigated and supported by Nayak et al. (2016); Kalita et al. (2016). The AE- index mostly exceeded 500 nT and increased often up to 1,500 nT during the day, which indicates the possibility of intense Joule heating of the thermosphere, which can cause positive storm phase by two ways: 1) the uplift of the layer through enhanced meridional winds, and then lift the ionization to greater heights (Prölss and Zahn, 1974). 2) the downwelling of atomic [O] causing the increase of O/N2 ratio (see also in Introduction). Nava et al. (2016); Polekh et al. (2017) found also that the positive ionospheric effects at middle and low latitudes are related to different inputs of energy (as we see in AE-index; Figure 1B), and as they described the high latitude heating of the thermosphere travelled toward the equator with the velocity of 460 m/s during 17 March (along 120°E, see Polekh et al., 2017). Besides, Zhang S.R. et al. (2017a) found PPEF signatures on dayside over American sector, appearing as poleward/upward ion drift, and the observations showed meridional wind equatorward surges during also daytime hours. 4.3 Deep electron density depletion during the night of 14 November and 17 March Our findings are in good agreement with the results of previous articles like Nayak et al. (2016), who examined the equatorward motion of the MIT in the American sector (reached up to 40° N latitude) and Huang et al. (2018) also observed obvious westward disturbances in the zonal wind over central China (Asia, 38.7°N, 111.6°E). From 22:00 -02:00 UT strong westward neutral wind appeared, and at 03:00 UT a poleward wind surge of ~100 m/s appeared due to the poleward Coriolis force arising from the significant westward wind amplitudes (see Zhang S.R. et al., 2015; Tulasi et al., 2016; Huang et al., 2018). These were found to be driven by strong SAPS westward ion drift of >500 m/s peaking in the MIT and were accompanied by > 50 m/s upward ion drift in the E−and F-layers during SAPS periods at 22:43 UT at subauroral and midlatitudes (Zhang S.R. et al., 2015; Zhang S.R. et al., 2017a). But at lower latitudes poleward winds might not be driven by SAPS, most probably these wind changes are associated with travelling atmospheric disturbances (TADs) originated from the disturbance source region in the southern hemisphere (Huang et al., 2018). of MIT lasts until 04:00 UT. Our findings are in good agreement with the results of previous articles like Nayak et al. (2016), who examined the equatorward motion of the MIT in the American sector (reached up to 40° N latitude) and Huang et al. (2018) also observed obvious westward disturbances in the zonal wind over central China (Asia, 38.7°N, 111.6°E). From 22:00 -02:00 UT strong westward neutral wind appeared, and at 03:00 UT a poleward wind surge of ~100 m/s appeared due to the poleward Coriolis force arising from the significant westward wind amplitudes (see Zhang S.R. et al., 2015; Tulasi et al., 2016; Huang et al., 2018). These were found to be driven by strong SAPS westward ion drift of >500 m/s peaking in the MIT and were accompanied by > 50 m/s upward ion drift in the E−and F-layers during SAPS periods at 22:43 UT at subauroral and midlatitudes (Zhang S.R. et al., 2015; Zhang S.R. et al., 2017a). But at lower latitudes poleward winds might not be driven by SAPS, most probably these wind changes are associated with travelling atmospheric disturbances (TADs) originated from the disturbance source region in the southern hemisphere (Huang et al., 2018). 4.3 Deep electron density depletion during the night of 14 November and 17 March According to the observed rTEC values, as we move into the night at 22:00 UT (note that in reality most possibly this happened ~01:00 UT based on the aforementioned 3 h delay in rTEC), the zone with depleted electron density moves towards the equator, and even the positive phase region has been pushed back in the Athens- Rome line. By 00:00, the depleted zone moved even lower, at this time both Rome and Athens go into negative phase based on the foF2 data, indicating that the low mid-latitude region is also under the influence of MIT. The location of the MIT reached the 44.6° magnetic latitude (or maybe even below 42°) at 0.72 h MLT based on Swarm electron density and temperature measurements (Figures 10, 11) and the method described in Heilig et al. (2022). This sharp MIT minima was also accompanied by increased electron temperature with more than 5000 K (Figure 10), which was also observed by Liu et al. (2016), Zhang S.R. et al. (2015, Zhang S.R. et al. (2017a) during 16–19:30 UT on 17 March, which was presumably caused by strong frictional heating due to large plasma drifts. The equatorward motion of the minima of MIT is indicating that the plasmasphere was shrinking (see also Supplementary Figure S4), and the stations (JR, PQ, SO) went outside the plasmasphere where the ionosphere was not filled any more from the plasmasphere leading to extremely decreased electron density (detailed in Section 2). The severely depleted ionosphere was conjugated to the depleted outer magnetosphere (plasma trough) (see also Heilig et al., 2022). The F-layer drift measurement (at PQ) detected an intense ~500 m/s speed westward drift from appr. 20–02:00 UT (see Figure 12B) which is associated with the MIT formation mechanism. The effect After the observation of Zhang S.R. et al. (2017a) the SED plume over America (Millstone Hill) 20–24 UT can be linked to the positive phase over Southern Europe (AT and RO) from 18–22 UT: the westward winds could bring the plasma from Europe to North America leading to the detected SED plume over there (see Figure 4; Figure 7B) within a few hours, however, PPEF may also play some role in its formation (Liu et al., 2016). Investigation of Liu et al. (2016) and model results of Lu et al. (2020) confirm this possibility. 4.3 Deep electron density depletion during the night of 14 November and 17 March caused by plasmasphere-ionosphere coupling due to the enhanced downward ExB drift (leading to a maintaining effect by the plasmasphere), which in the meantime led to a strong erosion of the plasmasphere. For the 2012 storm the drift measurements from PQ during that night shows the presence of a strong westward motion of F-layer plasma (v ~ 350 m/s) indicating the presence of the ionospheric trough (MIT) (see also Heilig et al., 2022). Thus we can conclude based on foF2 and drift data that the equatorward motion of the MIT region (so the PP footprint) was the main cause of the detected sharp electron density decrease in foF2, GNSS TEC and rTEC data during the night of the geomagnetic storm main phase at SO, PQ and JR stations. Another contributor was possibly also the depleted O/N2 ratio, GUVI measurements show significant depletion above Europe during daytime, which could corotate with Earth (by westward winds) into the night sector, causing long-lasting negative phase (see Figure 8). During the 2015 St. Patrick’s Day storm both foF2 and TEC decreased at all stations during the night. In the rTEC maps, a very strong positive phase is visible at 18 UT on 17 March at lower latitudes, its boundary located along the line connecting the Black See and N-France (shown by yellow in Figure 7B). SO, PQ are in the green area, indicating that there is no deviation from the quiet level, as confirmed by the foF2 value (Figure 4). At the same time, JR is in negative phase, the foF2 shows significantly decreased electron density. It should be noted that the start time of the negative phase in TEC (and rTEC) was delayed by 3 h comparing it with the foF2 parameter at the same location. Based on the Swarm data (Figure 11) the MIT has already reached that latitude and it moved further to lower latitudes later, so it possibly caused the negative effect at JR. Previous studies indicated that the afternoon appearance of MIT may be linked to a plasmaspheric plume (see Heilig et al., 2022). 4.3 Deep electron density depletion during the night of 14 November and 17 March Interestingly, a strong positive-phase region appeared at high latitudes (over Scandinavia), which can represent the poleward boundary of the MIT. The data show positive deviation, partly because the electron density was quite low in that region during QDs, while during the subsequent storm a quite significant increase took place in association with the equatorward movement of the MIT. To speak about the lower latitudes, previous studies found the cumulative effect of enhanced meridional wind and changes in the electric fields, during night of the main phase at the 30–40°N zone. At low latitudes (like RO and AT) the detected variations in the ionosphere were governed by the superposition of disturbed electric fields (DDEFs), and the PPEF, which triggered the drop of foF2 in the evening on 17 March and the morning on 18 March (see Astafyeva et al., 2016; Jin et al., 2017; Polekh et al., 2017). For the St. Patrick‘s Day storm, the ionospheric effects of PPEF to low latitudes and the ionospheric disturbance dynamo (DDEF) were examined and discussed by several other papers: Le Huy and Amory-Mazauider, (2008); Lu et al. (2012); Abdu et al. (2013); Nava et al. (2016); Nayak et al. (2016); Ramsingh et al. (2015); Ram et al. (2016); Kalita et al. (2016). This could contribute to the nighttime electron density decrease at lower latitudes (AT, RO). Frontiers in Astronomy and Space Sciences 4.3 Deep electron density depletion during the night of 14 November and 17 March Significantly eroded ionospheric plasma was detected during the nighttime hours in the main phase of the geomagnetic storms (from 18:00 UT on 14 November 2012 and from 20:43 on 17 March 2015) with short duration fade-out of the layers in the ionograms at some stations (detailed in section 3.1). The foF2 and TEC data show a clear negative phase at JR, PQ and SO stations in the 2012 November storm. On the contrary, a strong positive phase mainly in foF2 but slightly also in TEC at AT and RO stations was detected during the whole night of 14 November The rTEC maps of storm 2012 (Figure 7A) show the variation during the nighttime hours (18–4: 00 UT) on 14 November These rTEC maps show clearly how the reduced electron density region moves from the higher latitudes equatorward, reaching its minimum latitude around midnight along the Athens-Rome line. Between 00:00 and 02:00 UT, the depleted electron density region is very nicely drawn out, and it can be seen that JR, PQ and SO stations are fully located within it. In the meantime, the low-midlatitude Athens-Rome region remains in the positive phase throughout the night. Based on our observations and the findings of previous studies like Yizengaw et al. (2005), we can assume, that this nighttime positive phase at AT and RO region, is For the 2012 November storm, the GUVI data show a slight increase in the O/N2 over Europe on 13 November around LT noon (Figure 8), which could contribute to the positive phase in foF2 (Figure 3). There was a second peak (after the SSC) in AE-index with ~600 nT, which here indicates also the Joule-heating of the thermosphere. The generated processes here also could contribute to the positive phase similarly to 2015 storm by two ways. Unfortunately, there is no digisonde drift episode, which could strengthen the presence of uplifting of the layer or the enhanced equatorward meridional winds. This assumption needs verification also with Horizontal Wind Model 2007 (HWM07, Huang et al., 2018). Kumar and Kumar (2022) found that the PPEFs were contributors in the electron density increase during the day of 13 November, 2012. Interestingly, this day is the main phase of the ionospheric storm, and normally it develops on the course of the Frontiers in Astronomy and Space Sciences 17 frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. of MIT lasts until 04:00 UT. frontiersin.org 4.4 Recovery phase During the recovery phase (on 15–17 November 2012 - Dstmin = −40;-27;-13nT; Kpmax = 1,33; 2,77; 3; AEmax = 119; 310; 328 nT and 18–20 March 2015 - Dstmin = −200;-99;-81 nT; Kpmax = 6; 5;4,77; AEmax = 1,043; 1,134; 611 nT) all station’s TEC and foF2 data showed a positive phase in the 2012 storm case, while 18 frontiersin.org frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 they showed a significant negative phase in the 2015 St. Patrick storm case. Yizengaw et al. (2005) examined a geomagnetic storm from 31 March, 2001 (Dstmin = −380 nT) and found also an extended, long-lasting negative phase during the recovery phase. The reason for the long-lasting negative phase during storm 2015 can be the great extension of composition disturbance zone to low latitudes as suggested by Danilov (2013 and references therein). These statements are also confirmed by our analysis of GUVI data. On 18 March, 2015, a quite deep depletion was observed in O/N2 ratio over Juliusruh (Figure 9) accompanied by foF2 decrease (Figure 4). The effect also re-occurs on 19 March, moreover the foF2 parameter data show a slight depletion also at PQ, SO and RO on 20 March. Nava et al. (2016), Huang et al. (2018) and Chernigovskaya et al. (2021) also found this depletion in electron density (18–20 March). They found that, during the recovery phase the important role in dynamics of the midlatitude ionosphere may belong to the wave-like thermospheric disturbances of molecular gas, propagating westward for several days causing electron density decrease along the trajectories of propagation. Besides, after the investigation of Kumar and Kumar (2019) another contributor to formation of the negative phase can be related to DDEFs. As for the 2012 storm, a negative phase changed to a positive one, which is believed to be caused by travelling atmospheric disturbances (TADs) (Danilov, 2013). These are generally brought by the same storm-time equatorward meridional wind discussed above, and have velocity of about 440 m/s (Mansilla, 2003). These processes uplift the layer to greater altitude, where the loss rate is lower, beside there is downwelling in atomic [O], resulting in an increase in electron density. 4.4 Recovery phase On 16–17 November a slightly increased O/N2 was observed by GUVI (marked with yellow in Figure 8), which could contribute to the daytime positive effect in foF2 around 10–15 UT at all investigated stations (Figure 3; see also article of Illes-Almar et al., 1987; Pirog et al., 2006). they showed a significant negative phase in the 2015 St. Patrick storm case. Yizengaw et al. (2005) examined a geomagnetic storm from 31 March, 2001 (Dstmin = −380 nT) and found also an extended, long-lasting negative phase during the recovery phase. The reason for the long-lasting negative phase during storm 2015 can be the great extension of composition disturbance zone to low latitudes as suggested by Danilov (2013 and references therein). These statements are also confirmed by our analysis of GUVI data. On 18 March, 2015, a quite deep depletion was observed in O/N2 ratio over Juliusruh (Figure 9) accompanied by foF2 decrease (Figure 4). The effect also re-occurs on 19 March, moreover the foF2 parameter data show a slight depletion also at PQ, SO and RO on 20 March. Nava et al. (2016), Huang et al. (2018) and Chernigovskaya et al. (2021) also found this depletion in electron density (18–20 March). They found that, during the recovery phase the important role in dynamics of the midlatitude ionosphere may belong to the wave-like thermospheric disturbances of molecular gas, propagating westward for several days causing electron density decrease along the trajectories of propagation. Besides, after the investigation of Kumar and Kumar (2019) another contributor to formation of the negative phase can be related to DDEFs. As for the 2012 storm, a negative phase changed to a positive one, which is believed to be caused by travelling atmospheric disturbances (TADs) (Danilov, 2013). These are generally brought by the same storm-time equatorward meridional wind discussed above, and have velocity of about 440 m/s (Mansilla, 2003). These processes uplift the layer to greater altitude, where the loss rate is lower, beside there is downwelling in atomic [O], resulting in an increase in electron density. On 16–17 November a slightly increased O/N2 was observed by GUVI (marked with yellow in Figure 8), which could contribute to the daytime positive effect in foF2 around 10–15 UT at all investigated stations (Figure 3; see also article of Illes-Almar et al., 1987; Pirog et al., 2006). 4.4 Recovery phase is the best for portraying, assessing and understanding the overall behavior of the near-Earth thermal plasma (ionosphere and plasmasphere) (Mendillo, 2006). It has to be mentioned here that TEC plots used for this study (on Figures 5, 6) are based on a TEC grid map (not from individual GNSS stations). The plotted values are taken at a grid point (closest to each ionosonde station) of these TEC maps, so it is a smoothed interpolated value that can also be a reason for that it is less sensitive than the foF2 measured at individual stations. In future studies, we found important to compare these results with some existing models/simulations in order to analyze and test the aforementioned processes: with NRL ionosphere/plasmasphere model SAMI3 (Huba et al., 2017), the thermosphere-ionosphere- electrodynamic general circulation model (TIEGCM, see the article of Lu et al., 2020), Horizontal Wind Model 2007 (HWM07, Huang et al., 2018), Global Self-consistent Model of the Thermosphere, Ionosphere, and Protonosphere (GSM TIP, Ratovsky et al., 2019) and also with the simulations of Fuller-Rowell et al. (1994) and Forbes and Roble et al. (1990). It is important mainly for the 2012 storm case where we do not have Swarm satellite measurement, and Fabry–Perot interferometer neutral wind observations to confirm our assumptions about the possible ionospheric effect drivers. Frontiers in Astronomy and Space Sciences 5 Conclusion During the evolution of two ICME-related geomagnetic storms, we investigated the extent of disturbances in the ionosphere. Our supreme aim was to find connections between geomagnetic storm induced processes in the ionosphere and to determine the evolution of the effects over Europe through a meridional chain of digisondes. We used various measurements (GNSS TEC, GUVI, Swarm, digisonde drift data) to find the possible cause of the detected features. The results of this study are summarized below: The 2015 storm was followed by a second SSC by an HSSWS happened on 21 March at night. During that day, a negative phase was detected at all stations in both TEC and foF2 but not any disturbance was observed in GUVI data. One day later (Dstmin = −56 nT; Kpmax = 6.33; AEmax = 1078 nT) a slight positive phase was present both in TEC and in foF2 at all stations. During the remaining days in the recovery phase, foF2 data do not show significant departure from the QD values, while in TEC a slightly positive effect was recorded. The possible reason for the late recovery phase behavior may be linked to the fact that the examined European stations were then inside the plasmasphere during the daytime. Thus the effect of the composition change could have been suppressed by other factors. Another possible reason could be that the HSSWS caused electron density increase during the first 2 days. Nava et al. (2016) found that the HSSWS event is added some energy into the magnetosphere, extending the lifetime of the recovery phase. [1] On 13 November 2012: the thermospheric composition changes and PPEFs contributed to the evolution of the daytime positive phase at all stations. Drift measurements from Pruhonice indicate the presence of the ionospheric trough (MIT) between 20:30–10:00 UT, reflected in the westward motion of the F-layer plasma with a velocity of 350 m/s, which was the main cause of the depletion in foF2 during the postmidnight sector at JR, PQ and SO. [2] On 17 March 2015, a daytime positive ionospheric storm developed at all stations except at JR. We can conclude that the negative phase at JR is a consequence of the equatorward motion of the MIT during the daytime. By GUVI data we assume that the composition change possibly had no effect. 5 Conclusion We assume that the possible cause of the positive phase was mainly connected to the Joule-heating of the auroral thermosphere, which lifted up the plasma through enhanced equatorward winds to altitudes where chemical loss rate of ions are slow. Besides, after previous studies, one of the cause was most possibly the PPEFs. The observed virtual height variation and the vertical drift data along with previous studies also confirms the uplifting scenario. [1] On 13 November 2012: the thermospheric composition changes and PPEFs contributed to the evolution of the daytime positive phase at all stations. Drift measurements from Pruhonice indicate the presence of the ionospheric trough (MIT) between 20:30–10:00 UT, reflected in the westward motion of the F-layer plasma with a velocity of 350 m/s, which was the main cause of the depletion in foF2 during the postmidnight sector at JR, PQ and SO. [2] On 17 March 2015, a daytime positive ionospheric storm developed at all stations except at JR. We can conclude that the negative phase at JR is a consequence of the equatorward motion of the MIT during the daytime. By GUVI data we assume that the composition change possibly had no effect. We assume that the possible cause of the positive phase was mainly connected to the Joule-heating of the auroral thermosphere, which lifted up the plasma through enhanced equatorward winds to altitudes where chemical loss rate of ions are slow. Besides, after previous studies, one of the cause was most possibly the PPEFs. The observed virtual height variation and the vertical drift data along with previous studies also confirms the uplifting scenario. We detail the observed diversities between foF2 and TEC during the daytime and nighttime of the two storms in the Supplementary section. The differences between the behavior of the foF2 parameter and TEC can be linked to the fact that TEC includes the plasma not only from the topside ionosphere (from above the height of foF2 parameter - hmF2) but also from plasmasphere. It is generally accepted that about 2/3 of the integrated TEC comes from the region above hmF2 (Mendillo, 2006). Due to this fact TEC 19 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 and AE-index are from OMNIWEB (http://omniweb.gsfc.nasa. gov/form/dx1.html) with a resolution of 1 h and 1 min, as well as from WDC Kyoto (https://wdc.kugi.kyoto-u.ac.jp/dst_final/ index.html). International Q-days (QD) list (https://wdc.kugi. Acknowledgments We thank the data centers (OMNIWEB and WDC for Geomagnetism, Kyoto) and the Széchenyi István Geophysical Observatory at Nagycenk, Hungary, Athens (AT138), Rome (RO041), Pruhonice (PQ052) and Juliusruh (JR055) station for supplying high quality ionosonde and digisonde data for the research. The GUVI instrument was designed and built by The Aerospace Corporation and The Johns Hopkins University. The Principal Investigator is Dr. Andrew B. Christensen and the Chief Scientist and co-PI is Dr. Larry J. Paxton. GNSS RINEX files for the GNSS-TEC processing are provided from many organizations listed by the webpage (http://stdb2.isee.nagoya-u.ac.jp/GPS/ GPS-TEC/gnss_provider_list.html). Also we are grateful for the reviewers for their valuable advices, which improved our article. The GUVI data used here are provided through support from the NASA MO&DA program. [11] In future studies, we found important to compare these results with some existing models/simulations in order to analyze and test the aforementioned processes. Funding This article is supported by the NKM55/2018 MTA mobility tender. Global GNSS-TEC data processing has been supported by JSPS KAKENHI Grant Number 16H06286. The contribution of VB was supported by OTKA, Hungarian Scientific Research Fund (grant no. PD 141967) of the National Research, Development and Innovation Office and by Bolyai Fellowship (GD, no. BO/ 00461/21). Furthermore, her work was supported by the GINOP- 2.3.2-15-2016-00003 project. [8] By the 2012 storm” recovery phase, the variation from the daytime positive to negative phase in foF2 and TEC is probably due to the change in the neutral composition. [9] The foF2 and TEC data are similar in their main patterns during the two investigated geomagnetic storms. [10] The combined analysis of Swarm, rTEC, drift and foF2 measurements give another method beside the existing ones of previous researchers to monitor the evolution and movement of the ionospheric features like MIT (the ionospheric footprint of the plasmapause), SED and SAPS. This can deepen our understanding of the processes during different geomagnetic storms, and could be use in the future in space weather prediction models. Frontiers in Astronomy and Space Sciences Author contributions KB was the main organizer and writer of this article, and responsible for the manual correction of all ionosonde data used for the analysis. VB contributed to the structure of the research, the basic concept, checked the workflow and improved the manuscript. BH performed the analysis of Swarm satellite data, added figures. JU selected and plotted the GNSS TEC data. DK manually corrected the digisonde drift measurements and wrote the description for it. ÁK supported the first author with ideas and fundings. All members contributed substantially to science discussion and manuscript development. [6] Swarm data together with the digisonde drift measurements support our hypothesis that the extreme decrease in foF2 and TEC at night of 17 March is related to the equatorward motion of the MIT along with the intense SAPS westward ion drift, as MIT went down to 44.6° (or even below 42°) geomagnetic latitude at 0.72 h MLT. This condition resulted in an even pronounced electron density drop in the ionosphere above SO and PQ. The decreased O/N2 ratio also contributed to the depletion effect. [7] After the observation of previous studies, the SED plume over America (at subauroral latitudes) between 20 and 24 UT on 17 March can be linked to the positive phase (SED) from 18–22 UT in foF2 and rTEC over Southern Europe (RO and AT): the westward winds could bring the plasma from Europe to North America leading to the detected SED plume over there. 5 Conclusion kyoto-u.ac.jp/qddays/), geomagnetic coordinate calculator (https://wdc.kugi.kyoto-u.ac.jp/igrf/gggm/index.html), GIRO scaled digisonde parameters (https://giro.uml.edu/didbase/ scaled.php) and for drift data (https://giro.uml.edu/driftbase/), GUVI data available at http://guvitimed.jhuapl.edu/data_fetch_ l3_on2_gif, Swarm satellite constellation, Langmuir Probe data are available at: (https://earth.esa.int/eogateway/missions/ swarm/product-data-handbook/preliminary-level-1b-plasma- dataset). [3] On 14 November 2012: in foF2 data the negative phase is extremely significant at all stations from 6 UT till the afternoon hours. Based on the GUVI data, this effect is related to a very strong decrease in O/N2 ratio during the day. [4] During the night of the 14 November, 2012 we found that the presence of the midlatitude ionospheric trough (MIT) was the primary reason for the formation of the negative phase at JR, PQ, SO stations. The rTEC maps and the drift measurements at PQ also confirms the presence of a nighttime trough. In addition, based on the GUVI data the daytime compositional disturbance zone seems to extend equatorward during the night, contributing to a more pronounced electron density drop. [5] The location of MIT minima (coincides with the ionospheric footprint of the PP) is likely reached the latitude of Sopron at night of 14 November according to the data. On the contrary AT and RO remained inside the plasmasphere, appearing as increase in foF2. 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Identification of ATP binding residues of a protein from its primary sequence
BMC bioinformatics
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BioMed Central BioMed Central Identification of ATP binding residues of a protein from its primary sequence Address: Institute of Microbial Technology, Chandigarh, India Email: Jagat S Chauhan - jagat@imtech.res.in; Nitish K Mishra - nitish@imtech.res.in; Gajendra PS Raghava* - raghava@imtech.res.in * Corresponding author Email: Jagat S Chauhan - jagat@imtech.res.in; Nitish K Mishra - nitish@imtech.res.in; Gajendra PS Raghava* - raghava@imtech.res.in * Corresponding author Email: Jagat S Chauhan - jagat@imtech.res.in; Nitish K Mishra - nitish@imtech.res.in; Gajendra PS Raghava* - raghava@imtech.res.in * Corresponding author Received: 6 August 2009 Accepted: 19 December 2009 This article is available from: http://www.biomedcentral.com/1471-2105/10/434 This article is available from: http://www.biomedcentral.com/1471-2105/10/434 © 2009 Chauhan 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. © 2009 Chauhan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: One of the major challenges in post-genomic era is to provide functional annotations for large number of proteins arising from genome sequencing projects. The function of many proteins depends on their interaction with small molecules or ligands. ATP is one such important ligand that plays critical role as a coenzyme in the functionality of many proteins. There is a need to develop method for identifying ATP interacting residues in a ATP binding proteins (ABPs), in order to understand mechanism of protein-ligands interaction. Results: We have compared the amino acid composition of ATP interacting and non-interacting regions of proteins and observed that certain residues are preferred for interaction with ATP. This study describes few models that have been developed for identifying ATP interacting residues in a protein. All these models were trained and tested on 168 non-redundant ABPs chains. First we have developed a Support Vector Machine (SVM) based model using primary sequence of proteins and obtained maximum MCC 0.33 with accuracy of 66.25%. Secondly, another SVM based model was developed using position specific scoring matrix (PSSM) generated by PSI-BLAST. The performance of this model was improved significantly (MCC 0.5) from the previous one, where only the primary sequence of the proteins were used. Conclusion: This study demonstrates that it is possible to predict 'ATP interacting residues' in a protein with moderate accuracy using its sequence. The evolutionary information is important for the identification of 'ATP interacting residues', as it provides more information compared to the primary sequence. This method will be useful for researchers studying ATP-binding proteins. Based on this study, a web server has been developed for predicting 'ATP interacting residues' in a protein http://www.imtech.res.in/raghava/atpint/. Page 1 of 9 (page number not for citation purposes) Open Ac Research article Identification of ATP binding residues of a protein from its primary sequence Jagat S Chauhan, Nitish K Mishra and Gajendra PS Raghava* Open Access Background binding sites is a micro-environment where ATP is cap- tured and hydrolyzed to ADP, releasing energy which is utilized by the protein to "do work" by changing the shape of the protein and/or making the enzyme catalyti- cally active. These proteins are powered by the hydrolysis of ATP and convert this chemical energy for mechanical work [1]. Many ATP Binding proteins are transmembrane Adenosine-5'-triphosphate (ATP) is an important mole- cule in cell biology as an energy molecule and coenzyme. This molecule interacts with large number of proteins dur- ing cellular activities and plays a crucial role in various biological reactions. ATP binding proteins (ABPs) have a binding site that allows ATP molecule to interact. This Page 1 of 9 (page number not for citation purposes) Page 1 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 proteins and responsible for transport of a wide variety of substrates (e.g. lipids, sterols) across extra and intracellu- lar membranes [2]. In summary, ATP binding proteins have important roles in membrane transport, muscle con- traction, cellular motility and regulation of various meta- bolic processes. out cross-validation (LOOCV) [4,20,21]. In this tech- nique one sequence is used for testing and remaining sequences for training, this process is repeated in such a way that each sequence is used once for testing. Though this is the best technique for evaluation, it is time consum- ing and computer intensive. Thus, we have used 5-fold cross-validation in this study where sequences were ran- domly divided into five sets. One set was used for testing and the remaining four sets were used for training. This process was repeated five times in such a way that each set was used once for testing [9,22]. The final performance was obtained by averaging the performance of all five sets. Thus it is important to identify ATP binding proteins and 'ATP interacting residues' in these proteins. The experi- mental identification of residues that interacts with ATP in a protein is costly and time consuming. Thus there is need to use alternate techniques such as computational tech- nique, which have been used successfully for predicting function of proteins [3-12]. In past, methods have been developed for the prediction of polynucleotide (DNA/ RNA) interacting residues [8,13,14]. Support Vector Machine (SVM) In most of our studies including this one, we have imple- mented SVM using SVM light [23], which is freely down- loadable package from http://svmlight.joachims.org/. SVM is a machine learning approach based on structural risk minimization principle of statistics learning theory [24]. The main reason of using this package frequently by us is that it allows implementing various kernels and parameters. Position Specific Scoring Matrix (PSSM) We extract 360 ATP binding protein chains from SuperSite encyclopedia [18]. After removing the redundant sequences using the program CD-HIT, a total of 267 non- redundant PDB chains were obtained where no two sequences have more than 40% identity. In the next step, we examined these proteins using software Ligand Protein Contact (LPC) [19] and remove those proteins, which are not ATP binding proteins according to LPC. Our final dataset have 168 non-redundant ATP binding protein chains, available at http://www.imtech.res.in/raghava/ atpint/atpdataset p f g ( ) In this work, PSSM profiles were generated using PSI- BLAST [25] where a protein sequence was searched against SWISS-PROT dataset using E-value cut-off of 0.001. This profile contains the probability of occurrence of each type of amino acid at each position along with insertion/dele- tion. Hence, PSSM is considered as a measure of residue conservation in a given location. This means that evolu- tionary information for each amino acid is encapsulated in a vector of 21 dimensions where the size of PSSM matrix of a protein with M residues is 21 × M, where M is the length of the target sequence, and each element repre- sents the frequency of occurrence of each of the 20 amino acids and one dummy amino acid "X" at one position in the alignment [16]. Pattern or window size We have generated overlapping patterns (segments) of different window sizes from 7 to 25 for every ATP binding protein sequences. If the central residue of the pattern was a 'ATP interacting residue', then we assigned the pattern as positive pattern (ATP interacting) otherwise it was assigned as negative pattern (non-ATP interacting). To generate the pattern corresponding to the terminal resi- dues in a protein sequence, we have added (L-1)/2 dummy residue "X" at both terminals of protein (where L is the length of pattern) [9]. As an example, for window size 17, we have added 8 "X" before N-terminal and 8 "X" after C-terminal, in order to create M patterns from sequence of length M [16,17]. Finally we have obtained a total of 3056 unique windows/patterns of length 17 out of 3082 ATP interacting residues. In this study, a systematic attempt has been made to develop a highly accurate and reliable method for predict- ing 'ATP interacting residues' in a protein. Initially, Sup- port Vector Machine (SVM) based models have been developed using proteins sequence. In the past, it has been shown that the evolutionary information provided more information [16,17] than protein sequence, thus we have also used evolutionary information in the form of PSSM profile for developing a prediction method. All the models developed in this study were evaluated using five- fold cross validation technique. Background Saito et al [15] devel- oped a general method for predicting nucleotide-binding sites in a protein, which successfully predicts 31% ATP binding sites (not ATP interacting residues). To the best of our knowledge, no prediction method has been devel- oped for detecting specifically the residues interacting with ATP from a protein sequence. Thus, there is a need to develop method for predicting 'ATP interacting residues' in a protein in order to understand protein-ATP interac- tion. Page 2 of 9 (page number not for citation purposes) Average accessible surface area The accessible surface area is the surface area of a protein that is accessible to another protein or ligand. The average accessible surface area scale values of each amino acid were obtained from Janin et al [32]. Accuracy y Percentage of correctly predicted ATP interacting and non- interacting residues Five-fold cross-validation Evaluation of a newly developed method is a major chal- lenge for researchers. One of the commonly used tech- niques for evaluating a model is jack-knife or leave-one- Page 2 of 9 (page number not for citation purposes) Page 2 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 http://www.biomedcentral.com/1471-2105/10/434 Hydrophobicity The hydrophobicity effect is often a major contributor of binding affinity between a protein and its ligand. All Hydrophobicity calculations were obtained from Fauchère and Pliska scale [26]. Sensitivity This parameter allows computation of the percentage of correctly predicted ATP interacting residues. Sensitivity TP TP FN = + Residue interface propensities The residue interface propensity is an important feature of protein binding sites that shows the propensity of each amino acid residues in the interface area. Residue interface propensities for each of the 20 amino acids were com- puted from Jones and Thornton [30]. MCC TP TN FP FN TP FP TP FN TN FP TN FN = − + + + + ( )( ) ( )( ) ( )( )( )( ) Where TP is the number of correctly predicted ATP inter- acting residues, TN is the number of correctly predicted non-interacting residues, FP is the number of non-inter- acting residues predicted as interacting residues and FN is the number of interacting residues wrongly predicted as non-interacting. Specificity p f y This parameter allows computation of the percentage of correctly predicted non-ATP interacting residues. Specificity TN TN FP = + Polarity Specificity TN TN FP = + Polarity is a separation of electric charge leading to a mol- ecule having an electric dipole. It results from the uneven partial charge distribution between various amino acids in a protein. We have used Grantham R polarity scale values [28]. Solvation potential Accuracy TP TN TP FP TN FN = + + + + The solvation potential is an important parameter of pro- teins that gives an idea about the preference of amino acid residues to be exposed to solvent or buried in the inter- face. For calculation of solvent potential for each amino acid, we have used Jones et al scale [29]. Matthews's Correlation Coefficient (MCC) Net charge The surface of a protein has a net charge that depends on the number and identities of the charged amino acids, and on pH. At a specific pH the positive and negative charges will balance and the net charge will be zero. Net charge of amino acid obtained from Klein et al [31]. All the parameters described above are threshold depend- ent parameters, thus performance of a model depend on threshold. In order to provide the comprehensive view of performance of a model, we have calculated these param- eters on different threshold (range from +1 to -1). Beta-Sheet Sensitivity TP TP FN = + Many nucleotide-binding proteins having a P-loop or phosphate-binding loop, is an ATP binding site motif. It is a glycine-rich loop preceded by a beta sheet. Thus the Beta-Sheet may be important feature in the ATP binding protein. It is obtained from Chou and Fasman scale [27]. Matthews's Correlation Coefficient (MCC) It is the statistical parameter to assess the quality of predic- tion and to take care of the unbalancing in data. An MCC equal to 1 is regarded as a perfect prediction, whereas for a completely random prediction this value becomes 0. Evaluation Parameter For evaluation of the performance of methods, we have used standard parameter that is routinely used in this type field. Following is a brief description of the threshold dependent parameters which was used for evaluation. In this study we have used following seven important structural feature as SVM input feature - Area under the ROC Curve (AUC) All the measures described above have a common draw- back that their performance depends on threshold selected. A known threshold independent parameter is Receiver Operating Curve (ROC). It is a plot between true positive proportion (TP/TP+FN) and false positive pro- All the above features, parameter, scale values were taken from http://www.genome.jp/aaindex[33] and used as input features in SVM. Page 3 of 9 (page number not for citation purposes) Page 3 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 portion (FP/FP+TN). We have used SPSS package to plot ROC and calculate AUC. against database of ATP-binding proteins. In order to eval- uate the performance of BLAST on dataset used in this study, we have searched each ATP binding protein chain against remaining ATP binding protein chains. It was observed that only 71 ATP interacting protein chains showed similarity (BLAST hit) with other ATP binding protein chains. Thus, BLAST cannot be used to predict any ATP interacting residues in 97 ATP binding protein chains out of 168 chains in our dataset. In order to evaluate per- formance of BLAST on those protein chains which, showed similarity, we randomly picked 10 proteins, which have similarity with other ATP-binding protein chains. Even on these proteins, the performance of BLAST was very poor, where the sensitivity was 44% and the probability of correct prediction was 43.37%. This result suggests that BLAST is not suitable for predicting ATP interacting residues in a protein. SVM Modules using single sequence It has been shown in previous studies on nucleotide inter- acting proteins that they perform best for 17-window size (pattern length) [16,9]. Thus we have used pattern length 17 for developing our prediction model. All possible over- lapping peptides of 17 amino acids were generated from ATP binding proteins/chains, a peptide/pattern is assigned ATP interacting or positive if the residue at its center is ATP interacting otherwise it was assigned as negative. After clas- sifying them as positive and negative patterns, they were converted into binary patterns. The peptide of length N was represented by a vector of dimensions N × 21, where each residue is represented by a vector of dimension 21 (e.g. Ala by 1,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0; Cys by Composition analysis We have analyzed the composition of interacting and non-interacting residues by computing the amino acid composition of 17 amino acid pattern where the central residue was interacting or non-interacting and observed that the occurrence of Gly and positively charged amino acids Arg, Lys and His were significantly different in ATP interacting residue and non-interacting residues (Figure 1) with respective p-value 0.00186, 0.00862, 0.00254 and 0.07941. It can be inferred that the Gly and positively charged amino acids are important for the interaction with ATP. Beside above residues, significant composi- tional difference was observed for following residues Leu, Pro, Ala and Val with p-value 0.00511, 0.00011, 0.00049 and 0.02253 respectively (Table S1; see in Additional file 1). It shows that non-polar and hydrophobic amino acid residues such as Val, Leu are important in protein-ATP binding. We have used two-tailed unpaired T-test to check the significant in difference of amino acid compositions in binding and non-binding residues. Page 4 of 9 (page number not for citation purposes) SVM Module based on physico-chemical parameters SVM Module based on physico-chemical parameters In this study we have also developed SVM module using various physico-chemical features, which, are important for protein structure and function. Seven physico-chemi- cal parameters have been used for this (see methods sec- tion). We have normalized these parameters [36] before using them for developing SVM classifier. Performance of SVM Module based on physico-chemical parameters is shown in Table 4. As shown in Table 4, performance (maximum MCC 0.26) was lower than SVM module based on sequence. Prediction using BLAST One of the methods which is routinely used for predicting function of a new protein sequence is BLAST. It is a simi- larity based method and identifies segments/regions in the query sequences which are similar to the target sequence. This method can be used to predict ATP inter- acting residues in a protein by searching a query protein Percentage composition of ATP interacting and non-interacting residues Figure 1 Percentage composition of ATP interacting and non-interacting residues. Percentage composition of ATP interacting and non-interacting residues Figure 1 Percentage composition of ATP interacting and non-interacting residues. Percentage composition of ATP interacting and non-interacting residues Figure 1 Percentage composition of ATP interacting and non-interacting residues. Page 4 of 9 (page number not for citation purposes) Page 4 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 http://www.biomedcentral.com/1471-2105/10/434 We have achieved 66.25% accuracy with minimum differ- ence between sensitivity and specificity and MCC 0.33 by 17 window patterns (Table 1) at threshold 0.0. Normally we select a threshold where sensitivity and specificity are nearly equal, in order to make the balance between sensi- tivity and specificity. The performance of SVM model for window size 17 using single sequence is shown in Figure 2. We have achieved AUC 0.725 which was significantly better than random (AUC 0.5). 0,1,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0); contains 20 amino acids and one dummy amino acid "X". Our SVM module predict a score for each residue in protein (in range of -1.0 to 1.0), we define a threshold to discriminate ATP interacting and non-interacting residues. The performance of SVM module developed using a single sequence for win- dow size 17 is shown in Table 1. We have also tried various window sizes from 7 to 25 residues and observed that 17 window size patterns gave better performance (Table 2). Table 1: The performance of SVM model (learning parameter: g: 0.1 c: 2 j: 3) using amino acid sequence (The SVM parameter g (in RBF kernel), c: parameter for trade-off between training error & margin, j: cost-factor) Table 1: The performance of SVM model (learning parameter: g: 0.1 c: 2 j: 3) using amino acid sequence (The SVM parameter g (in RBF kernel), c: parameter for trade-off between training error & margin, j: cost-factor) SVM Modules using evolutionary information Table 1: The performance of SVM model (learning parameter: g: 0.1 c: 2 j: 3) using amino acid sequence (The SVM parameter g (in RBF kernel), c: parameter for trade-off between training error & margin, j: cost-factor) Thres Sen Spec Accuracy MCC -1 100 1.73 50.87 0.09 -0.9 99.87 2.88 51.37 0.11 -0.8 99.67 4.39 52.03 0.13 -0.7 99.25 6.51 52.88 0.15 -0.6 98.36 10.31 54.34 0.18 -0.5 96.89 15.78 56.33 0.22 -0.4 93.75 23.54 58.64 0.24 -0.3 88.94 32.9 60.92 0.26 -0.2 83.4 43.31 63.36 0.29 0 65.53 66.97 66.25 0.33 0.1 54.6 76.99 65.79 0.32 0.2 43.11 84.98 64.04 0.31 0.3 33.94 91.16 62.55 0.31 0.4 25.7 94.7 60.2 0.28 0.5 18.07 97.15 57.61 0.25 0.6 12.64 98.53 55.58 0.22 0.7 8.71 99.18 53.94 0.19 0.8 6.22 99.54 52.88 0.16 0.9 3.93 99.77 51.85 0.13 1 1.87 99.84 50.85 0.08 (Bold values indicate the point where sensitivity and specificity is roughly equal with maximum MCC.) In the past, it has been shown in several studies that evo- lutionary information gives more information about a protein than single sequence [34,35]. In this study the evolutionary information in the form of PSSM profile has been used for predicting ATP interacting protein residues. The PSSM profile for each sequence was generated using PSI-BLAST where sequence was search against of SWISS- PROT. Each element of PSSM matrix was normalized before using it as an input feature of SVM module. The performance of SVM module that was developed using PSSM, at different threshold, is shown in Table 3. We have achieved maximum MCC 0.51, with accuracy 75.25% at threshold -0.1. These results indicated that the evolution- ary information was very important for predicting ATP interacting residues as performance increase significantly from MCC 0.33 to 0.51. The performance of SVM model based on evolutionary information is shown by ROC plot in Figure 2 which indicates an improvement from AUC 0.725 to 0.823 and thereby clearly suggesting that SVM model based on PSSM profile perform better than module based on single sequence. In the past, it has been shown in several studies that evo- lutionary information gives more information about a protein than single sequence [34,35]. In this study the evolutionary information in the form of PSSM profile has been used for predicting ATP interacting protein residues. (Bold values indicate the point where sensitivity and specificity is roughly equal with maximum MCC.) Discussion The ATP interacting proteins play a significant role in sig- naling pathways, in which ATP is used as a substrate by kinases that phosphorylate proteins. The identification of ATP interacting residues is difficult using experimental techniques. There is a need for developing computational techniques for identifying ATP interacting residues in a protein from its protein sequence. Saito et al [15] devel- oped a general method for predicting binding site using empirical scores system. Though this method allows Page 5 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 Table 2: The performance of SVM model using binary pattern of different window size patterns. Window size Threshold Sensitivity Specificity Accuracy MCC parameters 7 0 60.99 64.47 62.73 0.25 g:0.1 c:1 j:1 9 0 61.25 67.41 64.33 0.29 g:0.1 c:1 j:1 11 0 63.2 64.01 63.61 0.27 g:0.1 c:3 j:3 13 0 63.4 64.81 64.11 0.28 g:0.1 c:2 j:1 15 0 61.62 67.11 64.37 0.29 g:0.1 c:1 j:1 17 0 65.53 66.97 66.25 0.33 g:0.1 c:2 j:3 19 0 61.98 69.44 65.71 0.32 g:0.1 c:1 j:1 21 0 60.86 69.96 65.41 0.31 g:0.1 c:1 j:1 23 0 63.65 68.52 66.08 0.32 g:0.1 c:2 j:2 25 0 63.32 70.26 66.79 0.34 g:0.1 c:2 j:1 (Bold values indicate the values where accuracy highest and sensitivity and specificity are roughly equal) Table 2: The performance of SVM model using binary pattern of different window size patterns. (Bold values indicate the values where accuracy highest and sensitivity and specificity are roughly equal) section we obtained poor performance both in terms of sensitivity and probability of correct prediction. Thus the routinely used similarity search technique like BLAST is not suitable for this problem. In the next step, we examine motif-based techniques for predicting ATP interacting res- idues. We search motifs using FingerPRINTScan [38] in 168 ATP binding protein chains used in this study and got motifs only in 54 proteins. No motif was found in the remaining 114 proteins. These motifs only cover around 11% ATP interacting residues (Table S2; see in Additional file) and no common motif was found in ATP binding protein (Table S3; see in Additional file). These results shows that motifs based method cannot be used for iden- tifying of ATP interacting residues. detection of ATP binding sites on a protein with low accu- racy but provides no information about ATP interacting residues. Page 6 of 9 (page number not for citation purposes) Discussion There are methods, which allow identifying ATP interacting residues in a protein if its structure is known [19,37]. These methods are basically assignment method, which assign ATP interacting residues in a PDB file. In this study an attempt has been made to predict ATP interacting residue in a protein with high accuracy. One of the obvi- ous question arise can we used existing techniques for pre- dicting ATP interacting residues. First we used BLAST for predicting ATP interacting residues. As shown in result ROC plot shows performance of SVM modules developed using amino acid sequence and PSSM profile Figure 2 ROC plot shows performance of SVM modules devel- oped using amino acid sequence and PSSM profile. This study is a systematic attempt to understand and pre- dict ATP interacting residues in a protein First we analyzed ATP interacting residues and its neighbors, and found that there is a significant difference in interacting and non- interacting residues. This means ATP interacting residues can be predicted using any machine leaning techniques. It has been shown in previous studies that SVM perform bet- ter than other artificial intelligence technique particularly on small dataset. Thus SVM based model has been devel- oped for predicting ATP interacting residues in a protein from its primary structure and achieved reasonable accu- racy. As PSSM based evolutionary information provide better information [9], hence we also made an attempt to develop method using evolutionary information for pre- dicting ATP interacting residues. The performance of SVM module increases significantly when evolutionary infor- ROC plot shows performance of SVM modules developed using amino acid sequence and PSSM profile Figure 2 ROC plot shows performance of SVM modules devel- oped using amino acid sequence and PSSM profile. ROC plot shows performance of SVM modules developed using amino acid sequence and PSSM profile Figure 2 ROC plot shows performance of SVM modules devel- oped using amino acid sequence and PSSM profile. Page 6 of 9 (page number not for citation purposes) Page 6 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 mation is in place of single sequence. This demonstrates that evolutionary information is important for predicting ATP interacting residues. In this study we used window size 17; the question arises why we have used 17. Discussion Though window size 17 is frequently used in prediction of second- ary structure of interacting residues, it does not mean that window size 17 is applicable to each problem. One should try different window size in order to find out opti- mize window size for a given problem. We try various window sizes from 7 to 25 residues for predicting ATP for window size 17. Although accuracy of binary pattern of 25 window size is better than 17 but difference in sen- sitivity and specificity is much higher. This means that window size 17 is most suitable for predicting ATP inter- acting residues. This is first study of this kind so it is diffi- cult to compare its performance with existing methods. We hope this study will be useful for researchers working in this area. There is a high probability that other researcher will work on this problem and will develop bet- ter method. Conclusion In this study we have develop method, for the first time, Table 3: The Performance of SVM model (Learning Parameter: g: 0.01 c: 4 j: 1) Using PSI-BLAST Profile Threshold Sensitivity Specificity Accuracy MCC -1 98.52 15.47 57 0.25 -0.9 97.93 20.43 59.18 0.29 -0.8 96.55 25.2 60.87 0.31 -0.7 95.07 30.68 62.88 0.34 -0.6 93.27 36.96 65.11 0.37 -0.5 90.87 43.59 67.23 0.39 -0.4 88.44 50.1 69.27 0.42 -0.3 85.48 56.34 70.91 0.44 -0.2 82 63.34 72.67 0.46 0 74.44 75.79 75.11 0.50 0.1 70.01 80.39 75.2 0.51 0.2 65.41 84.4 74.9 0.51 0.3 60.32 87.78 74.05 0.5 0.4 55.85 90.6 73.23 0.5 0.5 51.22 92.97 72.09 0.49 0.6 46.39 94.58 70.48 0.47 0.7 40.21 96.12 68.17 0.44 0.8 34.63 97.44 66.03 0.41 0.9 28.65 98.03 63.34 0.37 1 21.78 98.92 60.35 0.33 (Italic-bold values indicate the point where sensitivity and specificity is roughly equal and Bold values indicate point where maximum Accuracy and MCC.) Table 4: The Performance of SVM model (Learning Parameter: g: 0.001 c: 4 j: 1) Using seven physiochemical properties. Discussion (Italic-bold values indicate the point where sensitivity and specificity is roughly equal and Bold values indicate point where maximum Accuracy and MCC.) mation is in place of single sequence. This demonstrates that evolutionary information is important for predicting ATP interacting residues. In this study we used window size 17; the question arises why we have used 17. Though window size 17 is frequently used in prediction of second- ary structure of interacting residues, it does not mean that window size 17 is applicable to each problem. One should try different window size in order to find out opti- mize window size for a given problem. We try various window sizes from 7 to 25 residues for predicting ATP interacting residues and achieved maximum performance mation is in place of single sequence. This demonstrates that evolutionary information is important for predicting ATP interacting residues. In this study we used window size 17; the question arises why we have used 17. Though window size 17 is frequently used in prediction of second- ary structure of interacting residues, it does not mean that window size 17 is applicable to each problem. One should try different window size in order to find out opti- mize window size for a given problem. We try various window sizes from 7 to 25 residues for predicting ATP interacting residues and achieved maximum performance Discussion Threshold Sensitivity Specificity Accuracy MCC -0.9 93.68 18.71 56.19 0.19 -0.8 92.35 22.05 57.2 0.2 -0.7 90 26.85 58.43 0.22 -0.6 87.35 31.23 59.29 0.22 -0.5 84.3 36.72 60.51 0.24 -0.4 80.43 41.79 61.11 0.24 -0.3 76.32 47.35 61.84 0.25 -0.2 72.45 52.68 62.57 0.26 -0.1 68.25 57.78 63.01 0.26 0 63.18 62.95 63.06 0.26 0.1 57.95 68.08 63.01 0.26 0.2 52.55 72.75 62.65 0.26 0.3 47.15 77.05 62.1 0.25 0.4 41.56 80.86 61.21 0.24 0.5 36.56 84.77 60.66 0.24 0.6 31.79 87.42 59.6 0.23 0.7 26.99 89.57 58.28 0.21 0.8 23.15 92.55 57.85 0.22 0.9 19.44 94.34 56.89 0.21 (Bold values indicate point where maximum Accuracy and MCC.) Table 4: The Performance of SVM model (Learning Parameter: g: 0.001 c: 4 j: 1) Using seven physiochemical properties. Discussion Threshold Sensitivity Specificity Accuracy MCC -0.9 93.68 18.71 56.19 0.19 -0.8 92.35 22.05 57.2 0.2 -0.7 90 26.85 58.43 0.22 -0.6 87.35 31.23 59.29 0.22 -0.5 84.3 36.72 60.51 0.24 -0.4 80.43 41.79 61.11 0.24 -0.3 76.32 47.35 61.84 0.25 -0.2 72.45 52.68 62.57 0.26 -0.1 68.25 57.78 63.01 0.26 0 63.18 62.95 63.06 0.26 0.1 57.95 68.08 63.01 0.26 0.2 52.55 72.75 62.65 0.26 0.3 47.15 77.05 62.1 0.25 0.4 41.56 80.86 61.21 0.24 0.5 36.56 84.77 60.66 0.24 0.6 31.79 87.42 59.6 0.23 0.7 26.99 89.57 58.28 0.21 0.8 23.15 92.55 57.85 0.22 0.9 19.44 94.34 56.89 0.21 (Bold values indicate point where maximum Accuracy and MCC.) Table 3: The Performance of SVM model (Learning Parameter: g: 0.01 c: 4 j: 1) Using PSI-BLAST Profile Threshold Sensitivity Specificity Accuracy MCC -1 98.52 15.47 57 0.25 -0.9 97.93 20.43 59.18 0.29 -0.8 96.55 25.2 60.87 0.31 -0.7 95.07 30.68 62.88 0.34 -0.6 93.27 36.96 65.11 0.37 -0.5 90.87 43.59 67.23 0.39 -0.4 88.44 50.1 69.27 0.42 -0.3 85.48 56.34 70.91 0.44 -0.2 82 63.34 72.67 0.46 0 74.44 75.79 75.11 0.50 0.1 70.01 80.39 75.2 0.51 0.2 65.41 84.4 74.9 0.51 0.3 60.32 87.78 74.05 0.5 0.4 55.85 90.6 73.23 0.5 0.5 51.22 92.97 72.09 0.49 0.6 46.39 94.58 70.48 0.47 0.7 40.21 96.12 68.17 0.44 0.8 34.63 97.44 66.03 0.41 0.9 28.65 98.03 63.34 0.37 1 21.78 98.92 60.35 0.33 (Italic-bold values indicate the point where sensitivity and specificity is roughly equal and Bold values indicate point where maximum Accuracy and MCC.) Table 4: The Performance of SVM model (Learning Parameter: g: 0.001 c: 4 j: 1) Using seven physiochemical properties. Table 3: The Performance of SVM model (Learning Parameter: g: 0.01 c: 4 j: 1) Using PSI-BLAST Profile (Bold values indicate point where maximum Accuracy and MCC.) for window size 17. Although accuracy of binary pattern of 25 window size is better than 17 but difference in sen- sitivity and specificity is much higher. This means that window size 17 is most suitable for predicting ATP inter- acting residues. This is first study of this kind so it is diffi- cult to compare its performance with existing methods. We hope this study will be useful for researchers working in this area. There is a high probability that other researcher will work on this problem and will develop bet- ter method. Page 7 of 9 (page number not for citation purposes) Additional file 1 Additional file 1 Table S1. P-value for compositional difference in ATP interacting residue and non-interacting residue. Table S2, S3. Motifs based analysis. Fprint- Scan AccNumber list and its frequency. Probability of percentage coverage of FprintScan in ATP interacting residue. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-10-434-S1.DOC] 17. Kaur H, Raghava GPS: A neural-network based method for pre- diction of gamma-turns in proteins from multiple sequence alignment. Protein Sci 2003, 12:923-929. Table S1. P-value for compositional difference in ATP interacting residue and non-interacting residue. Table S2, S3. Motifs based analysis. Fprint- Scan AccNumber list and its frequency. Probability of percentage coverage of FprintScan in ATP interacting residue. Click here for file g 18. Bauer RA, Günther S, Jansen D, Heeger C, Thaben P, Preissner R: SuperSite: dictionary of metabolite and drug binding sites in proteins. Nucl Acids Res 2009, 37:D195-200. p 19. Sobolev V, Sorokine A, Prilusky J, Abola EE, Edelman M: Automated analysis of interatomic contacts in proteins. Bioinformatics 1999, 15:327-332. 20. Chen C, Chen L, Zou X, Cai P: Prediction of protein secondary structure content by using the concept of Chou's pseudo amino acid composition and support vector machine. Protein & Peptide Letters 2009, 16(1):27-31. Authors' contributions JSC created dataset and developed the SVM models and NKM re-checked these models and datasets. JSC, NKM created the backend web server and the front end user interface. GPSR conceived the project, coordinated it and refined the final manuscript drafted by JSC and NKM. All authors have read and approved this manuscript. 13. Jeong E, Chung IF, Miyano S: A network method for identifica- tion of RNA-interacting residues in protein. Genome Inform 2004, 15:105-116. 14. Jeong E, Miyano S: A Weighted profile based method for pro- tein-RNA interacting residue prediction. In Lecture notes in com- puter science Volume 39. Edited by: Corrado P, Luca C, Stephen E. Berlin/Heidelberg: Springer; 2006:123-139. g p g 15. Saito M, Go M, Shira T: An empirical approach for detecting nucleotide-binding sites on proteins. Protein Engineering, Design & Selection 2006, 19:67-75. Additional material 16. Kaur H, Raghava GPS: Prediction of β-turns in proteins from multiple alignments using neural network. Protein Sci 2003, 12:627-634. References 1. Bustamante C, Chemla YR, Forde NR, Izhaky D: Mechanical proc- esses in biochemistry. Annual Review of Biochemistry 2004, 73:705-748. 2. Hirokawa N, Tekamura R: Biochemical and molecular charac- terization of diseases linked to motor proteins. Trends in Bio- chemical Sciences 2003, 28:558-565. 3. Garg A, Bhasin M, Raghava GPS: Support vector machine-based method for subcellular localization of human proteins using amino acid compositions, their order, and similarity search. J Biol Chem 2005, 280:14427-14432. J , 4. Chou KC, Zhang CT: Review: Prediction of protein structural classes. Critical Reviews in Biochemistry and Molecular Biology 1995, 30:275-349. 5. Chou KC, Shen HB: Cell-PLoc: A package of web-servers for predicting subcellular localization of proteins in various organisms. Nature Protocols 2008, 3:153-162. g 6. Chou KC, Shen HB: Review: Recent progresses in protein sub- cellular location prediction. Analytical Biochemistry 2007, 370:1-16. Web Server 8. Ahmad S, Gromiha M, Sarai A: Analysis and prediction of DNA- binding proteins and their binding residues based on compo- sition, sequence and structural information. Bioinformatics 2004, 20:477-486. We developed a web server "ATPint" using CGI-Perl 5.8.4 script to predict of ATP interacting proteins, which is avail- able at http://www.imtech.res.in/raghava/atpint. This server allows users to predict ATP interacting proteins using PSSM based SVM models. User can select any threshold within -1 to +1 by default it is 0.2. The predic- tion result presented in graphical form where the pre- dicted ATP interacting and non-interacting are displayed in different color. 9. Kumar M, Gromiha M, Raghava GPS: Prediction of RNA binding sites in a protein using SVM and PSSM profile. Proteins: Struc- ture, Function, and Bioinformatics 2007, 71:189-194. 10. Shen HB, Chou KC: EzyPred: A top-down approach for pre- dicting enzyme functional classes and subclasses. Biochem Bio- phys Res Comm 2007, 364:53-59. p y 11. Xiao X, Wang P, Chou KC: GPCR-CA: A cellular automaton image approach for predicting G-protein-coupled receptor functional classes. Journal of Computational Chemistry 2009, 30:1414-1423. 12. Chou KC: Review: Structural bioinformatics and its impact to biomedical science. Current Medicinal Chemistry 2004, 11:2105-2134. Competing interests 7. Chou KC, Shen HB: MemType-2L: A Web server for predicting membrane proteins and their types by incorporating evolu- tion information through Pse-PSSM. Biochem Biophys Res Comm 2007, 360:339-345. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Industrial Research (CSIR) and the Department of Biotechnology (DBT), India for supporting this study. Industrial Research (CSIR) and the Department of Biotechnology (DBT), India for supporting this study. its protein sequence using SVM based model. It was observed that the evolutionary information (PSSM) based SVM modules perform better than the single sequence based modules. Though it has been shown in number of previous studies that the evolutionary information is important for predicting the structural component of a protein, first time we have demonstrated that the evolu- tionary information is also important for predicting ATP interacting residues. One of the major features of this study is that we are providing web service for predicting ATP interacting residues in a protein. Our web-server; ATPint allows users to identify ATP binding residue using the best model trained on our data set. This server will help the experimental biologist to predict ATP interacting residue from its primary sequence and avoid the number of essential experiments. Conclusion In this study we have develop method, for the first time, for predicting ATP interacting residues in a protein from Page 7 of 9 (page number not for citation purposes) Page 7 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 Page 8 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 (page number not for citation purposes) BMC Bioinformatics 2009, 10:434 BMC Bioinformatics 2009, 10:434 http://www.biomedcentral.com/1471-2105/10/434 http://www.biomedcentral.com/1471-2105/10/434 http://www.biomedcentral.com/1471-2105/10/434 22. Mishra N, Kumar M, Raghava GPS: Support vector machine based method for predicting Glutathione S-transferases pro- teins. 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Jones DT, Taylor WR, Thornton JM: A new approach to protein fold recognition. Nature 1992, 358:86-89. g 30. Jones S, Thornton JM: Principles of protein-protein interac- tions. Proc Natl Acad Sci 1996, 93:13-20. 31. Klein P, Kanehisa M, DeLisi C: Prediction of protein function from sequence properties: Discriminant analysis of a data base. J Biochim Biophys Acta 1984, 787:221-226. J p y 32. Janin J, Wodak S: Conformation of amino acid side-chains in proteins. J Mol Biol 1978, 125(3):357-86. p J ( ) 33. Du P, Li Y: Prediction of protein submitochondria locations by hybridizing pseudo-amino acid composition with various physicochemical features of segmented sequence. BMC Bioin- formatics 2006, 7:518. 34. Kaur H, Raghava GPS: Prediction of beta turns in proteins from multiple alignment using neural network. Protein Sci 2003, 12:627-634. 35. Kuznetsov IB, Gou Z, Li R, Hwang S: Using evolutionary and structural information to predict DNA-binding sites on DNA-binding proteins. Proteins 2006, 64:19-27. g p 36. Jones S, Thornton JM: Analysis of Protein-Protein Interaction Sites using Surface Patches. J Mol Biol 1997, 272:121-132. 37. Ting G, Yanxin S, Zhirag S: A noval statistical ligand-binding site predictor: application to ATP-binding site. Protein Engineering, Design & Selection 2005, 18:65-70. g 38. Zdobnov EM, Apweiler R: InterProScan - an integration plat- form for the signature-recognition methods in InterPro.". Bioinformatics 2001, 17(9):847-848. Acknowledgements p ( ) 21. Ding H, Luo L, Lin H: Prediction of cell wall lytic enzymes using Chou's amphiphilic pseudo amino acid composition. Protein & Peptide Letters 2009, 16:351-355. We are grateful to Dr Alok Kumar Mondal for proof reading of manuscript. The authors are thankful to Hifzur Rahman Ansari for his help in developing web-server. We are grateful for funding agencies Council of Scientific and Page 8 of 9 (page number not for citation purposes) Page 8 of 9 (page number not for citation purposes) Page 8 of 9 (page number not for citation purposes) BMC Bioinformatics 2009, 10:434 Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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https://openalex.org/W2808942715
https://link.springer.com/content/pdf/10.1007%2Fs00384-018-3108-5.pdf
English
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Meta-analysis of the prognostic value of CpG island methylator phenotype in rectal cancer
International journal of colorectal disease
2,018
cc-by
4,497
International Journal of Colorectal Disease (2018) 33:995–1000 https://doi.org/10.1007/s00384-018-3108-5 International Journal of Colorectal Disease (2018) 33:995–1000 https://doi.org/10.1007/s00384-018-3108-5 REVIEW REVIEW REVIEW 1 ABMU Singleton Hospital, Sketty Lane, Swansea, Wales SA2 8QA, UK Abstract bst act Purpose The pathological and prognostic importance of CpG island methylator phenotype (CIMP) in rectal cancer, as a sub- population of colorectal cancer, is unknown. A meta-analysis was preformed to estimate the prognostic significance of CIMP in rectal cancer. Methods A systematic search was performed of PubMed, Embase, MEDLINE, PubMed Central, and Cochrane electronic databases for articles pertaining to CIMP and rectal cancer. Articles were analysed and data extracted according to PRISMA standards. Results Six studies including 1529 patients were included in the analysis. Following dichotomisation, the prevalence of CIMP- positive tumours was 10 to 57%, with a median of 12.5%. Meta-analysis demonstrated the pooled odds ratio for all-cause death for CIMP-positive tumours vs CIMP-negative tumours was 1.24 (95% CI 0.88–1.74). Z test for overall effect was 1.21 (p = 0.23). Heterogeneity between the studies was low (X2 5.96, df 5, p = 0.31, I2 = 16%). A total of 15 different loci were used for assessing CIMP across the studies, with a median of 6.5 loci (range 5–8). Conclusions No significant association between CIMP and poor outcomes in rectal cancer was demonstrated. There was a high degree of heterogeneity in CIMP assessment methodologies and in study populations. Rectal cancer datasets were frequently not extractable from larger colorectal cohorts, limiting analysis. Keywords Methylation . CIMP . Rectal cancer . Biomarkers Keywords Methylation . CIMP . Rectal cancer . Biomarkers Meta-analysis of the prognostic value of CpG island methylator phenotype in rectal cancer R. F. Kokelaar1 & H. Jones1 & J. Beynon1 & M. E. Evans1 & D. A. Harris1 Accepted: 14 June 2018 /Published online: 20 June 2018 # The Author(s) 2018 Introduction techniques and the judicious use of neoadjuvant chemoradia- tion (nCRT) [3]. The potential for organ preservation is also increasingly accepted due to the introduction of minimally invasive surgery and ‘watch-and-wait’ strategies of non- operative management [4–7]. Despite advances, there is how- ever an inability to determine which patients may benefit from one treatment modality or another based upon anything other than traditional radiological and histopathological staging [8, 9]. As the era of personalised medicine advances, there is an increasing need for molecular biomarkers that will aid deci- sion making in the preoperative phase. Colorectal cancer (CRC) is a significant health problem due to high prevalence and mortality, representing the third most common cause of cancer death in the USA [1]. Rectal cancer (RC) as a sub-population of approximately 30% of CRC poses additional significant mortality, morbidity, and management challenges, due to the anatomical confines of the bony pelvis and proximity of significant neurovascular structures and oth- er organ systems [2]. The management of rectal cancers is currently undergoing a paradigm shift; previously inoperable locally advanced and locally recurrent rectal cancers are in- creasingly rendered operable due to improved operative The molecular and genetic aberrations that underlie CRC carcinogenesis are complex and not fully understood, al- though there is a consensus that there are divergent processes responsible for tumour development at different sites through- out the colon and rectum [10]. DNA methylation is one epi- genetic process implicated in CRC, as well as other cancers, and CpG island methylator phenotype (CIMP) has drawn in- terest as a potential mechanism underlying both carcinogene- sis and as a potential biomarker [11, 12]. CIMP, however, has * R. F. Kokelaar rory.kokelaar@gmail.com Int J Colorectal Dis (2018) 33:995–1000 996 primarily been associated with carcinogenesis in the right co- lon that is characterised by hypermethylation and microsatel- lite instability (MSI) (the serrated pathway), rather than the traditional chromosomal instability pattern typical of other sites in the colon and rectum [13–15]. Despite the preponder- ance in the right colon, CIMP tumours are known to occur in the rectum, although the clinical significance of this molecular tumour type occurring at this site is poorly understood, al- though some authors have suggested they represent a poor- prognostic subgroup [16, 17]. Statistical analysis Analysis was conducted using RevMan statistical software (v 5.3. Pub: The Cochrane Collaboration). Heterogeneity was calculated as the chi-squared value (X2, df −1) and the I2 statistic [24]. Overall effect was calculated by Z test with sig- nificance set as p < 0.05, based on meta-analysis employing Mantel-Haenszel odds ratio (OR) for CIMP status and 5-year overall survival rates with 95% confidence intervals (CI). Definition of CIMP No consensus definition of CIMP exists across the published literature. Studies variable report a binary CIMP +ve or -ve, or groupings of CIMP-high(-H), CIMP-intermediate(-I), CIMP- low(-L), or CIMP-negative(-N). For the purposes of this re- view, dichotomisation of different CIMP classifications was performed so to classify results into CIMP +ve (including CIMP +ve, CIMP-high, and CIMP-intermediate groups) and CIMP -ve (including CIMP-low and CIMP -ve/-N). This pro- cess is keeping with that of other authors [21]. Results All types of study were included in the analysis, although reviews, meta-analysis, and book chapters were excluded. Exclusion criteria were single-locus or gene methylation stud- ies, studies where DFS and OS outcomes were not reported, and studies where the rectum was not defined as in identifiable cohort within colorectal cancers. Studies where surgery was not performed with curative intent were also excluded. Electronic search yielded 203 original articles, once duplica- tions had been removed. No additional studies were found in the grey literature. Following review of abstracts, 160 articles were excluded on the basis of not relating to colorectal cancer (2), CIMP not basis of analysis or not assessed (89), single locus/gene methylation only (3), survival outcomes not assessed (59), and being review articles (7). The remaining 43 articles then underwent full manuscript screening for eligi- bility, where a further 37 were excluded on the basis of the colon and rectum being assessed as a single cohort (13), and the rectum not being defined as an individual cohort separable from the left or distal colon (24) (Fig. 1). The remaining six studies were included, and baseline and CIMP/outcomes data extracted (Table 1) [16, 17, 25–28]. Introduction Many of the studies that have examined the role of methylation in RC or indeed CRC have focused on a single gene locus or have relied upon small cohorts, making outcomes interpretation challenging [18, 19]. The aim of this paper is to review and meta-analyse the prognostic value of CIMP in adenocarcinoma in the rectum. institution, country, total number of patients, sex, TNM stag- ing, method of determining CIMP, and methodology. Numbers and/or percentages of patients expressing CIMP was extracted, as were DFS and OS. Search protocol Two reviewers independently assessed the quality of included studies using the Newcastle-Ottawa (N-O) scale [22]. A score of 6 out of 9 criteria fulfilled on assessment of selection, com- parability, and outcome was used as a cutoff for inclusion in the analysis, which was itself conducted according to PRISMA guidelines [23]. An online search was performed to retrieve original research articles where CIMP was assessed in rectal adenocarcinoma specimens, and where outcomes data were assessed (disease- free and overall survival (DFS and OS)). PubMed, Embase, MEDLINE, PubMed Central, and Cochrane databases were searched using the Boolean terms (CpG island methylator phenotype OR CIMP) AND (cancer OR carcinoma OR ade- nocarcinoma OR tumor OR tumour) AND (colorectal OR rectal) AND (prognosis OR outcome). A cutoff for inclusion was January 2018, results were compiled in a reference man- ager, and duplicates were removed. The grey literature was examined for additional contributions. Study design and search strategy was registered pre-emptively at PROSPERO (registration number CRD42018099569) [20]. Data extraction Two reviewers independently applied the exclusion criteria to retrieved abstracts, and discrepancies were agreed by consen- sus. Data was extracted by one author from full-text manu- scripts, and each dataset was verified by an independent re- viewer. Baseline data for each study included author, date, Int J Colorectal Dis (2018) 33:995–1000 997 Retrieved Arcles: 203 Screened for eligibility: 43 Analysis: 6 Excluded based on full text assessment: 37 Large bowel assessed as single cohort: 13 Rectum not separated from le / distal colon: 24 Excluded based on tle/abstract: 160 Not colorectal cancer - 2 Non-CpG methylaon focus of analysis – 89 Single gene methylaon only – 3 Not related to prognosis – 59 Fig. 1 Consort diagram showing selection criteria for inclusion in the analysis Jo (II–IV). The approximate mean age of the patients was 62– 65 years, and 68% were male. Three studies specify that all patients were nCRT naïve, two make no statement regarding nCRT prior to tissue sampling, and one specifies all patients received nCRT (Table 1). Excluded based on tle/abstract: 160 Not colorectal cancer - 2 Non-CpG methylaon focus of analysis – 89 Single gene methylaon only – 3 Not related to prognosis – 59 CpG island methylation status was assessed in a median of 6.5 loci (range 5–8), with a total of 15 different genes employed across all studies. CIMP characterisation is de- scribed in all papers, two employing a +ve/ve strategy and the others employing variable strategies of CIMP-H/-I/-L/-N (Table 2). Following dichotomisation, the prevalence of CIMP +ve tumours ranged from 10 to 57%, with a median of 12.5%. Two studies reported a positive association between CIMP and overall poor survival, and four reported no association. Meta-analysis demonstrated that the pooled OR for all- cause death for CIMP +ve tumours versus CIMP -ve tumours was 1.24 (95% CI 0.88–1.74). Z test for overall effect was 1.21 (p = 0.23) (Fig. 2). Heterogeneity between the studies was low (X2 5.96, df 5, p = 0.31, I2 = 16%). Single and mul- tivariate analysis of individual genes used in assessing CIMP did not demonstrate any association with outcomes. Pooled analysis of clinicopathological factors assessed in relationship to CIMP and survival was not possible due to inconsistent reporting and non-extractable data. Meta-analysis demonstrated that the pooled OR for all- cause death for CIMP +ve tumours versus CIMP -ve tumours was 1.24 (95% CI 0.88–1.74). Data extraction Z test for overall effect was 1.21 (p = 0.23) (Fig. 2). Heterogeneity between the studies was low (X2 5.96, df 5, p = 0.31, I2 = 16%). Single and mul- tivariate analysis of individual genes used in assessing CIMP did not demonstrate any association with outcomes. Pooled analysis of clinicopathological factors assessed in relationship to CIMP and survival was not possible due to inconsistent reporting and non-extractable data. Fig. 1 Consort diagram showing selection criteria for inclusion in the analysis The median N-O score for the included studies was 7 (range 6–7). No studies were excluded on the basis of quality assessed by this metric. Five studies were based on retrospec- tive cohorts and one was based on a cohort extracted from an ongoing phase III clinical trial [17]. CIMP analysis was per- formed on resected specimens in all but one study (Jo [17]; pre-treatment biopsies) and all employed poly-locus method- ologies. Two studies assessed methylation in CRC but had extractable primary outcome data for a RC sub-cohort, al- though extractable clinicopathological data was often not available for this sub-cohort [16, 27]. The remaining four studies only assessed RC. Discussion This study demonstrates the significant heterogeneity in methods used to assess CIMP in RC, but that across a range of gene panels, CIMP has not been shown to be associated with worse overall survival in rectal cancer. Confidence inter- vals for individual studies were broad and each range crossed OR 1.0. Of the two studies that did report an increased risk of death, the 5-year survival rates were reported as 63.2 and 25% respectively, although numbers in the second group was ex- tremely small (n = 5). DFS was not reported consistently or extractably across studies and was therefore not meta- analysed, although again, there was conflicting data presented between studies. Jo reports that although there is no difference The six studies included 1529 patients with a mean sample size of 254 (range 78–864). Only one paper reported a cohort of less than 100 (Kim; 87). Each of the studies included pa- tients with AJCC stage I–IV tumours, except Williamson and Table 1 Characteristics of included studies Reference No. of patients Study interval Age range (mean) Men (%) AJCC nCRT N-O score Samowitz et al. 2009 [24] 864 1997–2001 30–79 (nr) nr I–IV nr 6 Jo et al. 2011 [17] 150 2004–2006 nr (61) 71 II–IV No 6 Bae et al. 2013 [16] 168 2004–2006 36–87 (62) 67 I–IV No 7 Williamson et al. 2017 [25] 160 2002–2011 nr (65) 71 II–IV Yes 7 Kim et al. 2017 [26] 87 2006–2007 31–88 (65) 59 I–IV nr 7 Kokelaar et al. [27] 100 2010–2013 24–89 (71) 70 I–IV No 7 nr not recorded/extractable Int J Colorectal Dis (2018) 33:995–1000 998 Table 2 CIMP methodologies Reference CIMP markers CIMP classification CIMP association with outcome Samowitz et al. 2009 [24] hMLH, MINT1, MINT2, MINT31, CDKN2A CIMP-positive vs CIMP-negative CIMP-high poorer survival (p < 0.040) Jo et al. 2011 [17] SOCS1, RUN3, NEUROG1, IGF2, CACNA1G CIMP-positive vs CIMP-negative No statistical significance (p > 0.050) Bae et al. 2013 [16] hMLH1, CDKN2A, SOCS1, RUNX3, NEUROG1, IGF2, CACNA1G, CRABP1 CIMP-high vs CIMP-low vs CIMP-negative CIMP-high poorer survival (p = 0.019) Williamson et al. 2017 [25] hMLH1, MINT1, SOCS1, NEUROG1, THBD, HAND1, ADAMTS1, IGFBP3 CIMP-high vs CIMP-intermediate vs CIMP-low No statistical significance (p > 0.050) Kim et al. 2017 [26] SOCS1, RUN3, NEUROG1, IGF2, CACNA1G CIMP-high vs CIMP-low vs CIMP-negative No statistical significance (p > 0.050) Kokelaar et al. Discussion [27] hMLH1, MINT1, SOCS1, NEUROG1, THBD, HAND1, ADAMTS1, IGFBP3 CIMP-high vs CIMP-intermediate vs CIMP-low No statistical significance (p > 0.050) The factors accounting for the differences in these obser- vations are likely to be multifaceted, although differences in patient cohorts and lack of statistical power make interpreta- tion difficult. In this analysis, one study relied upon a popula- tion set drawn from a randomised trial of nCRT, and thus may not be representative of the wider population [17, 29]. There was also a wide geographical variation in datasets, representing populations with likely significant differences in clinical factors such as body mass index and smoking sta- tus. Clinicopathological variables may account for some if the variation in results was not able to be sub-analysed due to data being non-extractable, frequently because it was not consis- tently presented across the studies or was not presented sepa- rately for rectal cancers within a larger colorectal cohort, thus making multivariable analysis impossible. These factors are keeping with the wider experience in CRC methylation re- search [18]. Despite none of the studies being excluded based on their respective N-O scores, the overall quality of the in- cluded studies was only good–fair, with a median score of 7 out of a possible 9. in OS, there was a statistically significant relationship between poorer DFS and CIMP-positive tumours (p = < 0.010), in agreement with Bae and Kim, who also report a worse DFS with CIMP-H (p = 0.042 and p = 0.018, respectively). This finding is however directly contradicted by Kokelaar, where pooled CIMP-H and CIMP-I were not associated with DFS (p = 0.10). in OS, there was a statistically significant relationship between poorer DFS and CIMP-positive tumours (p = < 0.010), in agreement with Bae and Kim, who also report a worse DFS with CIMP-H (p = 0.042 and p = 0.018, respectively). This finding is however directly contradicted by Kokelaar, where pooled CIMP-H and CIMP-I were not associated with DFS (p = 0.10). Within the broader context of CRC, CIMP has been demonstrated to be significantly related to both DFS and OS. A systematic review and meta-analysis by Juo analy- ses 33 studies and extracted data representing 10,635 pa- tients, finding that the OR for DFS and OS was 1.45 (95% CI 1.07–1.97) and 1.43 (95% CI 1.18–1.73), respectively, for CIMP-positive tumours on the basis of pooled dichotomised analysis [18]. Fig. 2 Pooled analysis between CIMP and OS References 1. 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Conflict of interest The authors declare that they have no conflicts of interest. 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 appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Discussion The reported prevalence of CIMP in the included studies was 4.6 to 46.5%, with a median of 18.2%. Despite the pooled analysis demonstrat- ing a worse prognosis for DFS and OS, 8 out of 11 and 13 out of 19 studies in this meta-analysis failed to identify any significant relationship between DFS and OS, respectively. 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